THE EFFJ!

X:T OF HEAT ON WOOL

AND RELATID PROTEINS

A thesis submitted to

The University of New South Wales

for the degree

of

Doctor of Philosophy

by

Jeanette G. Norman

School of Textile Technology,

The University of New South Wales. August, 1970.
 ACIGWW LEl>G l~~LEll 'IB

'l'he author wishes to acknowledge with sincere thanks the

invaluable advice and assistance given in this work by ASsociate

Professor c. H. Nicholls.

The author also thanks her colleagues for helpful discussions

and assistance and her husband for his continual encouragement.

The Australian Wool Board is thanked for providing funds for

equipment which assisted in the execution of this work. A

Commonwealth Post-Graduate Award was provided for the duration of

this study.
 This is to certify that the work described in this thesis was
done by me in the School of Textile Technology-, The University of

New South Wales, and has not been submitted previous!y for any other
University degree or award.

Signed:
 CONTJ!NTS
Page
CHAPTI!Ji I. INTRODUCTION
1. The Chemistry of Proteins 1

2. The Action of Heat on Proteins 13

CHAPTJ!R II. SP~IFIC CH!MICAL REACTIONS CAUSED BY H&\T

1. Preliminary Experiments 20
2. Reactions Involving the Peptide Bond 24
3. Decanposition of Cy"stine 30
4. Reactions Involving Tryptophan 42
5. Reactions Involving the Hydroxy Side Chains 52
6. Reactions Involving the Free Amino Group 59
7. The Effect of Metall.ic Ions 72

CHAPT!R III. ISOLATION AND ID!ffi'IFICATION OF THE YEI..LCM CO!PON!NTS

1. Introduction 80
2. &lzymatic Hydrolysis 81
3. Mild Acid Hydrolysis 83
4. Chromotographic Separation 83
5. Analysis of Fractions 86
6. Amino Acid Analysis 88
7. Absorption Spectra 90
8. Fluorescent Spectra 91
9. Fo:nnation of Pyrroles 93

CHAPTI!Ji IV. SlJttMARY 105

 Page

APPINDIX 100

RBFBRBNC!S 112
 ABSTRACT

The effect of dry heat on a variety of proteins has been investigated

and attempts made to relate colour changes with specific reactions.
Bvidence showing that the configuration of the protein stron~ influences
the extent of degradation of protein bound amino acids has been obtained,
so that generalisations from one protein to another and the use of model
compounds are not always valid.
Amino acid analyses show that cystine, tyrosine, serine, threonine
and tryptophan are significantly degraded by heat. OXidation contributes
to the degradation of tryptophan and the amide bond.
A variety of inter- and intra-chain reactions occur on heating
although not all contribute to yellowing. Condensation of the -amino
gr-oup of lysine with a free carboxyl group appears to be the first step
in the formation of pyrrole type compounds which cause yellowing.

Degradation of tryptophan also contributes to the heat yellowing of wool.

The extent of yellowing may be reduced by treatments with dimethyl
sulphate, p-nitrophel\V'l acetate or by certain metallic ioos. B>cplana-

tions for the effectiveness of each treatment are proposed.

Procedures to isolate yellow products from heated wool are described
along with analytical data of the fractions obtained.
Chapter I: INTRODUCTION

1. The Chemistr:,y of Proteins.

Proteins are a class of naturally occurring compounds of high

molecular weight, which are an essential constituent of tissues of plants
and anillals and are thus very widespread. Included among the proteins
are the enzymes which selectively catalyse most of the chemical reactions
essential for normal cellular function; some of the hormones which
regulate metabolic processes; antibodies elaborated by an organism to
counteract agents harmful to it; some of the bacterial toxins; con-
tractile proteins such as ~osin of the muscle; respiratory proteins
such as haemoglobin and proteins for protective purposes such as hair and
homs. Proteins are used widely in industry in plastics, adhesives and
paints; the textile industry uses vast quantities of wool, silk and
mohair and in the past fibres formed from casein and zein.
In nature they seldom occur as a pure protein but usually as a
mixture of proteins, the complexity of which varies according to the
aource. Those found in the cells of animal tissues and micro-organisms
are made up of a variety of proteins whereas others such as casein or
lysozyme contain a much smaller number with one component predominant.
lven insoluble proteins such as wool are not homogeneous, however
separation into the various component proteins is so difficult that these
proteins are usually treated as entities.
The solubility of proteins in water varies within wide limits.
Same proteins dissolve easily in salt-free water, others dissolve only
in the presence of certain concentrations of salts; a third group is
insoluble in water but soluble in mixtures of water and ethanol, while
a fourth, the scleroproteins do not dissolve in &nT solvent. This
differing solubility has been chosen as a basis for classification of
proteins into albumins, globulin-like proteins, prolamines and sclero-
proteins. Classifications of this kind are more or less artificial
and are of restricted value. Thus some haemoglobins are alJllost
insoluble in the absence of salts but promptly dissolve 'When salts are
added. The solubility of proteins also depends on the pH of the
solution, with minimum solubility occurring at the isoelectric point.
Klemental analy'ses of proteins have shmm them to consist of carbon,
hydrogen, nitrogen and, by inference, oxygen;. in addition sane contain
aulphur or phosphorus. Hydrol,ysis of prot.eins yields a mixture or
e1 amino acids of formula NH2 .CEm.COOH and at least twenty-five different

amino acids have been isolated.

In 19~ 8nil Fischer! and Franz Hofmeister2 independently advanced

the hypothesis that in proteins the e1 amino gi-oup of one amino acid and
the e1 carboxyl group of a second amino acid are joined, with the
elimination of a molecule of water, to fom an amide linkage. The
product of such condensation is a •peptide" and an amide linkage joining
two amino acids is a "peptide bond" or "peptide linkage•.

N112•1H-CO.NH.r.CC>al
R1 Rz
Amino Acid Peptide Bond
 Since this theory was first proposed, considerable evidence
supporting it has accumulated.
1. Most proteins contain relatively few titratable amino or
carboxyl groups canpared to the number liberated by total hydrolysis
of the protein. During hydrolysis these groups appear in equal numbers,
as would be expected during the hydrolysis of amide linkages.
2. If a protein is partially degraded by acid h,Ydroly-sis it is
possible to isolate compounds in which two amino acids are joined by a
peptide bond.
3. Campounds containing peptide bonds give a characteristic
purple colour when treated in alkaline solution with copper sulphate.
This is termed the "biuret reaction" because it is given by a substance,
biuret NH2 •CO .NH. CO .NHz • The colour deepens as the number of peptide
bonds in .a series of synthetic peptides is increased, and proteins
produce an especially deep blue-violet colour.
,4.. Bnzymes lmown to cataly-se the bre&kdC>lCl of proteins to amino
acids (e.g. pepsin, trypsin) also h,ydroly-se peptide bonds in simple
synthetic peptides.
In accepting the peptide bond hypothesis it is still necessary to

consider how amino acids such as ly-sine, arginine, glutamic and aspartic
acids are linked in proteins. The present evidence indicates that in
most cases the l.-amino group of lysine, the gu.anidino group of arginine
and the imid&zole group of histidine do not take part in amide linkages
with other amino acids. This conclusion is based in part on the results
of titration experiments and in part on the finding that these groups are
available for reactions \fflich would not be observed if the groups were
substituted. S1ro1Jarl,y, the ~ and ~ carboxyl groups of the diacid
amino acids are not usually involved in peptide bonds. HOM!lver, a
number of small peptides have been isolated \'mere these groups are
involved3, a typical example is glutathione or y- glut,aivlcysteinylg].,ycine.
It should also be noted that in many proteins sane of the side chain
acids exist in the amide form i.e. g].utamine fran g].utamic acid and
asparagine from aspartic acid.
To understand the structure of proteins and the activity of
substances such as enzymes, it is necessary to understand the internal
bonding which gives proteins their characteristic shape. Mirsk;r and
Paulinlf suggested that a major factor in conferring its characteristic
folding upon an extemed peptide chain is the presence of eydrogen
bonds between the peptide CO and NH groups. Although these bonds are
quite weak individually, (approx. 5 kcal/mol), if a molecule has many
eydrogen bonds, they will reinforce one another, and thus produce a
stable structure. Bvidence for the existence of such bonds in poly-
peptides and proteins has cane mainly fran infra-red spectroscopy and
bond distance measuranents fran X-ray diffraction.
Another important stabilising bond believed to exist in proteins
is the so called •salt linkage", which is an ionic attraction between
negatively' charged coo- groups aoi positiYely charged NH3+ ones, e.g.
-coo--- H3...,_· Bvidence for salt linkages in wool includes the
fact that a reduction in work required to stretch a wool fibre occurs
\'men the pH of the so.lution in which the wool is iamersed, is removed
fram the iso-ionic point. Additional evidence is given by the fact
that the swelling of wool imnersed in water is a mininum if the pH of
the solution used is close to the iso-ionic point of the protein.
When proteins are placed in aqueous solutions both salt linkages
and hydrogen bonds are broken, however, the structure is now stabilised
by "hydrophobic bonds", which are due to the presence of numerous non-

polar side chains which aggregate micelle-1.ike in the interior of the

protein molecule in an h,Ydrophobic environment. 'Scheraga and Schrier5
provided evidence for the existence of hydrophobic bonds by study"ing the
influence of alcohol/water mixtures on proteins. They found that the
temperature at which the helices of ribonuclease "melt" is higher in
aqueous solutions than in aqueous alcoholic solutions; and furthermore,

that alcohols which lower this conversion temperature the more effectively,
have longer unbranched alkyl residues.
Van de Waals 1 forces also exist in proteins and have a stabilising
effect.
A phenomenon peculiar to proteins and still a srurce of cmtroversy
is "denaturation". This has been defined6 as "an.Y non-proteolytic
modification of the unique structure of a native protein, giving rise to
definite changes in chemical, ph,Ysical or biological properties•.
The coagulation of egg white by boiling is a classical example of
denaturation. Ammg the maey causes of denaturation are treatments
with high temperatures, organic solvents, ver:, high or very low pH,
ultra-violet radiation, solutions of £:tt urea, detergents and mechanical
action. Denaturation is usual.ly observed as a reduction in solubility,
 6.

increase in viscosity, increase in both the reactivity and number of

reactive groups such as -sH, loss of biological activity and inability
to crystallise.
The explanation most widely accepted at present is that denaturation
involves the breaking of hydrogen bends and unfolding of the peptide

cha.in. According to this hypothesis "reversible denaturation" occurs

vien the chain has only slightly unfolded and can be refolded into its

original fo:nn. As the degree of unfolding increases the probability

of exact refolding decreases until finally "irreversible denaturation"
has occurred. Unfolding ot the chains would allow reactive groups to
become more accessible and hence would explain their apparent increase
upon denaturation. It has also been suggested? that denaturing causes

a spatial redistribution of the polar and non-polar gi-oups, resulting

in the transfer of non-polar, hydrophobic groups to the surface of the

particle and thus causing a decrease in solubility. Denaturation is

therefore a property of the protein as a whole and cannot be attributed
to any specific group.
The reactivity of a protein is influenced by both its physical
structure and its amino acid canposition. The peptide chain may be
regarded as the "backbone• of the protein and is relatively inert to
most chemicals. HCMever, it is very succeptible to hydrolysis by acids,
alkalis am sane enzymes. Blackbum, Phillips and Carter8 claim to
have 0-Gieth,ylated the peptide bond and oxidation of the bcmd has been
reported9. Condensations involving the peptide bond and certain side
chains have also been reportedlO ,11.
 The side chains carried by the polypeptide backbone of the protein
var:, in size and reactivity and may be grouped according to their
chemical. nature.
1. Amino Acids containing hydrocarbon side chains •
These include glycine, alanine, valine, leucine, iso-leucine and
phenylalanine. They are non-polar, relatively chemically inert and
are involved in the fonnation of hydrophobic bonds.
2. Amino Acids containing basic side chains.
These are the dibasic, monoacidic acids arginine and lysine which
contribute strongly basic terminal guanidino and amino groups respectively,
and histidine which has a weakl,y basic imidazole group.
3. Amino Acids yielding acid side chains.
This group includes the monobasic, di-acidic acids, glutamic and
aspartic acids; which yield side chains wi. th a teminal carbmc;yl group.
In many proteins sane of these carboxyl groups are in the acid amide

form, i.e. glutamine and asparagine.

4. The aromatic amino acids.
These are tyrosine and tryptophan, al.though the latter is actual.ly'
heteroc;rclic in nature. Both have very reactive side chains and are
important 'When considering the chemical reactivity of maey proteins.
The fluorescent properties of proteins are chiefly due to these amino
acids.
5. The 8-HY'drax.y amino acids.
Two amino acids, serine ani threanine are included here. Both
have reactive, aliphatic hydroxy side groups 'Which are undoubtedl;r
 a.

involved in many protein reactions. The calcium phosphate group in

casein is believed to be linked through serine.
6. The sulphur-containing amino acids.
This group comprises cystine, cysteine, and methionine, of which
cystine is the most important. Cystine is the only' naturaJ.l.y- occurring
amino acid able to link two peptide chains together, which it does by-
means of the disulphide bond to give the follow.i.ng linkage:

I I
NH C• 0
I I

Cystine occurs abundantly in the keratins and is important in

detemining their properties.

In the present work a number of peptides and proteins have been

studied arv:i these will be discussed more fully. Their amino acid
camposition is sho~ in Tabler.

1. Glutathione.
The only relatively simple peptide available for the study of
cysteine is glutathione or cY•glutamylcysteinylglycine. This peptide
is unusual in that one of the peptide links is through the K carboxy-1
of glutamic acid. It is easil,Y obtained in an oxidised fom and since
cystine is the most reactive camponent in the resulting peptide, this
 TABLI I

The Amino Acid Composition of Selected Proteins lx:pressed as @JDS•

Amino Acid/100 pm. dry protein,

Amino Acid Caseinl2 Gel.atinl2 Zeinl2 IJ"sozyme63 si.1Jtl2 Woo164

arginine 4.1. 9.4 1.8 13.1 0.9 10.5

histidine 2-4 1.0 1.7 1.1 0.3 0.9
lysine 7 .8 5.7 o.o 5.9 o.6 2.8
tyrosine 6.7 o.6 5.2 3.7 10.0 6.4,
tryptopha n 1 .4 o.o 0.1 8.7 0.4 o.8
phenylala nine 5.1 2.6 6.4, 3.1 1.3 3-4
cystine 0.3 0.1 1.0 8.o o.o 11.3
methionine 3 -4 0.9 2.3 2.0 o.o 0.7
threonine 4 .o 2.2 3.0 5-4 1.3 6.6
serine 7.3 4 .o 7.7 7.0 13.9 9.0
leucine 9.9 4 .o 23.7 7.0 o.8 7.6
isoleucin e 6.3 1.9 7 .3 5.2 1.0 3.1
valine 6.5 3.2 3.0 4 .8 2.9 5.0
glutamic acid 23.8 ]2.6 26.6 4.3 2.0 15.0
aspartic acid 6.1 7.1 5.6 16.0 2 .4 6.7
glycine 1.9 27.0 o.o 5.7 37-4 5.2
alanine 27-2 9-4 11-4 6.0 22.2 3.7
proline ]2.7 17 .5 10-4 1-4 7 .3
hydroxyproline 0 14.9 o.o o.o
 10.

foi,n is very useful for studying the reactims of protein bound cystine.

2. ~-
Zein is a member of the group of proteins lmown as prolamines.
It is soluble in an aqueous solution of 50-9~ a.lcohol but is insoluble
in absolute alcohol or water. Zein is a pa.le yellow protein containing
no l,ysine, very little arginine or histidine and negligible tryptophan.
Lack of basic side chains means zein cannot form many salt-links or
amide cross-links with di-acidic acids while lack of tryptophan removes
the possibility of tryptophan breakdown during degradation.
3. Casein.
Casein is a mixture of proteins of molecular weight ranging from
75,000 to 375,000. It is obtained from milk and contains most of the
more reactive amino acids. It is water-soluble and in many cases can

be used as a water-soluble substitute for wool in studying specific

reactions. The hydr()JC1'1 goup of ser.ine is believed to be bound to a
phosphate group and casein is thus one of the proteins incorporating a
non-protein group.
4. Lysoz.yme •
IQsozyme is a white, crystalline, highly' water soluble pCMier
obtained in an almost pure form fran egg white. It has a ID0l.ecular
weight of about 14,000 and contains relatively large quantities of
cystine, tyrosine and tryptophan. IQsozyme is one of the few proteins
whose tertiary structure is lmown.

5• Gelatin.
Gelatin is a water soluble protein extracted from collagen and
 u.

has an amino acid content very similar to collagen. Although it

contains considerable quantities of basic groups, gelatin contains no
tryptophan, cystine or cysteine but is rich in praline and hydroxy--
proline. It is an unusual protein in that it contains a number of
different crosslinks and groups such as aldehydes3 not normal.l,y
encountered in proteins. Despite this, its amino acid composition
makes it a most useful protein for heat degradation studies. An

interesting property of gelatin is its ability to form gels containing

onl,y 2 or 3 iPS• of gelatin but 70 or 80 iPS• of water. These gels
have wide industrial. applications.
6. Silk.
Silk is a simple, fibrous protein produced in cont:inuous filament
form by the larvae of silk worms. Raw silk conta:ins two proteins lmown

as fibroin and sericin, the latter is removed during processsing and in

most cases the term "silk• refers to the fibroin onl,y. On hydrolysis,

fibroin yields fiftem amino acids, of which the amino acids glycine,
alanine, serine and, to a lesser extent, tyrosine, predominate.
Fibroin contains negligible cystine and hence no disulphide bonds to
link peptide chains. During hydrolysis, no amnonia is evolved which
indicates that none of the free carboxy-1 groups are in the amide form.
Because of the relatively few bulky side chains, it is possible for the
main chains of fibroin to approach each other closely, and form hydrogen
bridges from one chain to another via the polar amide groups. Thus a
solvent such as cupriethylene-diamine, with a strong capacity for
breaking hydrogen bonds, will dissolve silk.
 COli'TIC A~ C£LL5

Fig. 1: Schematic diagram of the morphologic al structure of

a wool fibre.
 12.

7• Wool.
Contrasting to the relatiYe simplicity of silk, wool is one of the
more complex proteins, due, in part, to its complex morphological.
structure. As shown in Fig. I the fibre consists of an outer 1'Yer of
scales lmown as the cuticle, surrounding a network of fibrill.ar cells
termed the cortex. In finer fibres the cortex itself is devided into
two, the ortho appearing more reactive than the para, being less highly
keratinised. Kl.ectron microscopy has shown that the fibrils in the
cortex can be further divided into microfibrils. It is generally
considered that the components of the wool fibre are held together by a
cement-like substance lmown as the matrix, the canposition of 'Nhich is
quite different to that of the microfibrils.

Since the canponents of wool fibres can only be separated with diffi-

culty, chemical analyses are genera.1.ly carried out on the intact fibre and
the result obtained includes contributions fran a.11 components. The re-
lative proportions of the various canponents will therefore influence the
composition of the fibre. Sane arnnonia is released during eydrolysis;
this is presumed to cane from the amide groups of the dicarboxylic acids.
Wool contains about lQC cystine, which, b.r fonaing covalent link•
between two peptide chains is partly responsible for its being insoluble
in solutions such as cupriethylenediamine. Wool also CCl'ltains a high
percentage of basic and acid grwps, so provided these are sited
relatively close to each other, salt linkages ~uld be expected to fem.
Additional. polar groups are provided by serine, threonine and tyrosine.
Both tyrosine and tryptophan provide aromatic, reactive groups "'11ch
significantly influence wool•s chemical. reactivity.

2. The Action of Heat on Proteins.

When a protein is subjected to dry heat, it undergoes a series of
changes dependent on both the nature of the protein and the conditions
of heating. Most of the publications on the effect of heating soluble
proteins has been directed towards biological rather than chemical.
changes, and, as a result, have little relevance to the present work.
H<Mever, considerable work has been done on fibrous proteins, especially
11«>01, and many of these conclusions also apply to soluble proteins.
Woodmanseyl-3 showed that heating at 100°c in air, reduced the
ability of wool fibres to absorb water. Others have since obtained
similar results but have not put forward a satisfactory explanation.
Some authors14,l5,l6 ,l7, have reported a decrease in the urea-bisulphite
solubility of wool heated for several hours at temperatures as low as
60°c, this was generally attributed to the conversion of cystine
disulphide bonds to lanthionine. However, Zahn and Kessle~ later
showed there was no correlation between insolubility and lanthionine
f o:nnation, independent of pretreatment. Lindley and Phillips19 suggested
that heating may cause intrachain disulphide bonds to be converted to
interchain bonds which were less accessible to the reagent, but offerred
no proof of this theory. The formation of new, l\Ydrogen bonded
structures M'lich were resistant to urea-bisulphite solution has been
suggested by both 5wan20 and 5pea1anan21.
zam22 heated dry horsehair at various temperatures for 24. hours
 14.

and found it became progressively less able to contract in ,C sodium

bisulphite or in 20.C aqueous phenol and found also a significant
reduction in acid ccmbining properties and suggested that this may be
caused by formation of new amide bonds between free amino and carbaxyl
groups. Mazingue and Van Overbeke23 al.so reported modification of the
free amino and carbolc;yl groups in heated wool and silk and again proposed
formation of amide crosslinlcs. It was also found24 that the apparent
lysine content in heated casein (unhydrolysed values) was much lower
than those obtained on hydrolysis, supporting the theory that amide
link■ are formed during heating and broken during hydrolysis. Since
proteins contain so .many amide bonds it is difficult to obtain direct
evidence for or against this theory; it is, however, another possible
explanation for the changes in urea-bisulphite solubility of heated wool.
Changes in solubility on heating have also been reported for the
soluble proteins. Unfortunately, these proteins can also exhibit
solubility changes as a result of denaturation and few workers have
attempted to differentiate between changes in solubility- due to denatura.-
tion and those caused by actual chemical changes occurring on heating.
Food chemists25,26,27,28,29, have reported that heated proteins were
less susceptible to enzymatic attack and harder to digest30,3l. Since
they al.so report losses in "avilable" lysine and up to lQC reduction in
free amino content without any- loss of lysine in the hydrolysate of
heated proteins, formation of new amide bands would al.so seem a possible
explanation for these changes in soluble proteins.
Si~icant cystine losses occur when proteins are heated, deccmposi-
 15.

tion products reported have included cysteic acid32,32a, cysteine33,

lanthionine34.,35,36, hy'drogen sulphide1 3,37, sulphuric acid and its
salts37, free sulphur38 and even non-identifiable, volatile sulphur
caapounds37, while Asquith et a140 have reported l,ysino-alanine and
~-aminoalanine among the decomposition products of heated wool.
Alt.hough considerable disagreement exists as to the decomposition

products, most authors agree that temperature and moisture content are
highly significant. Woodm&nseyl.3 reported production of hydrogen
sulphide at 100°c while Kasarda and Blacltl'1 claimed no significant
volatile products were produced at temperatures below 130°c. Most
papera35,36 in wu.ch an increase in lanthionine content has been
reported used conditions of heating at 16o0 c or higher. Several
aut.hora'-2,43 have reported that in com.pletelJ"' anhydrous conditions,
protein bound cystine is stable to heat but even O~ moisture is
sufficient to promote disulphide degradation.
A second amino acid suspected of being unstable to heat is tyrosine

but again reports vary. Bell, Clegg and Whewen'.33 report that at 200°c
there is a gradual loss in tyrosine content of wool. Others have
suggested that the heating of proteins in air causes the oxidation of
tyrosine to quinonesU,4.5, 5, 6-dihydrox;yindole-2-carbQlCY'lic acid32a or
melanins46. Barut and Leveau47 proposed the foi,natian of d.ihydrox;y-
phenylalanine mile Pinte et a148 reported no change in tyrosine content
of silk heated at lOOoC and Nortan49 found tyrosine stable in -wool
heated at 130°c.
Graham and Statham53 have famd protein bound tryptophan unstable
 16.

to heat but little work appears to have been carried out to identity
the decanposition products.
The effect of dry heat on the peptide bend of both proteins and
n,ylon has received .more attention recently. All papers9,51,52 have
agreed that oxidation is the main cause or peptide breakdown during
heating, indeed Valko and Ckicklis51 .maintain that in the canplete
absence of oxygen, heating causes solely a condensation reaction in
nylon.
The heat stability or the hydroxyamino acids, serine and threonine
has not received a great deal or attention, possibly because or the
difficulty in obtaining reliable analyses. Norton and Nicholls53 found
protein bound threonine stable to heat under neutral conditions and
assumed serine would behave similarly. H<Mever, Horio et a139 found
6 hours heating at 13<>°C in sealed tubes caused a 5% loss in serine which
was increased to 7°" destruction at tanperatures of 170°c or higher.
za11n32 also reported serine losses dependent on temperature and found
very little serine remained in wool after treatment at 2oo<>c.
Weclawowicz et a136 obtained similar results for wool but surprisi.ng].y,
found serine in silk Dll.ch more stable, no explanation for this discre-
pancy was offerred. Both Speakman and Janowski54 and Meybeck and
Meybeck9 suggested serine was dehydrated to c,-am1no acrylic acid residues
by heat but since neither group included an,y serine analyses, their
ccnclusions are suspect.
Norton4.9 reported that decarbox;vlation appeared to be the initial
reaction in the heat decanposition of a number of free amino acids
including cysteine, methionine, serine and arginine. He concluded,
however, that decarboxy-lation was less likely in proteins where most
carbdQ'l groups were involved in peptide bond formation.
()le of the most easi]¥ observed si~s of heat degradation of proteins
is the appearance or a yell.ow colour. Mazingue and Van Overbeke55
studied a series or proteins and found that those with a higher basic
group content ye11owed far more rapidly; they attributed this yel1owing
to the rormation or amide cross-links. Pinte et al48 proposed a
similar mechanism to explain the yellowing of silk under canpare.ble
conditions. Neither group explained how ad.di tional amide bonds could
cause yell.owing.
Mazingue and Van Overbeke23 and Norton and Nicholl.s53 reported that
acetylation of the free amino groups si~ificantly- reduced yellowing.
Norton and Nicholls53 also found a close correlation between the heat
yellowing of dry, neutral wool and the loss in £. -amino groups. Since
blocking free carboxyl groups was as effective in reducing yellowing as
blocking free amino groups, they suggested the formation of amide cross-
links leading to oxidised pyrroles could be responsible for yellowing.
Norton"-9 also suggested decanposition of tryptophan and praline could
contribute to yellowing.
Janowski and Speakman54. attributed yellowing to the formation of
unsaturated compounds from serine and threonine residues and proposed
that treatments "'11.ich promote the N - 0 peptidy'l shift l!IOuld be most
effective in reducing yellowing. However, their paper contained no
serine ana]Jrses and ID&lV of the reagents used were non-specific for
 18.

tyrosine and serine, being capable of reacting al.so with free amino and

carbo1cyl groups, so their conclusions are questianable.

Whether cystine is involved in heat yellowing has long been a source

of controversy. Several papers53,54,57 have rePorted that modification

of the disulphide bend in wool has no effect on yellowing, Me,ybeck and

Me,ybeck9 however, strongly disagree. The,y attributed the heat yellowing

of 1«>01 to the conversion of cystine to pyruvic acid, and for proteins

lacking in cystine, the conversion of serine to pyruvic acid. 'l'he,y

also suggested that the auto-oxidation of al.anine and glycine to pyruvic

and glyoxylic acids respectively contributed to yellowing, bit did not

rePort any cystine or pyruvic acid analy-ses on unheated wool. Moreover,

it is more likely that the pyruvic acid would be formed during the

hydrolysis of the heated protein, rather than during the actual heating

process.

Pinte et a148 suggested that yellowing was due to nitric oxides

produced fram oxidation of the free amino groups. However, if this were

so, one would expect similar oxidation to occur to the free amino acids,

many- of which are stable to heat.

The role of aerial oxidatim in heat yellowing has been largely

ignored. Most authors33,54 have reported only slight reductions 1n

yellowing Nien samples are heated under nitrogen. Whether it is actual.ly

involved in yellowing, aerial oxidation is believed by man,y51,52 to

contribute to the degradation of various groups in the protein.

Many attempts to stabilise proteins, especially wool, against

yellowing have been made. Barnes58 used aqueous solutions of metal.lo-

thioeyanate complex ions to reduce yellowing. He claimed that they
formed cross-links at the amino groups, leaving the disulphide bonds
intact. ICirst59 also used metallic ions, especially zinc, and suggested
that they reduced yellowing by forming -S-Zn-S- bridges at those places
which tend to fom unsaturated compounds during heating. Harris60
removed the disulphide groups by cross-linki.ng with dibromoethane
following reduction with thioglycollic acid but achieved only a slight
reduction in yellowing. Martin and Spea1anan61 found certain unsaturated
compounds, especially mal.eic anhydride, reduced yellowing; they attributed
this to crosslinking with thiols derived from the thermal breakdown of
disulphide bonds. This contrasted with reports from others53,54,57 who
foum modification of the disulphide bend had no effect on yellowing.
Bell, Clegg and Whewell.33 examined yellowing of wool at 200oC and
proposed that sulfonation of tyrosine and the hydraxy- groups of serine
and threonine was the rnost effective aetftod of reducing yellowing. More
recently, Bell, Hutchinson & Whew81162 reported that the precipitation
or :i.JlaolJable Alt ■ of zinc, cadaiua and aluminium in the fibre reduced
the loss in tensile strength and rate of yell.owing caused by heating in
the range 160°c - 2oo<>c. These temperatures are well. above those used
in the preaent work and it is likely that quite different chemical.
reactions are involved.
Since it is obvious that there is a considerable degree of conflict
as to the effect of dry heat on proteins, it was felt that a reappraisal.

of the topic was desirable.

 20.

Chapter II: SPICIFIC ~ICAL RllCTIONS CAUSID BY HMT

1. Preliminarz Blcperiments.
When a protein in solid form is exposed to heat for an extended
period the most obvious effect is a change in colour. Depending on a
number of variables including time and temperature of heating, pH,
moisture content and its amino acid composition, the protein will exhibit
changes in colour fran white through pale yellow to brown. Bictended
heating at temperatures above 2()()0C can cause charring.
In the present work &l.l heating was carried out in an a.ir-..draught
oven at 130°c ! 0.5. This temperature was selected as high enough to
cause significant degradation, yet was still below 150°c which both Zahn32
and Norton and Nicholls53 have reported is a critical temperature for

keratin, above which both decanposition and weight losses are accelerated.
It should be emphasised that the term "dry heat", to which reference is
made repeatedly, is a relative term and refers to air dry rather than
absolute dryness. In most cases the air dry protein was heated in an
a.ir-..draught oven so that much of the water vapour present was able to
escape to the air. In all probability a 11ttle moisture remains in the
protein during heating.
Five proteins, wool, silk, casein, gelatin and zein were initially
selected for this study- because of their wide variations in amino acid
canposition and as being representative of both fibrous and globular
proteins. Crystalline and pOll«lered proteins were spread in thin lay'ers
on glass plates for heating, while wool fabric and silk fibre were
 21.

suspended in the oven. In this way the five proteins were suanitted

to the same heating conditions so that analogies could be drawn between

them. The wool was in the form of an especiall,y prepared, plain woven
fabric which had not been subjected to any- alkaline treatment in manu-
facture. It was solvent extracted (ethanol and ether) to remove any-
gease etc. and dried at room temperature before being axandned. The
silk fibres were al.so solvent extracted before use. Casein, which was
obtained in power form, and zein, gelatin and lysozyme, all crystalline
in form, received no treatment before being examined. The pH of each
protein was checked by measuring the pH of an aqueous solution of the
soluble ones (aqueoos-ethanol. for zein) and an aqueous extract of the
fibrous ones; in all cases the pH range was 5 to 7 •
Changes in colour were determined by reflectance measurements on a
Col.or-lYe Colorimeter against a white vitreol.ite glass standard. The
discoloration was expressed in terms of a Yellm,mess Index (Y.r.)53
given by the expression Y.I. • X y Z x 100 where X, Y, and Z are the
colour coordinates given by the instrument. This expression gives a
value of zero for the standard, positive values for yellow samples and
negative values for blue. The values obtained for protein samples were
reproducible and correlated well with visual observation.
The effect of time of heating on colour, as given in Table II,
indicates that there are considerable differences in the ext.en\ of
yellowing.
 TABLI II

Effect on Colour (in Y.I. Units) of Proteins Heated in Air at 130°C •

Time of Hetine; ~ ~ Gelatin Casein !!in

(Hours)
0 19 13 25 8 35
0.3 22 14 35 14 35
0.75 23 14 38 15 36
1.0 24 15 39 16 36
2.0 26 16 39 17 36
24.0 40 21 43 30 39

Whilst the vast difference between the extent of yellowing of wool

(100.C increase in 24 hours) and silk (5QC increase in 24 hours) may

perhaps be explained in terms of amino acid compositions which are quite

different, the enomous difference between wool and casein (37'J/, in 24
hours) cannot be similarly explained, so the amino acid sequence and/or
configuration, must be important.
For proteins rich in l.y-sine, (e.g. wool, gelatin, casein) most
yellowing occurred in the first hour and the rate then decreased. An

extended period of 24 hours heating was examined to see if yellowing

reached a limiting value but this did not appear to be so, as Table II
shows.
&lch obvious colour changes that occurred in 24 hours of heating
might be expected to alter the absorption curves of the proteins in the
visible and ultraviolet regions. Both casein and gelatin are water
 1
z
-------+-- ---+-----t -----+----
Z.90
-----
..300
zso Z60 280

WAV.£1.LNGTH

Fig. 2: Ultra-violet absorption spectra of heated Casein and

Gelatino

Legend 1 - Casein.
2 - Gelatin.
soluble and although their solubilities were greatly' reduced by the heat
treatments, it ws still possible to obtain 0~ solutions qr- dissolving
in warm w.ter. Absorption spectra were measured on a Cary Modal 15
Spectrophotaneter and are shown in Fig. 2 where it is evident that
absorption in both visible and ultra violet regions was increased by the
heat yell.owing, but no new absorption peaks appeared.
Attempts were made to obtain thin films of casein and gelatin wdch
could be ftXAlll1ned by infra-red spectroscopy. Sol.utions of both proteins
were spread carefully' on a surface of mercury and allowed to dry.
However, all films that were strong enough to be handled proved too thick
for infra-red analysis. Instead Nujol mulls were used to prepare samples
of casein and gelatin for recording infra-red spectra on a Perkin Elmer
Model 337 Infra-Red Spectrophotaneter. Again, despite strong colour
changes as a result of heating for 24 hours at 130°c, no change was
detected in the infra-red absorption spectra. Proteins contain many
absorbing groups and it is not altogether surprising that protein absorp-
tion should swamp &n.Y absorption by the yellow material. In all cases

a general, overall absorption pattern with no distinguishing peaks was

obtained. It would appear that too small a quantity of protein was
sufficiently modified by the heat treatment to be identified by infra-
red analysis.
Since the absorption spectra of the heated proteins provided so
little information, a stuey of individual. chemical. groups within the
protein was undertaken.
2. Reactions Involving the Peptide Bond.
Despite the number of investigations9,l3,14,l8 that have been

carried out on the effect of dry heat on proteins, the role of the
peptide bond has been largel,y neglected. Slch neglect may be due, in
part, to the difficulties in determining any changes in peptide b011d
concentration in a material so rich in such bonds. However, despite
the difficulties, several interesting papers48,5l have been published
which suggest that this bond is not as stable as is often assumed.
Until recently, breakdown of the peptide bood has generally been
regarded as a simple, h,ydrolytic reaction as follows:

r + _y
R-
u- r -r-«- > R - I- T- T- ff -
0 H R 0 OH H R 0

!"20
+

//
0
°f2 y
R - C
\
OH
+
R-r-i-r-,r-
OH H R 0

Under the experimental conditions employed, in which the samples

of proteins were heated in an oven with air circulation at 130°c, the
moisture content would be comparatively low, since heating in air will
remove most of the unbound and loosely bound water. However, some
moisture is almost certain to be trapped within a fibre btmdle, and
indeed, during this investigation, several small bundles of wool were
fowid to be distinctly clamp to the touch in the centre after being heated
for 24 hours at 130oC. It is therefore possible that some peptide bond
l\vdrolysis could occur under the experimental. conditions used in this work.
Viscosity measurements have long been accepted as an indirect
technique for determining breakdown of the peptide bond of soluble
proteins, since viscosity is directly related to molecular weigllt.
Theoretically, the relationship can be expressed by the equation tl, • I<M°'
where 11, is the intrinsic viscosity, M the molecular weight, and K and a.
are constants depending on the properties of the solute and solvent
molecules and their interactions. It can be shown that69 the value of
a. would be approximately 2 for rod-like solute molecules and about unity

for spherical. ones, while for randomly kinked linear molecules the index
a. wi1J. have values less than unity.

When peptide bmd breakdown occurs, the molecular weight will

decrease, resulting in a corresponding reduction in viscosity. It is,
however, possible that at the same time that peptide bends are being
broken, other interchain linkages may form; this means that the molecular
weight may- increase, remain unchanged, or decrease, depending on the
relative extent of each reaction.
Another complication that must be ccnsidered when interpreting
viscosity measurements of protein solutions is that of denaturation.
Man.:, proteins exhibit definite changes in viscosity due to the unfolding
of molecular chains, without any concomitant change in molecular weight.
In effect, denaturation causes changes in the value of a. by causing

changes in the shape of the solute molecules. However, if used carefully

 26.

under standardised conditions, viscosity measurements are still useful

as a means of detennining the overall effect of a treat.ment on molecular

wei31t.
For accurate and meaningful viscosity measurements, it is essential
that the protein is completely soluble in a solvent which is stable,

non-degradative and inert towards the solute, so no solute-solvent

complexes or aggregates fo:nn. For practical reasons it is desirable
that the solvent is relative],y simple to prepare. In practice, these

conditions are seldom canpletely satisfied. Most solvents have an

adverse effect on proteins if they are left standing for any length of
time and some interaction between protein and solvent invariably occurs.

However, most difficulties due to degradation and interaction can be

overcome by accurate timing fran the manent the protein and solvent are
mixed until the measurements are completed, and by careful maintenance
of constant temperature. While such procedures do not prevent some
errors they do serve to keep them relatively constant.
Since wool cannot be dissolved without undergoing considerable
degradation, the initial investigations were ca?Tied out with casein.
Casein ismluble in water but is also subject to denaturation, so the
strong denaturing agent, 8-1 urea, was used as a solvent. Immediately
casein was mixed with this solution it would be denatured, so any
viscosity changes detected, could be attributed to heat induced chemical
reactions and not mere],y to heat denaturation.
Silk was selected as a second protein for examination. Silk can
be dissolved in concentrated aqueous solutions of inorganic salts,
notably the thiocyanates of lithium, sodium and calcium. Cupriethylene-
diarnine has been widely used as a solvent but it was found unsatisfactory

for the present work since it was difficult to prepare to the required
concentrations, appeared to cause some degadation and in many cases
failed to dissolve completely the silk. A solution of 9a( formic acid
containing 2!C calcium chloride95 which readily dissolved the silk and
caused no noticeable degadation in thirty minutes standing was found to
be the most satisfactory for this study. A time of twenty minutes fran

the moment of mixing to completion of viscosity measurements was found

to be ample.
'!be third protein examined was zein which is insoluble in either
pure -water or pure ethanol but quite soluble in a 50C aqueous solution
of ethanol.
To measure the viscosities, Ostwald viscometers of 3.0 mls volume
were clamped into a water bath thermostatted at 30°c. This temperature
was selected as sufficiently above ambient for easy control yet not high
enough to promote extensive reactions between the protein and the solvent.
To reduce the time for the protein solution to reach equilibrium tempera-
ture, the solvent was also maintained at 300C.
Triplicate samples of the protein, previously dried under reduced
pressure over phosphorus pentoxide for J..8 hours, were weighed accurately
into volumetric flasks and the solvent added to give o.~ solutions.
The mixture was shaken for a set period, (5 minutes for silk, 10 for

other proteins) to hasten dissolution, then exactly 3.0 mls were pipetted
into the viscometers. After standing for a further 5 minutes to reach
 28.

temperature equilibrita, the times of flow of the solutions were measured.

The relative viscosity (l, R of each solution was determined from the ratio
of time of now of solution to time of now of solvent. The relative
viscosity q R is related to the intrinsic viscosity of the solution
by the equation rt R • to where q0 is the intrinsic viscosity of the
solvent. Use of relative rather than intrinsic viscosity avoided the
need to calibrate viscometers and was sufficient for this work. Since
different solvents were used, no direct comparisons can be made between
the proteins, but the effect of heating on the relative viscosity of a
particular protein does show a trend in each case.

TABLE III

The Effect of Time of Heating on Relative Viscosity of 0.19

solutions of Selected Proteins.

1'.!!!! (Hours) ~ Casein ~

o.o 1.1+8 1.04. 1.10
0.17 1.1+6 1.(1'/ 1.10
1 1.45 1.10 1.(1'/
2 1.43 1.18 1.06

3 1.42 1.19 1.04.

24 1.29 Insol. 1.02

Fran Table III it is obvious that heating has differing effects

on the three proteins. In the cases of silk and zein, there is a
marked reduction in viscosity which indicates peptide breakdown. A
 2 6
3 4

Fig. 3: Apparatus for heuting under nitrogen.

Legend 1 nitrogen cylinder.

2 scrubbing solutions.
3 copper heating coil.
4 sample container.
5 oven.
b lead acetate solution.
 29.

reduction in the viscosity of silk solutions following heating of the

fibres has also been recorded by Pinte et a1.1+8 and Rochas and Pierret?O.
If peptide breakd<Ml occurs in these two proteins, it is reasonable to
assume it would do so in others, yet casein shows an increase in viscosity
which suggests other reactions 1111st be occurring simultaneously. These
will be discussed later.
Since silk had shown such significant decreases in viscosity on
heating, it was decided to canpare the effects of heating in air and in
an atmosphere of nitrogen to see if oxidation was an important factor
in peptide breakdown. Accordingly, a sample of silk was placed in a
glass container connected to a cylinder of nitrogen by means of a copper
heating coil. Both heating coil and the tube containing the sample were
placed in the oven, as shown in Fig. 3. A second silk sample was hung
in the air in the oven. After 48 hours of heating at 130°c, the samples
were examined for yellowness and viscosity changes. The results are
sh<Ml in Table I'l.

TABLE I'l

Bffect on Yellowness and Viscosity of Silk heated at 130<>c for

Given Times in either Air or Nitrogen.

~ !k Nitrogen (dry) Nitrogen (moist)

(Hours) L.L. _..R L1:. _..R Lb. ....!
0 14 1.48 14 l-48 14 1-48
2 16 1-43 15 1-45 15 1-45
48 33 1.20 20 1.34 20 1.32
 The absence of ox;rgen results in a significant reduction in both
I.I. and relative viscosity changes so that ox;rgen is imPortant in both
peptide breakdown and heat yellowing of silk. However, some yellowing
occurs in the absence of air and reactions other than oxidation must be
considered; hydrol,ysis is probable. Since moisture is necessary for
qydrol,ysis and the hot gas passing over the silk could have had a drying
effect, the experiment was repeated with the nitrogen bu'bbl.ing through
two jars of distilled water before entering the heating coil. The
results (see Table IV) indicate that there is no significant difference
between samples heated under moist and dry nitrogen and it appears that
oxidation is more important than moisture in peptide breakdown. Rochas
and Martin71 reported that in the thermal degradation of n_ylon, the
presence of water vapour was far less important than that of OJcy"gen.
Since oxidation apparently accounts for onl,y half the peptide
breakdo\cl observed, and hy'drol,ysis plays a minor role, a further reaction
must be involved, the nature of which has not been ascertained.
Fran this investigation it can be seen that oxidation of the peptide
bond is highly probable when proteins are heated in air, but in many
cases the breakdmm is compensated for by other reactions not yet
considered.

3. Decomposition of C:ystine.
A number of investigations 9 , 22 ,32,32a, 42 into the modification of
proteins by dry heat have coocentrated on the behaviour of peptide
bound cystine. Although most authors agree that disulphide decanposition
 31.

occurs when cystine-containing peptides and proteins are subjected to dry

heat, considerable controversy exists as to the products or degradation
and to their influence on yellowing.

The present work was initiated to see if any relationship existed

between c7stine degradation and protein yellowing and to see if any or
the cystine decomposition products could be identified. Accurate
analyses or protein-bound c7stine have always provided same difficulty,

especial.ly' '114'len cystine degradation products are present. Until quite

recentl,y it has been necessary to hy-drol,yse the protein prior to deter-
mining the actual cystine content by colorimetric77 or gavim.etric78
methods. Conditions for hydrol,ysis needed very careful standardisation
to minimise cystine decanposition. If serine or tryptophan ware also a
coostituent of the original protein, the decanposition or cystine during
hydrolysis became a source of major error.79 In addition to the problems
posed by cystine degradation during hydrolysis, it has been shown68 that
disulphide reformation~ also occur, especially if the protein has been
subjected to an,y oxidising treatments, e.g.

lstimation of cysteine is even more complex since it is extensively

decomposed during hydrolysia79 •
Because of these problems, methods for determining cystine and
cysteine in the intact 'llft)()l have been developed. Leach•a68 technique
involves the quantitative absorption or mercury ions by the cysteine
component of an insoluble protein. The protein is treated with a
solution containing a reducing agent and mercuric chloride for 24 hours,
after which the mercury remaining in the solution is determined pol.aro-
graphically. In the present work a similar technique -was used but the

mercury remaining in the solution was determined by atamic absorption

spectroscopy. Both methods require triplicate samples and their
reproducibility is of the same order. Provided care is taken to prevent
"WOol fibres clogging the aspiration unit of the atomic absorption spectro-
phot01Deter, this technique has the advantage of giving a more direct
reading and is quicker. For cysteine determinations concentrated urea
solutions were used to swell the wool fibre and in this case it was
necessary to dilute an aliquot before aspirating, otherwise the urea
caused clogging.
The effect of heat on proteins with differing cystine contents are
shONn in Table V •

TABLE V

The Effect on Yellowing and Cystine Content caused by Heating

Various Proteins in Air at 1300C.

I!!! ~ ~ Gelatin
(Hours) y .I. i C,stine Y.I, ! Cystine L1.:. g c,:stine
0 20 n.3 13 Negligible 25 Negligible

1 26 n.1 15 39
2 28 11.0 16 40
24 4.0 10.1 21 4.3

It is inmediately apparent that there is no obvious relationship

between yellowing and cystine ccntent since certain proteins, e.g. silk
and gelatin, W'lich contain negligible cystine yellow considerably'.
This, however, does not necessarily' eliminate the possibility of cystine
degradation contributing to yellowing, but simply emphasises it is not
the only mechanism.
Having verified that cystine in wool was degraded by heating at
130°c, the question of degradation products was considered. Thin
layer chromatography was used in attempts to detect any new amino acid
in hydro]J"sates of wool that had been heated at 130°c in air for several
dqs. Hydrolysis was carried out with 6N hydrochloric acid in sealed
tubes at no0 c for 20 hours. The acid was removed by repeated evapora-
tion at reduced pressure and finally the hydrolysate was dissolved in
water to give a solution containing approximately LLtmole of cystine per
ml. The hydro]J"sate was stored at 3°c.
Thin layer chromatography plates were prepared by shaking a mixture
of 30g. of silica gel in 60 mls of distilled water for exactly two
minutes. The slurry was then applied to 20 cm x 4,0 cm glass plates
in a unifo:nn layer of 0.3 nm thickness. The plates were air-dried for
ten minutes, then oven heated at no 0 c for a further 45-60 minutes.
The hydro.lysate was applied along with separate samples of cystine,
cysteine, cysteic acid and lanthionine. After conditioning, the plates
were placed in the eluent (phenol: ethanol: water: acetic acid: in
the proportions 75: 20: 20: 5) which was allowed to ascend to a height
of about 28 ans. The eluent was then evaporated fran the plates in an
air-draught oven at 50°c and the chranatograms were developed by spraying
with a~ solution of ninhydrin in acetone.
 C01Dparisons of hydrolysates of unheated and heated wool, showed an
extra spot on the chromatogi-am of the heated wool, corresponding to the
cysteic acid spot. Amino acid analyses using a Beckman Unichr01D amino
acid analy'ser also revealed cysteic acid present in the hydrolysate of
heated wool. The amino acid analyser also detected the presence of
methionine sulphoxide in the hydrolysate of heated wool. zahn32
reported detecting a weak cysteic acid spot in hydrolysates of wool
heated in air at l200C ~ile others36 , 43,56 have reported detecting
cysteic acid but in hydrolysates of wool heated at higher temperatures
(160-200oC).
Snmination of the hydrolysates by two dimensional chromatography
(butanol: acetic acid: water 40: 10: 10 followed by phenol: water
70: 20) failed to reveal any extra spots except that corresponding to
cysteic acid. Asquith et a140 reported detecting~ amino alanine and
ly'sineoalanine in hydrolysates of heated wool, however they heated their
11«>01 samples in sealed tubes for which different mechanisms are believed
to apply.
Unfortunately, attempts to relate quantitatively the losses in
cystine in terms of cysteic acid were unsuccessful. Quantitative
detenaination of cysteic acid by the intensity of the ninhydrin colour
fonned on the chromatogram was not possible because of poor separation;
such a technique is not very reliable even ~en good separation is
achieved. FUrthermore such small amounts of cysteic acid were present
that accurate analysis by the amino acid analyser was not possible.
Moreover, cystine may be reformed during hydrolysis fr01D sane of the
 35.

intermediate oxidation products including su1phoxides, sulphones and

sulphinic acid, which could be present. No method of even qualitative
analysis is available to distinguish between these partial oxidation
products in the protein. Cy"stine may be detennined in the intact
protein but losses determined in this way cannot be accounted for by
cysteic acid formation as estimated in the hydrolysate. However, the
detection of sane eysteic acid emphasises that oxidation occurs in the
dry heat degradation of proteins.

Since the thin layer chromatograms and the amino acid analysier
provided no evidence of lanthionine formation it is apparent that no
significant conversion of cy-stine to lanthionine occurred under the
cmditions of heating used (13<>°C for 24 hours). It must be observed
that the temperature used is well below those recorded in the litera-
ture32,36,56 at M1ich lanthionine fonnation has been detected (i.e.
160-200°c); it is therefore probable that different mechanisms or cystine
decanposition occur at these higher temperatures.
Since oxidation appeared to be the chief source of cystine decanposi-
tion, it might be expected that W00l heated under vacuum or oxygen-free
nitrogen would be less susceptible to degradation. Accordingly, lft>ol
was packed into a glass test tube which was evacuated to 10111D of mercury
by means of a high vacuum pump, and sealed. A. further sample was sealed

in a test tube without evacuation while a third sample was simpl.7 placed
in an unsealed tube. A.11 three samples were then heated at 1300C for

24 hours.
It was found that the sample sealed in the unevacuated tube was far
 36.

more severely' yellowed than was the sample left open to the air; the
sample heated under vacuum was even less yellow. These results probably
reflect the different moisture contents of the samples more than the
effect of aerial oxidation. The sample heated under vacuum would have
had most of its moisture removed along with the air by the vacuum pump

while the other sealed sample would have contained moisture both absorbed
in the wool and in the air itself. The third sample was heated open to
the air so that any water vapour present was able to escape to the
atmosphere without causing severe degradation.
Both sealed tubes smelled strongly or hydrogen sulphide when opened
and the gas evolved turned lead acetate solution brown. However, the
condition of the sealed, non-evacuated sample made it obvious that heating
in sealed tubes was not a satisfactory method for examining the effect of
heat in an oxygen free atmosphere and no further analyses were done on
these samples.
An alternative approach was to exam1n.e samples heated in a stream of
oxygen free nitrogen. Nitrogen was passed through two scrubbing solutions
of ammonium metavanadate/zinc amalgam before being connected to a heating
coll (see Fig. 3), the issuing gas was passed through a tube containing
lead acetate solution to detect hydrogen sulphide. Before heating, the
sample tube was evacuated, then filled with nitrogen several times, then
placed in the oven and connected to the nitrogen line. A control sample
was heated in air in the oven at the same time to all.ow comparative
analyses of samples heated in air and nitrogen. Since the nitrogen
first passed through aqueous solutions before reaching the wool it
contained some moisture; no attempt to control moisture content beyond
this was made.
The fact that no hydrogen sulphide was detected during the 24. hours
of heating suggests that degradation caused by heating in sealed tubes
involves a different mechanism than that occurring when heating is open
to the air or to a continuous gas flow. Horio et a139 have also
reported that heating wool in sealed tubes caused reactions "radically
different" to those occurring wen the heating is open to the air.
Table VI shows the effects of 24 hours heating in air and nitrogen
on the colour and cystine content of the wool samples.

TABLE VI

Bffect of Heating Wool in Air and Nitrogen for 24 Hours at 130°c.

.I!!!! Yell.owness Index Cystine (%) Cyssi.ne (%)

(Hours) A!£ Nitrogen Air Nitrogen !!£ Nitrogen
0 21 21 u.3 u.3 0.16 0.16
24 40 32 10.1 0.12 0.17

It can be seen that although heating under nitrogen reduces the

yell.owing slightly', it causes no significant difference in the rate of
cystine decomposition. The slight increase found for the cysteine
content of wool heated under nitrogen agrees with results previously'
reported by Bell et a133. Cysteic acid could not be detected by thin-
layer chranatography in the hydrol.y'sates of the sample heated under
nitrogen, confirllling that it is produced by aerial oxidation.
 The mechanism by which wool-bound cystine is degraded by heat was
not explained althou€tt Table VI shows it is not an oxidative process.
Meybeck and Meybeck9 attributed the heat yellowing of wool to the fonna-
tion of pyruvic acid from cystine and suggested the following equations
applied.

-CO-CH-NH-
1
c~
~ HBlT
s ~ 2-CO-c-NH- + H2S + S
I //
r2
-NH-CH-CO-
CH2l
H20
-co.co.CH3 + H~-
However, such a reaction leads to an increased number of free amino
goups in the wool, which, it will be shown later, is contrary to most
reports on the heating of wool. In addition, Meybeck and Meybeck9 failed

to report any analyses on unheated wool so their conclusions are open to

question.
The fonnation of dehydropeptides by cystine degradation is possible
but such compounds are too unstable to be detected as such; however, it
would be reasonable to assume a relationship between dehydropeptide
content of unhydrol.y-sed wool and the pyruvic acid content of the l\v'dro-
].ysate. Qual.itative analyses using 2,4,dinitropheny-lhydrazine8o,
revealed pyruvic acid present in the hydro].ysates of both heated and
unheated wool (see Table VII), but the reproducibil.ity was poor and the
 39.

TABLB VII

Pyruvic Acid Content of Hydrol.ysates of Wool heated at 130°c in

Air for Given Times.
Time (Hours) PYruvic Acid (~)
0 1

2 1.3
24 1.6

results appear to be influenced greatly by time of hydrolysis, time

required to e,,:aporate the acid fran the hydrol.ysate and even the time
taken to mix the hydrol.ysate and reagent. It is therefore, extremely
difficult to determine to what extent dehydropeptide fo:nnation occurs
during heating. There is also the fact that other amino acids (e.g.
serine) can yield deh,ydropeptides so any relationship between pyruvic
acid content and cystine loss may well be largely coincidental.
Since the results obtained with wool provided no satisfactory
explanation of the degradation of cystine by dry heat, the tripeptide
glutathione was examined. Glutathiane is normally' obtained in the
reduced form as f-gl.utamy'leysteiny'lglycine but can be arldised to the
disulphide form by 24 hours aeration in the presence of ferrous ions.
To study the effect of heat on both reduced and oxidised forms,
solutions of each were spotted anto separate silica gel chranatograp}v
plates and the plates heated for 24 hours. After heating, both spots
were yellow-brcn.n in col.our and fiuoresced strongly under ultra-violet
radiation. In attempts to detect any new products, two dimensional
separation was used with butanol:acetic acid:water 40:10:10 followed by

phenol:water 75•20. After the seccnd elution the plates were dried at
105°c and examined under ultra violet light, and again after development
with ninhydrin. The only fluorescent spot obtained was at the origin,
corresponding to the initial glutathione. However, after developing
with ninhydrin a second spot appeared, which had Rr values corresponding
to glycine am the typical glycine pinkish colour, indicating peptide
breakdown. Plates heated for 48 hours gave identical results.
When a sample of reduced glutathione crystals were heated on a

glass plate for 24 hours at 130°c, hydrolysed and examined chromato-

graphical.ly, no yellow spot was evident and the only ninhydrin positive
spots found were those corresponding to the three constituent amino
acids. Obviously, no ninhydrin positive substance was f onned in
sufficient quantity to be detected and the absence of any fluorescence
ccnfinned that the yellow material was unstable to hydrolysis.
The yellow material formed by heating glutathione on the plate
remained at the origin, as did the control glutathione. This suggests
that the main yellow product has a molecular weight and solubility
resembling the tripeptide, possibly a cyclisation product.
The formation of thiazole derivatives between the thiol group of'
cystein and the carbonyl carbon of the peptide has occasicned interest.81 •
Thiazol.es are generally prepared by a condensation of an~ halogenated
aldehyde or ketone with a thioamide82 •
HC ■ 0

I
CH2 Cl
+
However, 2-thiazoline is obtained by cyclisation of a foi,vl derivative
of 2 mercaptoethylamine in dehydrating conditions.

2-thiazol.ine

Similar reactions have been proposed for peptide bound cysteine81

and such a mechanism may explain the heat yellowing of glutathione.

,,,HS\
NH2, ~ \2
CH-(CH2)z - C - ; - f-
CO - NH:.. CHz - 000H
Hor£/ H H

J
NBz S - CHz
' CH-(CH2)2 - C
I I - CO - NH - CHz - COCJI
CH
HOOC
I ~I
N

Barton et a183 have isolated thiazoleamino acid from the antibiotic

bacitracin and have proposed condensation between cysteine and the
peptide to explain its formation. If some glutathione did exist as
the thiazole derivative

Hooe, s - CH
i.e.
' CH - (CHz)z - C
/ \\
C - CO - NH - CH2 - COCII
HzN/ ~N/
it wauld contain sufficient conjugation to exhibit some colour and pro-
bably fluorescence. Unfortunately, it has not been possible to isolate
a thiazole structure from gl.utathione, al.though the existence of a
thiazoline ring is accepted by manyl0,81,83,84. Whether such a ring in
the glutathione peptide could cause yellowing is highly speculative.
If such condensation does occur in glutathione, it may- well occur
in proteins whose structure is favourable. Lundgren81 postulated such a
react.ion being involved in the steam setting of wool, leading final.l,y to
the formation of an ionised amidine crosslink and regeneration of the
thiol group. No evidence that this actually occurs has been published.

+ ifl, ,,
- NH - C
II CH
N, # HN+ - CH - CH2SH
I
l
Thus,despite its lmown structure and low molecular weight, this
study of glutathione did not lead to a satisfactory explanation of the
behaviour of peptide bound cystine when exposed to dry heat. However,
it is obvious that such behaviour is complex and it may- be influenced
by the adjacent amino acids as well as by the conditions of heating.

4• Reactions Involving Tryptophan.

SUrprisingly little work has been published on the dry heat
stability of tryptophan, whether in the free fonn or protein bound.
Graham and Statham50 rePorted a reduction in the tryptophan content ef

wool heated in air at 1300C but failed to isolate any decanposition

products. They and Norton and Nicholls53 suggested tryptophan
degradation may contribute to the heat yell.owing of wool, but neither
group presented any conclusive evidence.
Since tryptophan is unstable to acid hydrolysis in the presence of
other amino acids, methods for its determination in the intact protein
Most methods depend on the production of an intense
colour when specific reagents canbine with the indole nucleus. Various
aromatic aldehydes have been used, but p -dimethylaminobenzaldehyde has
been found to be one of the most reliable67. Hydrolysis is carried out
slo~ in the presence of this reagent which is believed to canbine with
the tryptophan either before or as it is released fran the protein.
Although the actual mechanism of the reaction is not clearly understood,
it has been used reasonably successfully for tryptophan determinations
for some time.
Various modifications of this technique exist; the method used
in the present work was developed by Graham and Statham67 (see Appendix
C). Calibration curves with pure tryptophan were unreliable, so
lysozyme, whose structure and composition are lmown, was used as a
standard.
To see if any direct relationship existed between tryptophan content
and rate of yell.owing, the proteins wool, silk, lysozyme and gelatin were
heated and examined.
The results obtained, which are shown in Table VIII, indicate that
there is no simple relationship between tryptophan breakdown and yellow-
ing. Canparison of the extent of tryptophan degradation in the
different proteins heated under identical conditions shows that whereas
 TABLB VIII

The Bt'fect of Dry Heating at 1300C in Air on the Yellowness

and Tryptophan Cmtent of Selected Proteins.
Time of Wool !Jsozym.e Silk Gelatin

Heating (Hours) Y.I. % Try Y.I. % Try Y.I. % Try Y.I. % Try
0 19 0.91 13 9.17 13 0.51 25 N
I
0.67 23 o.86 19 9.08 14. 0.50 37 G
L
1 24 o.so 22 9.01+ 15 0.4.9 39 I
G
2 26 0.75 23 9.03 16 OJ.,8 40 I
B
24 40 0.70 30 s.97 21 OJ.,5 43 L
E

2'1C of the tryptophan in wool is degraded, only 2.~ of that in lysozyme

is affected. Wool and lysozyme have some-what similar amino acid canposi-
tions and there is no reason to believe that steric effects are greatly
different in the two proteins. Both contain cystine so the presence of
cystine can have little effect although it has been suggested92 that
cystine assists in the autooxida.tion of tryptophan. !Jsozyme contains
four cystine residues, one of Wtich is situated next to a tryptophan
residue, so if cystine was responsible for the heat degradation of
tryptophan, one would expect lysozyme to show marked tryptophan degr-ada.tion.
The observed degradation of silk bound tryptophan (approx. 10.C after
24 hours heating at 130°c) is certainly much lower than that which occurs
in wool, but is still four times greater than that occurring in lysozyme,

so steric effects do not provide a satisfactory explanation. It would

appear that wool has an amino acid sequence and peptide configuration
peculiar to itself which facilitates tryptophan degradation.
Some previous work 5o, 93, 94 has shown that tryptophan is one of the

most labile of the naturally occurring amino acids and under the condi-
tions of heating, may be susceptible to oxidation. Oxidation of
tryptophan usually results in disruption of the 2:3 carbon bond of the
pyrrole ring and/or substitution in the aromatic ring. Burke93 has
shown that tryptophan is oxidised by various reagents to formylkynurenine,
which Stewart94 found degraded rapidly on heating to yiel.d intensely

r f, /COOH
-4't/~C- C - CH
1~1 I \
tt (,If, NH2
tf NH.CHO

Formyl kynurenine

Since hydrolysis would dstroy such products, it has not been possible
to isolate them from heated wool. Most evidence that such ccmpounds as
foneylkynurenine are formed in wool is indirect, based on their isolation
from less canplex substances.
To see if oxidation was of importance in the heat induced degrada-
tioo. of tryptophan, samples of wool were heated under vacuum in sealed
tubes and also under a stream of oxygen free nitrogen. The analyses of
the wool samples before and after heating are shown in Table IX.
Whilst heating in a vacuum or under e itream of nitrogen diminishes
the degradation of tryptophan, it is apparent from these results that
 46.

TABLB IX

The Effect on r.r. and Tryptophan Content of Wool Heated for

24 Hours at 130°c in air, in Nitrogen and under Vacuwn.

Sample y • I. Tryptophan Content%

Before Heating 20 0.91
Heated in Air 40 0.70
Heated under Vacuwn 32 0.76
Heated under Nitrogen 31 0.77

the major portion of the tryptophan degradation in air ½£ • 701,

occurs independent of the atmosphere of heating. The increased degada-
tion which occurs during heating in the presence of air canpared to a
nitrogen atmosphere is not surprising in view of the well-known suscept-
ibility of tryptophan to aerial. oxidation. What is worthwhile noting
is the considerable degradation that occurs in the absence of air, and
which rust, therefore, be the result of a reaction which competes
strongly with oxidation.
'!be differences between the yellowness indices observed. for heating
in the presence and absence of air, indicate that yellowing is partially
dependent on oxidation; this will be discussed in greater detail later.
That part of this oxidation yellowing is due to tryptophan degradation is
not improbable.
A condensation reaction may contribute to the apparent reduction in
tryptophan content. If the indole nucleus is in the right configuration
 47.

relative to the peptide chain, it is possible to envisage a candensation

between the irdole nucleus and the amide group. One possible canfigura-
tion is shown below.

Hydrogen bonding could be expected to stabilise this cmformation and,

on heating, elimination of water may occur, followed by bonding between
the carbonyl carbon and the indole nucleus as illustrated,

H H '
NH H H I
,1'\ - - \cl Jo //' - _\I co
I~ If 11 "-ci/ d II I/ '-CH/
~/\H/ \
H /
L ~/\., 1 \
H ~
A
C C
M
CR
I
CHR
f
NH
I
NH
I I
The addition of a carbon-nitrogen double bond 'WOUld increase
conjugation, resulting in a shift in absorption to a higher wavelength,
hence a yellow colour. Obviously, combination with the aldehyde reagent
for the colorimetric estimation, would be prevented, so analyses would
 48.

record a reduction in tr,yptophan content. Such a reaction would also

explain the nature of the tryptophan rate curve. Residues in the right
conformation would react relatively quickly, then as fewer and fewer
residues were able to condense, the reaction rate would decrease.
Graham and Statham50 have similarly suggested that tryptophan in wool
exists in two fractions of differing reactivity, depending on environment.
Although such a condensation does not appear to have been proposed
for proteins before, there are many accounts1 02,l03,lot.. of such reactions
with simpler tr,yptophan derivatives. The reaction is based on the
Bischler-Napieralski reaction, which involves heating an acyferivative
of 2-phenylethylamine under strongly dehydrating conditions96

Sumpter and Miller9'7 fOWld a similar condensation occurred with deriva-

tives of tr,yptophan heated in the presence of phosphorus o:,cy-chloride
 During the past few years papers1 ~-l0l+ have been published reporting
variations of the above reactions and the use of various dehy'drating
agents. Although a temperature of 130°c is not extremely dehy'drating
canpared to conditions used in the above reports, it is quite probable
that the conformation of the protein is such that reactions similar to
those proposed are actually stericall.y favoured, so that less severe
conditions would be sufficient to pranote the condensation. Obviously,
such a reaction would depend largely on the spatial configuration of the

peptide chains, so that in some proteins no suitable confo:rmation is

present for such a reaction to occur. Unfortunately, isolation of this
type of canpound from proteins is virtually impossible, as hydrolysis
would tend to destroy them.
Another difficulty in obtaining results to relate heat yellowing
with tryptophan degradation is the problem of selectively modifying the
tryptophan residue without otherwise degrading the protein; at present
no satisfactory method is available. However, it was found that when
the free amino and carboxyl groups were modified by acetylation and
esterification respectively the extent of tryptophan degradation was
also reduced (see Table X). Since the treatments were believed to be
specific to the respective groups,this result was somewhat surprising.
As can be seen from Table X, the decrease in the percentage
tryptophan degradation for esterified wool is approximately 80.C 2 3=~;5
which corresponds closely to the tryptophan degradation produced by
heating in the absence of air. It seems unlikely that acetylation, and

certainly esterification, would modify the indol.e nucleus• Two

 50.

TABLB I

Effect of Acetylation with P-nitrophenyl acetate and Bsterification

with Methanol/HCl on the Tryptophan Content of Unheated Wool and
Wool Heated at 130°c for 24 Hours.
%Tryptophan

Treatment Untreated Wool Bsterified Wool Acetylated Wool

Before Heating 0.9a o.89,C 0.90
After 24 Hours Heating 0.70,, o.85 o.82
i deccmposition 23 4.5 8.4

possibilities migJ.,t be considered to explain these results:

(a) The product formed by the condensation between free amino and free
carbaxyl groups (prevented by above treatments) subsequently
interacts with the indole nucleus.
(b) Bither treatment reduces the number of stabilising salt linkages
allowing conformational changes to occur 'lllhich reduces the
possibility of intramolecular condensation.
'!he first of these two possibilities can be ruled out on purely
spatial considerations since the new amide bond formed is unlikely to
be sufficiently near an indole nucleus to interact with it. However,
the second proposal. is a distinct possibility and adds weigJ.,t to the
earlier suggestion that the conformation of a protein considerably
influences the heat degradation of its tryptophan component.
The results given in Table II have shown that t:ryptophan degradation
in wool heated at 13<>°C in air is partly due to the presence of OJCYgan,
so it is worthwhile to consider the effect of oxidation on free tryptophan,
as a similar reaction path may occur for protein bound tryptophan.
Accordingly, the free amino acid was spotted onto thin-layer chromato-
grap~ plates which were heated in air for 24 hours. After heating,
the tryptophan spot was brown yellow in colour and fluoresced strongly
'
under ultra-violet radiation. The plates were eluted in t11ro directions
using butanol:acetic acid:water: 40:10:10 followed by phenol:water
70:20. At least eight fluorescent spots could be detected under the
ultra violet lamp and after further development with ninhydrin and
ID'lrlich•s reagent, the number of products increased to twelve. This
contrasted with Norton•s49 11rork in which he reported ve-r:, little
chromatographic difference between unheated and heated tryptophan.
Stewart98 has reported that free tryptophan is very susceptible to auto-
oxidation and the results obtained in this investigation support his
argument. The free amino group apparently influences the reaction
indirectly, since when N-acetyl-tryptophan was heated under identical
conditions, only four spots were obtained. Two of these were identified

as N-acetyl-tryptophan and free tryptophan. Since sane degradation of

the N-acetyl group had occurred, it is difficult to sa;y if the other·
spots were degradation products of N-acetyl-tryptophan or of the free
tryptophan produced. However, if simple modification of the amino group
can influence the degradation of tryptophan to such a significant extent,
it is most unlikely that extensive auto-oxidation of tryptophan occurs
in proteins. It is therefore apparent that the degradation of tryptophan
in proteins bears little similarity to the degradation of the free amino
acid.
Whilst it is not possible to prove conclusively, the existence of
condensation products at this stage, it is at least certain that much
of the degradation of tryptophan caused by heating occurs independentl,y
of oxidation. 'J.'1e effect of tryptophan degradation on wool yellowing
is difficult to gauge with any certainty, in view of the complexity of
the independent reactions involved. However, the results presented
here have shown that the role of tryptophan degradation in heat yellowing
cannot be disregarded but an exact correspondence between the two does
not exist, due to the existence of at least two different mechanisms for
tryptophan degradation, and a number of independent yellowing reactions
not involving tryptophan.

5. Reactions Involving the Hydroxyl Side Chains.

The heat stability of protein bound serine, threonine and tyrosine
has not received a great deal of attention, possibly because of the
difficulty in obtaining reliable analyses unless an automatic amino acid
analyser is available. However, a number of authors33,44,46 ,4?,54 have
suggested that the degradation of these amino acids may contribute to
the dry heat yellowing of proteins.
Since serine is difficult to determine quantitatively by other
methods, canplete amino acid analyses of wool and gelatin before and
after heating at 130°c for 24 hours were obtained by means of a Beckman
Unichrom automatic amino acid anal,yser. Two samples of each protein
were h,ydrol.ysed and duplicate analyses were carried out for each
hydrol.ysate so that the values included in Table II are the mean of
four analyses. These results indicate that all three hydroxy amino
acid canponents of wool and gelatin undergo considerable degradation
when the proteins are heated at 130°c.
It is interesting to note that while wool and gelatin yellow to
similar extents, the amino acids serine and threonine in gelatin are
degraded far more than those in wool. This may be a result of different
amino acid canposition or differences in peptide configuration.
Weclawowicz et a136 found that 6 hours heating in air at 200oC destroyed
5()( of the serine in wool while serine in silk remained stable under the

same conditions. It thus appears that, as with tryptophan, the decan-

position of the hydroxy amino acids is influenced by peptide configuration.
According to Meybeck and Meybeck9 and Speakman and Janowski54, the
most probable decomposition products of protein bound serine and threonine
are dehydropeptides • Table XI shows that threonine is degraded far
more than serine W'lich is what would be predicted if the reaction is
regarded as dehydration of alcohols, the ease of which is in the order
tertiary) secondary ) primary.
Since dehydropeptides may also be fonned by the degradation of
cystine, the detection of pyruvic acid in hydrol.ysates of heated wool
cannot be regarded as evidence that dehydration of serine occurs.
Gelatin, however, contains negligible cystine, but pyruvic acid determina-
tions (Table XII) show that unheated gelatin contains considerable
pyruvic acid, arising, no doubt, from the alkaline treatment ccmnonl.y
 TABLB I I

Amino Acid Analyses of Wool and Gelatin Heated at 130°C for

24 hr. (in mol./g.)

Amino Acid Wool Gelatin

Control- Heated Control Heated
aspartic acid 510 510
threonine 630 540 190 120
serine l~0 930 350 250
glutamic acid 940 940
praline 540 520
glycine 710 680
alanine 440 440
half-cystine 950 860
Valine 480 475
methionine 25 20
isoleucine 250 255
leucine 640 640
tyrosine 280 280
phenylalanine 2l..5 210
lysine 230 215
histidine 65 55
amnonia 1020 1000
arginine 540 540
 55.

TABLE XII

Pyruvic Acid Content of Gelatin Before and After Heating at 1300C

for 24 Hoo.rs.

Gelatin :C Pyruvic Acid

Unheated 1.3
Heated 21+ Hours 1.5

used in preparing gelatin fran collagen. Heated gelatin does show a

slight increase in pyruvic acid content but, as discussed earlier
(page 39) the accuracy of the determination and the probability of
pyruvic acid reacting with other components of the hydro]ysate, make it
difficult to draw definite conclusions from these analyses. Thus,
while dehydration is the most probable reaction for the degradation of
protein bound serine and threonine, it is difficult to prove conclusively.
In Speakman and Janowski's54 investigation they used dimethyl

sulphate to block the hydraxy groups of silk and found this reduced
yellowing. In this work (see next section) it has been found that

dimeteyl sulphate meteylates free amino and free carboxyl groups as well
a;s l\YdrQ'XY'l and phenolic groups. Another reagent, p-ni trophenylacetate
blocks serine and the free amino groups of wool, but mild saponification
with O.lM eydroxylamine converts the 0-acetyl groups back to free ser1ne87.
Since the extent of heat yellowing of acetylated wool (by p-
nitrophenyl acetate, see footnote to Table XVII) before and after the
free hydroxyl groups had been liberated -was found to be almost identical.
(see Table XIII) it 1110uld appear that the blocJcl.ne oft.he hydroxyl aroups
 56.

of serine has little if any influence. Serine decanposition, therefore,

cannot be considered as an important factor in the heat yellowing of wool.

TABLE XIII
Bffect of Blocking Hydrox;yl groups on Heat Yellowing of Wool
(Y. I. Units).

Time of Acetylated Acetylated &

Heating (Hrs.) (OH Blocked) Saponified
0 u. 16
l 18 18
2 19 19
2J+ 26 27

The decomposition by heat of protein bound tyrosine has been

reported by sane authors32A,36,33,47 and denied by others48,49,54.

It is interesting that Weclawowicz et a136 reported 6Q( destruction of

tyrosine in wool when heated for 12.5 minutes at 200°c 'llilile Bell et a133
found only l2!C tyrosine loss in wool heated for 6 hours at the same
tsnperature. An explanation for this discrepancy may lie in the method
of analysis. Be.11 et a133 dete:nnined tyrosine colorimetrically using
Millon• s reagent which will combine w1 th phenols and possibly quinones
"1ile Weclawowicz et a136 obtained their anal,yses from an amino acid
a.na.lyser which would separate tyrosine from any of its derivatives.
It is therefore possible that sane modification of tyrosine occurs but
is not detected by colorimetric techniques.
 In the present 1«>rk tyrosine was determined by its reaction with

a.-nitros~naphthol as well as by an autanatic amino acid analyser.

The a.-nitros~naphthol detennination showed that no significant change
occurred in the tyrosine content of wool or silk after it had been
heated at 130oC for 2l+ hours whereas the amino acid analyser detected a
Jo,C loss in tyrosine in the same wool sample (see Table II). Thomaslll
has investigated the application of a.-nitros~-naphthol to the analysis
of a variety of substituted phenols and he concluded that the full. colour
was given by para substituted phenols in which the }vdroxyl group was
free and at least one of the positions ortho to the phenolic group was
not substituted; thus, 4-met}vlphenol and 2,1+-dimet}vl phenol gave
positive tests, while 2,4,6-trimethyl phenol did not. He also found
that 3,5-diiodotyrosine f!J1Ve a positive test, presumably at least one
of the iodine substituents is replaceable under the conditions used.
Accordingly it would seem that a negative test will be given only by a
di-ortho substituted species in lfflich the ortho substituents are stable
to the con:iitions of analysis. Therefore to account for the discrepancy
between the results obtained by the two methods of analyses used in this
investigation, it is proposed that heat treatment causes substitution of
the phenol ring but no disruption of the ring.
Proposed degradation products of tyrosine have included o- and p-
quinooes33,44, d.ih,ydroxyphenylalanine47, and 5:6 dih,ydroxy-indole-2-
carboxylic acid32A,l09. The reagent a,.nitros~naphthol would therefore
tail to distinguish between tyrosine, dihydroxyphen,ylal.anine and 5:6
dil\Ydrox;y-indole-2-carbox;yllc acid, but would not give a colour reaction
 58.

with quinones. Although Schirle and Meybeck109 have isolated the

melanin precursor 5:6 dihyd.roxy-indole-2-carbmry-lic acid fran oxidised
wool it is doubtful if sufficient oxidation occurs on heating at 1300C
in air for such a campO\Uld to fotm and no evidence for its formation was
obtained in this work.
It is possible that tyrosine condenses with the peptide goup in a
similar reaction to that proposed for tr.rptophan.

H
,/
H 'I C • 0

/c - ~
/:;;;::. ~--c ~N
\'/
di
'CHR
/
The phenolic ring would thus be more tightly bound to the peptide but
would still be able to condense further with a.-nitroso-~-naphthol, so
giving a positive tyrosine test. Hydrolysis of the protein containing
such a condensation product wou1d yield a substituted tyrosine and hence
would give a loss of tyrosine on the amino acid analyser. Such a

condensation reaction would be largely dependent on the peptide configura-

tion and could explain the different stabilities of protein-bound tyrosine
reported by Weclawowicz et a136 (silk bound tyrosine stable in conditions
which destroyed 60.C of wool-bound tyrosine). It may be that in silk the
confonnation prevents such a condensation occurring.
Attempts to modify specifically the tyrosine residue in wool and
to determine the effect en yell.owing were inconclusive. Wool treated
with freshly prepared diazomethane in ether, which is believed to react
 59.

with the tyrosine hy'drox;yl groups, ws found to yell.ow to a similar

extent as untreated wool. Unfortunately, even carefully prepared
diazomethane can cmtain some methylamine which, even if m1,y traces
of moisture are present, is converted to tetramethyl-anmonium hydroxide110
Wlich can severely' damage the wool • Other treatments such as dimethyl
sulphate in alkali part:i.al.ly block tyrosine residues but also combine
with other groups in the protein, so provide no evidence as to whether
tyrosine is involved in heat yellowing or not. In any case blocking of

the phenolic group might not restrict the cmdensation reaction proposed
above from occurring.
From this relatively' brief investigation it was cmcluded that

tyrosine is modified by heating. The final product is believed to be

formed by condensation with the peptide chain, while the formation of a
quinone and reactions involving disruption of the benzene ring are
considered unlikely. The conformation of the peptide is probably
important when considering the behaviour of peptide bound tyrosine to
heat. There is, however, no evidence that degradation of tyrosine
contributes to the discolouration.

6. Reactions Involving the Free Amino Group.

The effect of dry heat on the free amino group has received
considerable attention, both from the chemica122,23,49,53,55 and
nutritional24- 3l point of view. It has repeatedly been reported that
a reduction in free amino groups or available nitrogen occurs on
heating at 100°c, and this reduction has often been explained in terms
 40 1

wi..:
~
::,
...: .30
).:
,_
z
~

~
~
.3
4
s
~
~
~
C)
"
-4
Lt/
),..

0
l
(HOURS )

Fig. 4: Effect of time of heating on yellown ess.

Legend 1 - gelatin.
2 - wool.
3 - acetylat ed wool.
4 - casein.
5 - silk.
 60.

of crosslinks involving the free amino groups.

To determine the role of the free amino group the effect of heat
on several proteins of differing free amino content ws examined. Free
amino content was detennined by the ninhydrin technique6 5 developed by
Mazingue and Van Overbeke for intact proteins ( see Appendix A) • The
results of heating at 130°c on free amino content and yel1011ness are
shown in Table XIV.

TABLE XIV
The Effect of Heating Proteins in Air at 130°c on Yellowness and
Free Amino Content (in m.mol./lOOg. protein).
Time Wool Silk Gelatin Casein
Hours y .I. NH2 y • I. NH2 y. I. NH2 y .I. NH2
0 19 17.5 13 5.8 25 32.4 8 27.2
0.17 21 17.3 13 5.5 29 32-4 ll 26.4
0.33 22 17.2 .u. 5.4 35 32.1 .u. 26.1
0.75 23 17.1 .u. 5.1 38 31.9 15 25.7
1 24 17.0 15 4.9 39 31.8 16 25.5
2 26 16.7 16 4.5 39 31.8 17 24.8

24 40 16.5 21 5.0 43 29.2 30 23.2

'lbese results show that while there is no direct relationship between

yellowing am free amino content, those proteins richer in free amino

groups tend to yellow more. Both Mazingue and Van Overbelce55 and Norton
and Nicholls53 reported similar results for proteins heated in the range
of 100 - 1600c and suggested that cmdensation between the free amino and
 61.

free carboxyl groups had occurred. If such a condensation occurred,

the protein would contain an increased number of amide bonds, but with
the large number of peptide or amide bonds already present, a small
increase would be very difficult to detect chemically.

The decrease in free amino groups as heating proceeds (Table XIV)

is compatible with the formation of new amide bonds. A similar reduction

has been reported by various food chemists in experiments ranging from

baking peanut biscuits28 ,29, to spray- drying skim mi1.k25, but no one as
yet has completely accounted for such losses.

Unfortunately, the technique used for free amino analysis does not
distinguish between the f-groups of lysine and tenninal amino groups.
Table IV shows the distribution of amino groups in wool.

TABLE XV

Distribution of Free Amino Groups in Untreated Woo185.

Fonn m.mol ./lOOg. Wool

IQsine 19.3
Tenninal Amino 1.68 1.8
Tenninal N-Acetyl 5

Since 24 hours heating caused a reduction of 6'1, in the free amino

content of wool, it is possible that modification of either lysine or
tenninal groups may occur. Amino acid analyses during this investiga-
tion have shown that no si~ificant change in lysine content occurs on
heating in air at 130°c for 24 hours, so the possibility of deamination
of terminal. amino groups must be considered. However no significant
change in total nitrogen as a result of heating could be detected,
using the Kjeldahl teclmique as modified by Miller and Hougjlton86 •
Therefore there appears to be no significant amount of deamination
occurring.
If new peptide bonds are formed by condensation between free amino
and free carboxyl groups, increases in the viscosity of solutions of
soluble proteins and reductions in the urea-bisulphite solubility of

others could be expected. It has already been shown (Table III) that
solutions of heated casein are more viscous than solutions of the
unheated protein, while solutions of heated silk and zein are less
viscous than those of the unheated proteins. '!he decrease in the
viscosity of heated silk and zein may be related to their very low free
amino content and consequent inability to form new interchain peptide
bonds.
The changes in u.rea-bisulphite solubility of wool and gelatin after
heating for 24 hours at 130°c, detennined by the technique of Lees and
&lsworth16 , are shown in Table XVI.

TABLE XVI
Effect on Urea-Bisulphite Solubility of Wool and Gelatin of
Heating in Air at 130°c for 24 Hours.
Wool (% Sol.) Gelatin (% Sol.)
Before Heating 45 99
After Heating 19 15
% change 58 85
 63.

It is imnediatel,y obvious that a large reduction in urea-bisulphite

solubility- occurs when wool or gelatin is heated. This occurs whether
the protein is fibrous or globular so it is unlikely to be the result or
simple configurational changes. Similar resultsl.4,22 ,18,35 have been
frequentl,y reported but whether such results are due to the formation or
new amide bonds or other reactions is not certain. Certainly, such

bonds are stericall.y favoured by the existence of salt liks of the

form -NH3 ----ooc- in the protein, and peptide formation would account
for the changes in solubility, viscosity and digestability previously
recorded. The large change in the solubility of gelatin which contains
negligible cystine tends to contradict previous explanations20,21,35 in
terms or conversion or cystine to lanthionine.
Althcngh the formation of amide bonds probably occurs, it is difficult
to relate such changes to yellowing. Yet those proteins with the highest
lysine content yellow most, and correlations between extent or yellowing

and reduction in free amino groups have been reported for both woo153 and
eylon71.

Fram the results obtained at this juncture, it was felt that further
investigation into the role of the amino group was warranted, so methods
or modifying them were sought. Wool and silk were selected as sample
proteins for a number of reasons. They- have .markedly di! f erent amino
acid compositi0l'ls; both appeared to withstand the treatments without
degradation and remained in a form suitable for colour measurement.
On the other hand, attempts to acetylate the soluble proteins gelatin
and casein produced some degradation, (casein became a pinkish colour)
while the recovered proteins were in the fonn or hard, discoloured
lumps which were not su1 table r or colour measurement.
A ccmmonl.,y emplcyed reagent for blocking free amino groups is
l-fluoro-2-4-dinitrobenzene, but it is not specific for amino groups and
also colours wool a deep yellow, hence is unsuitable when yellowness is
being used as a measure or degradation. Nitrous acid has also been
used to remove the free amino groups but it, too, is non-specific and
causes degradation and discoloration or the protein.
Norton and Nicholls53 reported that acetylation by the I. G. Farben
technique (acetic anhydride in glacial acetic acid with concentrated
sulphuric acid as a catalyst) modified 65% or the free amino groups of
wool without causing discoloration oer- degradation. However Spealanan
and Janowski54 found this method caused a large increase in alkali
solubility and introduced a number of sulphonic acid groups into the
wool. In this investigation it was found that the alkali solubility

increased from li.% to 55% after acetylation, so other less severe

techniques were sought. Some of the treatments selected for examination
and their effectiveness. are shown in Table XVII.
From Table XVII it can be seen that only treatments with dimethyl
sulphate and p-nitrophe1'lacetate produced a si~ificant reduction in
heat yellowing; significantly, both treatments modified a considerable
proportion of the free amino groups • Moreover, in contrast to most or
the treatments viich caused additional yelloW'less, these reagents tended
to bleach the wool during treatment.
Although Phillips, Blackburn and Carter8 have reported that dimethyl
 TABLE XVII
Summary of Treatments Examined in Attempts to Block Free Amino
Groups of Wool.
Effect of Treatment on
Treatment No.* Wool Colour Free Amino Heat Yellowing
1. yellowed
2. yellowed
3. ~ increase in 3% reduction overall reduction of
yellowness ~ in yellowing after
6 hrs. at u.ooe
4. 2~ increase promoted yell.owing by
in yellowness 6'I, after 6 hrs. at
u.ooc
5. 12!( increase 2o,C reduction reduction of lo,( in
in yellowness yellowing
6. yellowness 50( reduction reduction in yellow-
reduced by lo,( ing or 40.C
7. yellowness in- increase in yellowing
creased by 4QC no change of 2QC after 2 hrs.
at u.ooe
8. yellowness 7~ reduction reduction in yellow-
reduced by 20.C ing of 3~ in 24 hrs.
at u.o0 c
9. no significant increase in yellowing
effect by 1~ in 6 hrs. at
u.o0 c
* Treatments
1. 1.5g wool in 32 ml.s 9ClC fonnic acid+ 5.3 ml.s acetic al'leydride:
6o0 c 1 hr. then room temp., 2 hrs •
2. 1.5g wool in 45 ml.s glacial acetic acid containing 6.6 ml.s tri-
nuoracetic acid: room temp., 4 hrs.
 66.

3. Phthalic anh,Ydriae in dioxan.

4. Toluene sulphoeyl chloride.
5. 1 ~ wool treated with 50 mls 5% cyanuric chloride in ~lene at
50°c for 6 hrs.·"
6. 1.5g wool added to 1 ml dimethyl sulphate in 30 mls borax buffer
pH9; room temp. 2 hrs; wash well in dilute acetic acid then
several times in water8 •
7. 2g wool in 50 mls O.lM NaHC03 + 50 mg of me-Cl in 50 mls acetone

3 hrs. at room temperature91 •

8. 5g wool added to 1.25g p-nitrophe1'fl acetate, 0.125 mls. glacial
acetic acid, 125 mls. dimethyl formamide: 2 hrs. at 600C:
wash thoroughly with acetone, dilute aumonia, dilute hydrochloric
acid, then water8'7.
9. Formaldehyde+ Na acetamide in water.

sulphate is a non-specific reagent, reacting with free amino, carbo:xyl,

hydroxyl and phenolic groups, it was decided to use this reagent for
further studies and to compare its effectiveness with that of p-nitro-
pheeylacetate which Milligan87 reported was specific to free amino and
hy'dro:xyl groups and that only very gentle saponification (O.lM hydroxyl-
amine) was necessary to remove the 0-acetyl groups from serine. Samples
of wool and silk were treated with either dimethyl sulphate or p-nitro-
pheeylacetate as described in .footnote to Table XVII and washed well.
For both treatments thorou~ washing was essential, as any unreated
reagent left on the fibre caused severe degradatia,. on heating. After
air-drying, samples or each were heated in air ror various times at
1300C • The degree of yellowing produced and the changes in free amino
content, as detennined by the ninhydrin method, are given in Table XVIII.

TABLE XVIII
The Yellowness {in Y.I. Units) and Free Amino Group Analysis
{in m.mol./lOOg. protein) of Acetylated and Methylated Wool
and Silk Heated at 130oC in Air.
Time of Untreated Acetylated Methylated Untreated Acetylated Methylatec
Heating Wool Wool Wool Silk Silk Silk
(hours) Free Free Free Free Free Free
y .I. NH2 y .I. NH2 y .r. NH2 y .I. NH2 y .I. NH2 y .I. NH2
0 19 17.5 14 4.1 17 8.7 13 5.8 10 3.0 12 3.2
0.33 22 17.2 15 3.6 18 8.2 14 5.4 ll 2.3 13 3.0
0.75 23 17.1 17 3.2 19 7.8 14 5.1 12 1.8 14 2.8
1.0 24 17.0 18 3.2 19 7.6 15 4.9 12 1.4 15 2.0
2.0 26 16.7 19 3.1 19 7.5 16 4.5 13 1.2 16 1.6
24.0 40 16.5 26 4.8 28 8.8 22 5.0 18 2.6 19 2.8

While the blocking or 7 ~ of the free amino groups caused a significant

reduction in the extent of heat yellowing of wool, the effect of similar
treatments on silk is less obvious, possibly due to the fact that silk
contains rew free amino groups at anytime and these may be sterical.ly
hindered fran taking part in the yellowing reactions. The remaining free
amino groups in both proteins arter acetylation or methylation still tend
to decrease as heating progresses. The analytical technique used
determines only the resultant free amino content and probably some groups
 68.

are disappearing 11mile others are formed by peptide breakdown or

decomposition of the acetyl grrup. Since the acetyl group apparently
remains stable 11men boiled in aqueous pyridine (for free amino determina-
tions) peptide breakdown is the more probable. Eventually, for both
proteins, the rate of fo:nnation of free amino groups appears to exceed
their rates of disappearance.
It is interesting to note that although acetylation and methylation
reduce the extent of yell.owing due to heat by sig}lificant amounts, there
is little change in the shape or the rate curves (see Fig. 4). The
yellowing which still occurs after treatments may be the result of
reactions involving the remaining free amino groups, the amino groups
produced by peptide or acetyl breakdown, and reactions completely
unrelated to the amino groups.
Attempts were made to block the remaining free amino groups in wool
by repeated treatments with p-nitrophenylacetate. It was found,
however, that whereas the initial treatment reduced the free amino
concentration from 17.5 m.mol./lOOg. wool to 4.3 m.mol./J.OOg. wool, a
second treatment only reduced this value to 4.1 m.mol./lOOg. and no

further reduction could be obtained by further treatments. This limiting

value is probably due to a certain proportion (approximately 2!!C) of the
free amino groups being located in areas of the fibre inaccessible to the
alky'lating reagent.
'lhat exposure to dry heat causes a reduction in the urea-bisulphite
solubility of certain proteins has alread1' been shCMi• If the cross-

links affecting solubility are formed from the free amino groups, then
blocking these groups should result in a smaller change in solubility.
To see if this was so, samples or untreated and acetylated wool and or
zein, which contains negligible lysine, were subjected to urea-bisulphite

solubility tests before and after heating at 130°c for 24 hours. The
results are shown in Table XIX.

TABLE XIX
Urea-Bisulphite Solubility" Before and After Heating for 24 Hours.
Untreated Wool Acetylated Wool Zein
(% Soluble) (% Soluble) (% Soluble)
Before Heating 45 38-4 9.1
After Heating 19 26.4 6.2
% Change 58 31 32

'!be acetylation treatment itself was fo\llld to reduce the urea-

bisulphite solubility or wool, possibly due to the treatment removing
small soluble peptides from the wool. Since acetylation also has a
bleaching effect which could be due to the removal or some yellowed
material, this would appear to be a likely explanation. In addition
the acetylation or amino groups would reduce the solubility or any
soluble proteins or peptides present.
It should be noted that acetylated wool still exhibits a change in
solubility or 3~ and zein, despite its negligible lysine content,
changes in solubility by 32%. While the comparison or these values to

those or untreated wool indicates that free amino groups contribute to

the changes in solubility or proteins as a result or heating, there also
 70.

appears to be additional reactions Nrl.ch do not involve the free amino

groups, causing a reduction in solubility. Since the change also occurs
in zein, it cannot be attributed to modification or cystine or tryptophan
residues but possibly to sane fonn of oxidation or condensation reaction.
It must be remembered, however, that considerable controversy exists aa
to the value and interpretation of urea-bisulphite solubilities, and,
at best, they are only indicative of changes and are not conclusive.
However, when the viscosity results in Table III are also considered,
there is evidence that, for proteins rich in lysine, heating causes an
interchain condensation reaction involving the free amino group.
I! the free amino groups are condensing with carboxyl groups,
modification of the latter should produce results similar to those
obtained when the free amino groups are blocked. Fraenkel-Conrat and
Olcott88 found that esterification with anhydrous methanol in the presence
or a catalytic amount of mineral acid was specific to the carboxyl groups
and caused no significant degradation. Wool was esterified in this
manner and after thorough washing and air-drying, no obvious degradation
had occurred. In fact, the wool was slightly whiter than before the

treatment and there was no significant change in free amino content,

indicating that little or no peptide damage had occurred. The extent
of esterification was determined by analyses for free carboxyl groups,
using a modification of the Alexan4er ar¥i Hudson89 technique.
Table II shows the effect or heat on untreated and esteriried wool
in terms or ye.llON1ess and free carboxyl content. lsterification was
fo\llld to reduce the heat yellowing of wool by about the same extent as
 71.

TABLI ll

&rtect or Heating Wool at 130oC on Yellowness, Free Amino and Free

Carbox;yl Content.

Time or
Heating Untreated Wool Esteritied Wool
(Hours) y .I. Free COOH Free COOH
m.mol ./lOOg. Y.I. m.mol./lOOg.
0 19 26.0 15 14..0
1 24. 25.0 19 13-4
2 26 25.0 20 13.0
24. 40 24.7 26 12.6

that found for acetylation.

Attempts to esterify more than 50;( or the carboocyl groups by
repeated treatments with methanol/acid were unsuccessful, despite the
fact that Fraenkel-Conrat and Olcott89 reported that they were abl• to
esterify 65',( of the free carboocyl groups of wool. The unesterified
carboocyl groups were found to be reduced by the heat treatment.
If yellowing is due to a reaction involving both free amino and
free carbc,Jcy'l groups, blocking either should be as effective in reducing

yellowing as blocking both. A sample of wool was therefore acetylated

with p-nitrophenyl acetate, then esterified by anl\Ydrous methanol
containing acid. Analyses showed ?(:$, of the free amino groups blocked
and 40C or the carbOlCYl groups. The sample was then subjected to 24.
hours heating at 130°c. The reduction in yellowing (Y. I. • 40 ~
Y. I. • 25) was equivalent to that obtained tor either treatment alane
 72.

(Y. I. • 4.0 ~ Y. I. • 26) and so supported the interaction theory, since

if different reactions were involved, the effects or the two treatments
should be additive. Norton49, too, obtained similar results for a
combined treatment and concluded that sane fom of interaction occurred.
While considerable indirect evidence supports the theory that inter-
action occurs between free amino and free carboxyl groups, it is ver:,
difficUl.t to prove it conclusively. Such crosslinking would account
for changes in viscosity4-9,53, solubilityl'+ and available nitrogen29.
The formation of these amide crosslinks will not result in discolouration,
but, as will be proposed later, could be the first or a series of reactions
leading to the formation of yellow products. It should be noted too,
that insolubility does not always correlate with yellowing; in this
investigation it was round that heating at so0 c for 4. days caused a
reduction of 3,,C in solubility but caused an increase of only five units
in yellowness. This suggests that the condensation reaction can take
place at a lower temperature than that required for the subsequent
yellowing reactions.

7• 'lha Sf fect of Metallic Ions.

The use or metallic ions, in particular zinc and aluminium, to
reduce the heat yellowing of wool, has been reportad58,59,60,62, but
no reasons for the choice of a particular metallic ion or a satisfactory
explanation for its action have been offerred. B>cplanations have
included the formation of -S-Zn-S- bridges at those places which tend
to form unsaturated groups59 during hydrolysis, and the formation or
 73.

crosslinks by zinc thiocyanate salts at the f'ree amino groups58.

'!he effectiveness of a variety of metallic ions in reducing heat
yellowing was ex:arn1ned to see if any satisfactory explanation for this
effect could be found. Wool samples 'Were agitated in aquerus solutions
(pH4 to 6.5) of a range of metallic ions (listed in Table XXI), then
air-dried and heated at 130°c. Insoluble salts such as zinc phosphate
were precipitated in the fibre by first immersing the wool in a solution
of soluble phosphate, followed by imnersion in zinc sulphate solution.
Treatment in the reverse order, i.e. zinc sulphate followed by phosphate,
was ineffective, a fact Bell, Hutchinson and Whewell62 had also noted.
This was attributed to the smaller zinc ion diffusing out of the wool
before the larger phosphate ion was able to diffuse into the fibre and
combine with the zinc. Zinc analyses of the treated samples confirmed
this by showing that little or no zinc remained in wool treated in this
manner.
'!he effectiveness of the various metallic ions examined in reducing
heat yellowing are shom in Table XXI.
By using solutions of different anions, e.g. zinc chloride, zinc
sulphate, precipitating zinc phosphate in the fibre, it was found that
the anion did not contribute to the reduction in yellowing.
That only metals in the groups 2A and 2B of the periodic table
(e.g. magnesium, calcium, barium, cadmium, zinc) and aluminium were
effective in reducing yellowing is significant and indicated that the
electronic structure or the metal is involved. Mercuric salts are
lm<Ml to attack the disulphide bond73, so this anomalous result was not
 74.

TABLB III
Bt.f'ectiveness of Metallic Ions in Preventing Heat Yellowing.

(Solutions Approx. ~ Metallic Ions)

Metallic Ion Bf.feet on Heat Yellowing

Na+ no effect
M,f+ slifllt reduction
ca2 + significant reduction
Ba2 + ver:, significant reduction
Mn2+ no effect
Co2 + blue colour of ion overshadowed any- effect
eu2+ promoted yellowing
Cd2+ significant reduction
Hf+ promoted yellowing
Pb2 + promoted yellowing
zn2 + significant reduction
n3• reduction
Fe2+ promoted yellowing

entirely unexpected.
The amowit of .metallic ion needed to produce maximum reduction in
yellowing WIS determined by inmersing wool samples in solutions of
different concentration• of metallic salts, am. by measuring the amount
of metal.lie ions absorbed by the wool using a Techtron Model AA-4 atomic
absorption spectrophotaneter. To determine the metallic ion uptake,
triplicate samples (5 mg.) of treated wool ware weighed out, ~dro]ysed
 75.

1n 18N sulphuric acid and diluted to 500 ml.s. Calibration solutians

were prepared rran A.R. grade metallic salts. The relationship between
metallic ian content and heat yellowing is shown in Table llII.

TABLB XIII

Effect or Metallic Ion Concentration on the Heat Yellowing or Wool

Heated 24 Hours at 130°c in Air.

Cadmium Zinc

% Cd2+ on wt. Change in Y.I. % zn2+ on wt. Change in Y. I.

or Fibre of Fibre
0 27 Y.I. units 0 25 Y. I. units
1 16 II
2 18 n

1.7 13 n l+ 11+ II

4.5 11 " 8 12
,,

It is apparent that a cadmium or zinc content of ~ metallic ion on

the weight of the wool is sufficient to reduce heat yellowing by al.most
6QC while even 1% metallic ion causes a significant reduction. The
metallic ions were not bound strongly to the fibre and repeated washing
in cold water was sufficient to remove all the ions, as verified by
metallic ion analyses or wool after several washinga.
'!he nature of the binding of metallic ions to proteins is far frcm
clear, suggestions have ranged from binding specifically at car~l
groups99 to coordination with amino and amide1o6 llhks and even to form
coordination type crosslinks of the type•HN_. Ni~ NH_lOO. It is not
unreasonable to suggest that the binding of a metallic ion with a
 76.

carbox;rl anion would prevent the latter condensing with other gi-oups
present, such as the free amino. In additian, coordination or meta.llic

ions with free amino goups would hinder their participatian in condensa-
tion reactions, resulting in a reduced loss of free amino groups as a
result of the heating treatment. Results given in Table XXIII show that
the presence or zinc ions do indeed reduce the loss of free amino groups
by heating. The free amino analyses were carried out by the ninhydrin
method (see Apperni.x A) on samples washed free of zinc ions after heating.

TABLE XXIII
The Effect of Zinc Ions on Yellowing and Loss of Free Amino Groups
Caused by Heating at 130°c in Air.

Time of Control Sample Sample Containing 6% Zinc

Heating Ion on Wt. of Wool
(Hours) y .I. Free Amino Y.I. Free Amino
m.mol./1.00g. Wool m.mol ./lOOg. Wool
0 19 17.5 19 17.5
0.17 21 17.3 20 17.4
1 21+ 17.0 21 17.3
2 26 16.7 23 17.3
21+ 40 16.5 30 17.1

Further confirmatory evidence was obtained by eY8D11n.ing the effect

an yellowing of the presence of zinc ians in conjunction with acetylation
of the wool. These results are given in Table XXIV.
 71.

TABLB llIV

The Effect on Yellowing of Wool Heated at 130°c in Air After

Treatments with Zinc Ions and p-Nitrophen_ylacetate.

Time of Yellowness Index

Heating
(Hours) Untreated Acetylated Wool containing Acetylated containing
6% zn2+ 6% zn2+
0 19 13 18 13
0.33 22 15 20 lJ+
1 24 18 21 18
24 40 26 30 24
Change in
Y.I. after
24 hours 21 13 12 11
heat

Since the canbination treatment gives no improvement in reducing

yellowing canpared with either acetylation or zinc ion treatment alone,
it appears that each treatment prevents yellowing by the same mechanism;
that is, by preventing the free amino group participating in cmdensation
reactions.
'!his explanation, however, does not explain why other metallic ions
(copper, ferrous, mercuric and lead) promote yellowing, as they should
be as effective as zinc or cadmium ions in co-ordinating with the carboJcy'l
and amino groups. One possible explanation is that Wlile they are, in
tact, reducing yellowing occurring by the nomal condensation mechanism,
they are pranoting yellowing by other means, possibly by a free radical
Fig. 5: Electron spin resonance spectra of heated wool.
 78.

mechanism since transition metals such as copper and iron are known to
initiate free radical reactims.
To see if free radical mechanisms were involved in heat yellowing,
a series of E.S.R. tubes were packed with 50 mg. fibre samples of
untreated, zinc treated and copper treated wool and evacuated to a
pressure of less than 0.l nm of mercury and sealed. The electron spin
resmance spectraneter was arranged so that the samples could be heated

in the cavity, which meant that the E.S.R. spectra could be obtained
N'lile heating (at 1300C) was actually occurring, so that an,y short-lived
radicals produced would then be detected. Spectra were obtained at
intervals during heating and representative samples are shown in Fig. 5,
where it can be seen that no identifiable free radicals could be detected
during the heating of the untreated or zinc treated samples. These
results are supported by a recent paper of Eaton and Keighleyl05 who
found no free radicals were fo:nned in wool by heating either in air or
nitrogen. The large E.S.R. response due to the copper ion present in
the wool prevented the detection of other radicals, hence no conclusive
evidence was obtained to support the theory that copper ions enhance
yellowing by a free radical mechanism.
Previously it has been shown that treatments which reduce yellowing
al.so tend to reduce tryptophan degradation. Table XIV shows the effect
of the presence of zinc ions on the tryptophan degradation caused by
heating wool at 130°c.
 TABLB IXV

The Bff ect of zn2+ on Tryptophan Degradation in Wool Heated at 130°c.

Time of Untreated Wool Wool containing &I, zn2 +

Heating (hrs.) on wt. of Fibre
y • I. y .I.

0 19 18 0.90
24 40 30 o.86

It is imnediately apparent that the metallic ion treatments which reduce

yellowing al.so reduce tryptophan degradation. .Again, steric hindrance
resulting from metallic ions co-ordinating with the amide bcnd106 and
preventing the proposed ccndensation between the peptide carbo:xyl and
the indole nucleus ~ be responsible. Alternatively the metal.lie ions
are able to disrupt the protein configuration sufficiently' to reduce
significantly' the tryptophan degradation, a result closely paralleling
those found for acetylation and esterification treatments. It is
difficult to visualise a metallic ion preventing disruption of the indole
ring. This proposal. again emphasises the importance of the protein
configuration in the degradation of peptide bound tryptophan in wool ■
 80.

Chapter III: ISOLATI~ AND ID•TIFICATION OF THE Yl[,IOV' CCMPONaJTS.

1. Introduction
The previous investigations had revealed that a whole series of
reactions were involved in the dry heating of proteins, only some of
which caused colour changes. It was therefore considered that the
only W83' in which the colour producing reactions could be elucidated
unequivt,cabl.y' was by separation and identification of the yellow
material.
Numerous attempts9,48,49 to isolate the yellow components from heat
damaged proteins have been made, but with little success. Such lack of
success can be attributed to a number of factors but especially to the
complexity of the protein itself. Furthermore, when yellowing occurs
the chromophoric material formed appears to be bound strongly to the
protein chain, since various attempts9,4S,49 to remove it b,r solvent
extraction have proved unsuccessful. Therefore, a satisfactoey method
for separating the yellow material from the protein is required before
any isolation and purification can be attempted.
The need for hydr~sis complicates arr:, work with proteins. Some
amino acids such as tl',Y'Ptophan and serine72 are partiall,Y destroyed
during acid or alkaline hydroly'sis; certain reactions such as the
partial oxidation of cystine are reversed to some extent73; there is
a strong possibility that during hydrolysis, reactions may- occur between
the various degr-adation products; and final.l,Y, it is a distinct
possibility that the chromophoric material 'llilich is being isolated will
 81.

be chemically modified or completely' destroyed during the isolation

process.
In the present work alkaline h,Ydroly'sis was considered unsuitable

since alkali is known to produce a series of yellowing reactions49,53

and it would be impossible to distinguish between the yellow material.
formed by heating and that due to the alkali. Both enzymatic l'\Ydroly'sis
and mild acid hydroly'sis were investigated.

2. &1.z.vmatic Hydrolysis
&,.zymatic l'\Ydrolysis is probably' the method least likely to destroy
the chrornophoric material. The general mechanism of enzyme action may
be stated as follows: the enzyme and its substrates combine to form an
enzyme-substrate complex, within which occur a series of atomic and
electronic rearrangements, the rearranged complex then dissociates to
give products and free, regenerated enzyme74. Unfortunately for this
work, most enzymes are specific to peptide bonds joining certain amino
acids only, so that no one enzyme will completely' break down a protein
to its constituent amino acids, and even with a carefully chosen combina-
tion of enzymes specific to different likages, sane of the bonds between
amino acids remain unbroken75. Apart from the fact that only partial
}\ydrolysis is obtained, there is also the problem of separating the
enzyme itself fran the hydrolysis products. Precipitation of the
enzyme with trichloracetic acid has been used as a method of separation,
but has the disadvantage of also precipitating any large peptides in the
hydroly'sate. Gel filtration and column chranatography are now the
ravoured methods or separation.
The eneymatic ~ol,ysis or unheated and heated casein was examined
using o.~ papain (on weight or protein) in an acetate buffer of pH5.6
ror 18 hours at 3?0 c. Papain is less specific than most enzymes and
was the most suitable or those available at the time. However, despite
its relative non-specificity, after 18 hours a considerable amount or
casein remained undissolved. The liquor was filtered fran the insoluble
protein and treated with trichloracetic acid to precipitate the enzyme
which was removed by centrifuging and filtering. The remaining solution,
~ich was yellow in colour demonstrating that the enzyme had not destrc,yed
the chranophore, was then treated with a series of solvents, (ethanol,
methanol, acetone, dimet~l formamide) each of which caused a fraction to
precipitate. &!lch precipitated fraction was yellow in colour and was
collected and subjected to thin-layer chranatographic analysis. These
analyses showed each fraction so obtained contained such a large variety
or products that the method was of little use in isolating the yellow
material. Separation on ion-exchange columns of Dowex: resin failed to
produce purer fractions. It appeared the degree or hydrolysis obtained
was not sufficient for isolation or the yellow canponent. However,
it was observed that heated casein appeared to resist enzymatic hydrolysis
more than unheated casein, bearing out the observations or others27,30,31
that heated proteins were less susceptible to enzymatic hydrolysis.
 83.

3• Mild Acid llydrol.ysis

Although mild acid hydrolysis is far fran satisfactory, it is less
specific than enzymes and if hydrochloric acid is used, there are no
problems in separating the hydrolysis products fran the acid. Samples

of wool heated at 130°c for several dqs were imnersed in 6N hydrochloric

acid at a temperature of ?OOC for 4 hours, with occasional shaking.
These conditions produce only partial hydrolysis but judging fran the

absorption spectra of the resultant hydrol.ysate, the chromophoric groups

are not destroyed. Furthermore, samples of unheated wool, hydrolysed
under the same conditions, produced a strong blue colour, indicating that
this method of hydrolysis did not produce yellow material. '!be usual
hydrolysis conditions used for amino acid analyses, viz. 24 hours at

100°c, produces a yellow to brown solution, irrespective of the original

state of the wool.
After the mild hydrolysis, the solution was filtered, then evaporated
to dryness under reduced pressure. Distilled water was added and
evaporated several times so that the final material, which was dark brown
and eygroscopic, was approximately neutral.

4. Chromatographic Separation
Separation of the various products in the mixture was achieved by
column chranatography. A series of columns were packed with various

Sephadex ion-exchange resins and small quantities of the brown material

1n a range of buffers applied to each. Sephade.x ion-exchange resins
were especially selected since a white material was necessary to allow
observation of yellow bands and so to assess the degree of separation
achieved. Many of the other ion-exchange resins available were yellow

to brown in colour which made observation of yellow bands difficult.

'lhe resins and buffer systems examin,ed are shown in Table IIVI.
For Sephadex G.100 a sodium chloride eluent of increasing ionic strength
was used, while for the others, gradient buffers were necessary.

TABLE XXVI
Resin and Buffer Systems Examined in Attempts to Separate
Chromophoric Material. from Acid Hydrolysate.

Resin* Eluent Result

Sephadex GlOO Sodium chloride O.O]M-+ 3'1 Poor separation
n nD.B TRIS-HCl Buffer pHlO _,. 2 Very little separation
n CM Phosphate Buffer pH4 - 10 3 bands separated
n CM Acetate Buffer pH3 --t: 10 3 n n

n SI Phosphate Buffer pH4-+ 10 6 bands poorly separated

II
SB Acetate Buffer pH3 _.., 10 7 bands well separated

* DIAi - diethylaminoethyl - weakly basic resin

CM - carbOJcymethyl - weakly acidic resin
SB - sulphoethyl - strongly acid resin

'lhe strongly acidic resin Sephadex SE was found to provide the best
separation ""1en canbined with a O.]M acetic acid/0.lM potassium acetate
buffer. '!he broWl mixture was applied in O.]M acetic acid solution,
and all except one fraction was absorbed onto the top of the column.
Fraction A was eluted with O.]M acetic acid, then the pH of the eluent
was gradually increased by the addition of potassium acetate and the
remaining bands eluted. The last two bands, (F and G) were eluted by
O.]M potassium acetate solution, after which O.]M potassium hydroxide
was used to cleanse the column before it was regenerated with o.J.M
acetic acid.
Bach fraction was evaporated to dryness under partial vacuum and
its colour and solubility noted. No attempt, apart from solvent
extraction, was made to separate the fraction from the buffer salts.
All fractions were found to be soluble in water, but solubilities in
other solvents varied. Early fractions were soluble in acetone or
dimeteylf ormamide, while others were soluble in ethanol. The last few
were soluble in water only. The differences in solubility of the
various fractions may have been due to different chromophores but since
such mild eydrolysis techniques were used, it is probable that the
chromophores were still bolllld in peptides which would be responsible
for the differing sol.ubilities between the fractions.
AlthougJ-l several of the fractions appeared to be oils, cooling
and scratching in various solvents such as etl'\Yl acetate or ethanol
eventually led to the fonnation of crystalline compounds. These
crystals remained stable 1'1tlile maintained in an aneydrous atmosphere
but exposure to the normal atmosphere for even a few minutes caused
reversion to the oily state. Barland, Stell and Wiseman46 encountered
 86.

similar difficulties lilen isolating melanins from oxidised wool.

5. Anal.ysis of Fractions
The colour, solubility and stability to roan atmosphere of the
crude fractions are summarised in Table XXVII.

TABLI XXVII
Sumnary of Properties of Unpurified Fractions.

Fraction Colour Solubility Stability to Air

A lmion yellow soluble in acetone very hygroscopic
B red brown partially soluble in
ethanol slightly hygroscopic
C mediwn brown II II hygroscopic
D orange brown insoluble in ethanol hygroscopic
g orange brown II II hygroscopic

F yellow brown partially soluble in

ethanol hygroscopic
G dark brown insoluble in ethanol stable

To see if the fractions were indeed peptides, small samples of each

were eydrol.ysed in 6N HCl for 18 hours at no0 c in sealed tubes. After
evaporation to rmiove the acid, samples of each hydrol.ysate, as well as
samples of each unhydrol.ysed fraction, were spotted onto individual thin
1.ay'er silica gel chranatographic plates and eluted in butanol: acetic
acid: water ~0:10:10, then dried and developed with n.inhydrin.
Table XXVIII shows that the original fractions themselves were not pure
and secmdly, that each fraction is indeed composed of one or more large
peptides.

TABLE XXVIII

Sumnary of Chromatographic Separation of Unpurified Fractions.

Fraction Before Hydrolysis After Hydrolysis

A one spot 10 spots

B two spots l2 spots poorly defined

C six It
12 It It It

D five It
15 It II It

I three " 15 It It n

F two n 16 n n It

G three" 14 It It n

Attempts to obtain a pure peptide containing the chromophore by

means of recrystallisation were undertaken. In most cases recrystall.i-

sation required a mixture of solvents such as dimethyl fomamide/etJ:\yl

acetate or water/ethanol. Usual.l.y two recrystallisations in this manner
were sufficient to obtain a fraction which contained only two or three
very similar compounds when examined by thin-layer chromatograpJ:\y.
These final fractions resisted all attempts at further purification by
recrystallisation. However, since some separation was achieved on
silica gel plates using an eluent of 7°" ethanol in water, separation

by means of a colWll'l of silica gel was attempted but no further purifica-

tion was obtained. It should be realised that at this stage one is

working with perhaps 5 mgpis of material. althougti the original. wool
sample was about 20 gns.

6. Amino Acid Anal.ysis

'Ihe recrystallised fractions were hydrolysed and subjected to a
more detailed analysis by means of a Beckman Unichrom automatic amino
acid analyser, the results of which are shown in Table XXIX. Since
the fractions were so hygroscopic, no attempts were made to measure
accurately the amounts of fractions hydrolysed, the analyses therefore,
only give relative amounts of amino acids. 'Ihese analyses immediately'
confirmed the conclusions drawn from the thin layer work, that the
fractions contained very large peptides.
'Ihe appearance of cysteic acid in each fraction emphasises that
oxidation is involved in the degradation process, whether or not it
causes yellowing.

Al>art frcm demonstrating that the chromophore was still bound to

large peptides which, no doubt, were responsible for differing solubility
and chromatographic properties, Table XXIX shows that each fraction
contained a significantly large proportion of amnonia. This suggests
that the chromophore is a compound which is destroyed by acid hydrolysis
to yield anmonia as one or its p,·incJ.pal. products; possibly a pyrrole
type canpound as will be discussed later. Veey little other significant
information was obtained fran these analyses.
 TABLE llIX

Amino Acid Composition of Fractions Separated from Heat Yellowed

Wool (in mol. relative to 100 mol. glutamic acid) •

Amin?S:on
Acid A B C D I F G

cysteic acid 10 12 7 2 4 6 6
aspartic " 40 59 103 30 37 58 58
threonine 10 23 14 50 27 11 25
serine 13 23 26 30 45 26 31
glutamic acid 100 100 100 100 100 100 100
proline 22 27 19 180 6 5 30
glycine 87 120 38 40 68 50 59
alanine 61 52 24 60 50 26 47
cystine 8 4 2 7 10 2
Valine 33 46 12 20 22 13 31
iso-leuoine 15 19 12 3 7 10 22

leucine 26 42 26 8 29 24 50
tyrosine 23 7 7 18 30
phenylalanine 23 11 19 8 12
lysine 4 19 11 I+ 4 I+ 50
amncnia 225 420 280 330 132 60 300
arginine 4 62
 zso .300 320 .360 400

WAV£L£/VGTH

Fig. 6: Ultra-violet absorption spectra of fractions isolated

from heat yellowed wool.

 90.

7• Absorption Spectra
Since the fractions showed quite marked differences in colour, the
visible and ultra violet absorption spectra of aqueous solutions of each
fraction were examined. Despite the colour differences each fraction
showed similar absorption properties in the visible region. However,
canparisoos of the ultra violet spectra showed significant differences
which are illustrated in Fig. 6 and sunmarised in Table XXX.
The slight shoulders detected in the 280-305nm range in all fractions
except A are probably due to absorption by tyrosine. This conclusion is
supported by the amino acid analyses which show small quantities of
tyrosine present in those fractions which absorb in this range. The
slight peak at 350nm in fraction F cannot be attributed to absorption by
aey of the amino acids revealed in the analyses, so it is possibly due to

the yellow component. However, it is difficult to understand why the

other fractions failed to show similar peaks unless at least two chromo-
phores are involved, only one of which absorbs at a specific wavelength.

TABLE XXX
Sunmary of U.V. Absorption Curves.
Fraction u.v. Curve
A Snooth curve
B Slight shoulder at 290nm
C Slight shoulder at 280nm
D Slight shoulder at 300nm

I Shoulder at 295nm
F Slight peak at 35Cni, shoulder at 290nm.
 91.

a. Fluorescent Spectra

Al.l of the fractions isolated from the heated wool samples were

found to be fluorescent. The fiuorescent spectra of aqueous solutions

or the fractions, obtained on a Farrand Spectrofiuorimeter are shown in

Fig. 7 and sunmarised in Table XIII. It is obvious that the fiuorescent

spectra of the various samples are very similar, which suggest that each

fraction contains a similar chromophore. It is interesting to note that

the excitation lll8J is 335nm, whereas the U. V. absorption spectra show

no distinct maxima at this wavelength. The material responsible tor

fiuorescence must therefore be one of a number of yellow canponents present.

TABLE XIII

Sumnar:, of Fluorescent nata of Fractions Obtained fran Wool Heated

Fraction Colour Eiccitation !mission

Wavelength Wavelength
(nm) (nm)

A lemcn yellow 335 415

B red brown 335 410
C medium brown 335 415
D orange brown 330 420
I orange brown 330 415
F yellow brown 335 410

For a compound to absorb and emit at these wavelengths, a reasonably

extensive degree of conjugation would be required. For example,

3-1\Ydra,cy-anthranillc acid absorbs at 320nm and emits at 415nm while
pyridoxamine absorbs at 335nm and emits at 400nm. Both these canpounds
are aromatic and are obviously well-conjugated. It wrul.d therefore be
expected that the compounds isolated in this work would also be extensively
conjugated and most likely, aromatic. The amino acid analyses of the
fractions given in Table XXII show that tyrosine and phenylalanine are
the only aromatic amino acids present and they are known to nuoresce
at different wavelengths (tyrosine gives an emission peak at about 305nm
while phenylalanine gives a weak emission at 290nm). In these fractions

their fluorescence appears to have been canpletely quenched and the

nuorescence spectra obtained must be attributed to the yellow.material
in the peptide.
The fluorescence spectra of a r ew soluble proteins were also
determined. Heat yellowed gelatin absorbed at 325nm and emitted at
410nm Wlereas unheated gelatin exhibited no fluorescence. Heat yellowed
]Jrsozyme behaved similarly, but unheated lysozyme al.so nuoresced, but
in the tryptophan region (350nm emission). Although neither glycylserine
nor gl.utathione nuoresced, they too, exhibited strCl'lg fluorescence after
heating at 130°c for 24 hours. That such a simple compound as
gly'eylserine should fluoresce limited the possible canpounds considerably.
No fluorescence was detectable Wlen the dipeptide glycylglycine was
heated, so it would appear that the initial reaction resulting in
fluorescence must occur at the serine residue, possibly deh,ydration to
fo:nn a dehydrc,peptide. Decarboxylation and deamination could conceivably"
occur but would also occur in glycylglycine.
 93.

Following the formation of a dehydropeptide, various condensation

and cyclisation reactions leading to conjugated systems can be postulated,
these will be discussed later. Glutathione can also fonn a dehydro-
peptide by elimination of }vdrogen sulphide or can condense to form
thiazole systems as has already been discussed.

9. Formation of PYrroles
Norton49 has suggested the formation of oxidised pyrroles to explain
the heat yellowing of wool. Although pure pyrrole is colourless and
does not fluoresce, on exposure to air it becanes first yellow then dark
brown in colour and fluoresces strongly. This dark colour is believed
to be due to the fonnation of 2,5 dieydroxypyrrole10'7 which is known to
fluoresce strongly. Substituted pyrroles vary in colour fran white to
dark brolffl and man,y can be obtained in cr:,stall.ine f o:nn. 'Dle fiuorescence
of sane pyrrole compomids was therefore eYNDjned and the results
sumnarised in Table XllII.

TABLI XXIII

s,mmar:, of Fluorescent Data of Selected Pyrroles.

Excitation Bnission
Compound Colour Wavelength Wavelength
(nm} (nm}

Freshly distilled pyrrole colourless

2,5-dil\V'droxypyrrole broNi 350 430
3,3 1-dipyrrole ketone yellow 'J?O 425
3-carboxy eteyl-4-meth,yl-
2,5-dicarboxy-pyrrole broNi 350 425
 94.

While it is obvious that these results do not prove that heated

proteins contain pyrroles, they do show a substituted pyrrol.e may- be
sufficiently conjugated to nuoresce at similar wavelengths to those
found for the heated wool fractions.
A generally accepted test for pyrroles is the fonnation of a red
colour when mixed with Ehrlich's reagent (p-dimethylaminobenzaldehyde in
hydrochloric acid). 'lbe reagent is not specific for pyrroles since it
also reacts with indoles, hydroxyindoles, aranatic amides and ureides.
However, the colrurs are so distinctive that in most cases the results
are quite definitive. Table XXXIII illustrates the variety of colours
the reagent forms.

TABLE XXXIII

Colour Reactions with Ehrlich's Reagent.

Canpound Colour
Pyrrole bright red
Bt.hy-1 2,4-dimethylpyrrole-
2,5-dicarboxylate bright red
Indole purple
Tryptophan purple
Benzamide blue
Benzopyrazine yellow
Aspergi.ll.ic acid yellow

'lbe presence or pyrroles in heat-yellowed polyamide (eylon 6 .6)

 95.

was demonstrated?l,lOO by the formation of a red-colour with lhrlich•s

reagent and the following series of equations formulated.

02
---;,, -NH-C-CH-(CH2)2-CH-C-NH-
II I I II
0 OH OH 0

J, Oz
-NH-C - C • CH - CH • C - C-NH- ~ -NH-C-C - ( CH2 )2-C - C-NH-
d I I II enol II II II I/
O OH OH O 0 0 0 0

l condense with NH2 from

neighbouring cnain

O CH - CH
,, II ,,
- NH - C - C
\ I C - ,,C - NH -

N 0
I
R
I

Wool is chemicalzy a much more complex polymer than n.vlon. However,

both polymers contain amide (peptide) bonds in the polymer chain and
have tenninal. amino and carboxyl groups. Hence similar reactions to
those occurring in n.vlon could possibly occur in wool.
Since most proteins contain tryptophan, which gives a blue colour
with the reagent, the detection in heated proteins of pyrroles by
lbrlich 1 s reagent is more difficult. However, gelatin contains no
tryptophan and after prolonged heating it was found to give a clear red
colour with Ehrlich's reagent, indicating the presence of pyrroles,
unheated gelatin gave no such colour.
R
A protein has the formula -NH-CO-bi-NH- and in no case does the
 96.

side chain R contain sufficient paraffinic groups for the reactions

suggested for the polyamide to apply. However, if amide bcod formation
occurs between the -amino group or ly-sine and one or the acid side
chains or the dicarbOXY"lic acid residues, reactions similar to those
postulated for the poly-amide might take place.
'I
NH
I
C■O
I
+ HOOC-CHz-CH
I
NH
) amide bond I
W fo:nnation
'
NH
I I
C ■O C■O
I I
CH-CHz-CHz-CHz-CH2~ - CO - CHz - CH +
I I
NH NH
J,,
' I
oxidise etc. I
canbine with NH2 from neighbouring chain
NH
I
C ■O CH - CH 0 C•O
I II
tl
CH-C C- NH - "C - CH2 - ICH
l ,; I
NH N NH
I I I
(CH2)4 - CH

This theory explains the previously observed importance or the free amino
group in yellowing and also accounts for the disappearance of carbOXY"l
groups.
After prolonged heating, casein, W'lich contains a high percentage of
lysine, ,>ve a reddish colour with Bhrlich•s reagent, indicating that
moat of the tryptophan had been destroyed and sane pyrrole formed.
Zein, llhich contains no lysine, darkened slightly in colour (pale yellow
to slightly deeper yellow) an prolonged heating, but gave a negative
reaction with Ehrlich's reagent, thus indirectl,y supporting the argument
that ly'sine is necessary for the formation or pyrroles in proteins.
SUrprisingly, some of the simpler peptides and model compounds al.so
~ve red colours with Bhrlich I s reagent • Serylgly-cine, after heating
in air at 130°c for 24 hours, at first gave a negative reaction with the
reagent, but after standing at room temperature for a further 24 hours,
~ve a distinct red colour. This suggests that heating caused the
formation of a canpound which was subsequently oxidised or cyclised to
form pyrrole type compounds. The following equations were fonmtl.ated
to explain such a reaction.

f2
r-CH20H

r•O > NH2

I
NH -HzO C • CH2
I VS-
'
cr2 -C02 c
~\{/ 3
s~

COOH 0 NH

J, condense
 98.

Certainly this proposal can be criticised, mainly because or the

improbability or a terminal. -CH3 group attaching to the free end of a
carbon-carbon double bond. However, such a reaction would be necessary
to account for pyrrole formation.
If decarbox;ylation does not occur as the initial reaction, condensa-
tion involving the free carbax;rl group may- occur. A condensation with

the free amino group could lead to the formation of a ureide type can.pound
which would also react with Ehrlich's reagent, but would give a yelJ.ow
colour. Since the reagent and solution are also yellow such a canpound
would be more difficult to detect by this technique. However, since
the final colour with Bhrlich 1 s reagent was red, this reaction is not a
satisfactory explanation of the yellowing or serylglycine.

NH2 HO 0
I \ II
CH-CH20H NH2 C
I I I
,-o A) C • CH2 CH2
NH
I
CH2
"0
~c - N
/
I
H

Ureide
An altenia.tive condensation involving the carboxyl group was
proposed by Norton49 1n his work on alkali yellowing. This involved a
 99.

reaction between the free carboxy-1 group and the free end of the carbon-
carbon double bond as shown in the equation

NH2

"/
C ~2
1- NH2"-
o-,c (j
6tc
I
C • CH
\
_/', 0 • C C • O
NH - CH2 OH
' NH I2
- CH
However such a compound does not react with !brlich 1 s reagent so is not
a satisfactory explanation for the present work.
When the fractions isolated from heated wool were treated with
J!hrlich 1 s reagent, not all fractions gave positive pyrrole tests (see
sumnary of results in Table XXXIV') • In some cases the colour formed
by the reagent was partially masked by the strong yellow colour of the
untreated solutions; however sufficient colour change occurred to
conclude that there was some reaction but not necessarily' a positive
pyrrole test. The fact that some fractions gave positive lbrlich 1 s
reagent tests indicate that substituted pyrroles are formed when wool
is subjected to dry heat for long (e.g. 1 week) periods. A negative
test for pyrroles but a yellow colour could be due either to the initial
solution colours or ureides. In view of the fluorescence discussed
earlier, ureides -would seem possible.
Pyrrole and many of its derivatives can be reduced by zinc/acetic
acid to pyrrolines. In fact this is a simple additi+action to a
 100.

TABLB XllIV

Sumnary or Colour Reactions Between Fractions fran Heated Wool

and ID'lrlich 1 s Reagent.

Fraction Colour Reaction

A yell.ow solution - no apparent reaction
B red colour - positive pyrrole test
C n II II n n

D slight reaction - pyrroles probably present

B red colour - positive pyrrole test
F slight reaction on standing - pyrrole probably present
G yellow solution - no apparent reaction

1,3 diene system. The wool fractions, substituted pyrroles and several
heat yellowed proteins were treated with zinc and hydrochloric acid and
the effect on their colour noted.
The results indicate that there are at least two chranophores
involved in yellowing: one is a relatively simple chranophore, possibly
containing a 1,3 diene system which can be reduced by a zinc/acid
treatment; the second chromophore is probably more complex, is certainly
more resistant to reduction, and is found in both wool and gelatin after
heating.
A.not.her canplication in isolating and identifying the yellow material
formed by heating is, that during heating, deh,ydropeptides may form.
As soon as these canpoWlds are subjected to acid hydrolysis they will form
 101.

TABLB XXXV

The Effect of Zn/HCl. Treatment on Colour of Selected Compounds.

Compound Effect of Zn/HCl. Treatment

Fraction A decolourised
n B parti.al.ly decolourised
n C decolourised

" D decolourised
II
E decolourised

" F decolourised

" G decolourised
heated gelatin partially decolourised

heated zein de colourised

heated l.ysozyme decolourised

3,3'-dipyrrole ketone (yellow) decolourised

3-carboxyethyl-~ethyl-
2,5-dicarb~-pyrrole (brown) decolourised

keto acids which may react with free amino acids.

I I
NH NH
I
CH-CH20H
I
C • CHz H+ - NHz + CH3-CO-COOH + NHz
I ~ I ~
C• 0 C• 0 pyruvic acid
I I
NH NH
I I

CH3-C-C-OH + NHz-CH-COOH --+ variety of products including

II I I Schiff bases HOOC - CH R
0 0 R• I

~
COOH-C-CH3
 Kuzen and Olsf!J'la76 have shown, for sample, that heating pyruvic
acid and glycine in solution leads to the formation of a variety of
products including pyrrolidine derivatives.

CH3 Me

'
C• 0 NH2 H2C - c,
I
2 \
C• 0
I
OH
+
,~
I

COOH
~ Ho,/
H
I
i, /c,
N
:
OH

'
,:12COOH

It is conceivable that the pyrrolidine could be dehydrated to a sub-

stituted pyrrole.

+ 2 H20

Since the fonnation of dehydropeptides and subsequently keto acids

during hydrolysis appeared probable, a mixture of pyruvic acid and glycine
was absorbed onto talc, evaporated to dryness and then heated at 130°c.
Even at room temperature a pale yellow colour formed and after heating
for 30 minutes, a brown-coloured material similar to the material fran
heated wool, was obtained. 'lbe brown material was extracted from the
talc with O.]M acetic acid and separated into fractions on an S.E.
Sephadex colwnn using the same buffers as were used for the wool extracts.
Five fractions were obtained, two of W1ich had absorption peaks at 265nm
 103.

and another at 240nm. One of the fractions obtained gave very positive
pyrrole tests and fluoresced at similar wavelengths (335 420nm) as the
wool fractions. When pyruvic acid alone was absorbed onto talc and
heated under similar conditions, it also produced a yellow brown colour.
However, no separation was obtained on the ion exchange column and the
heated pyruvic acid showed strong u.v. absorption at 320nm; it fiuoresced
at 510nm (emission) and f!J.Ve a negative pyrrole test.
Thus the possibility of pyruvic acid fonning and these reactions
occurring during hydrolysis, geatl.y adds to the difficulty in isolating
the yellow canpounds caused by dry heat but at present there appears no
practical. alternative.
Since the fo:nnation of pyruvic acid is dependent on the formation
of dehydropeptides, a series of compounds of similar structure was
examined to see if the formation of the dehydropeptide was indeed the
first step in one of the yellowing processes. The results are summarised
in Table XXXVI.
Fran this table it is obvious that a dehydropeptide is not necessary
for yellowing. The only difference between acetylalanine and a.-
acetimidoacr,ylic acid is the substitution of the unsaturated -C•CH2 group
for the -CH-CH3 of acetylal.anine, yet both yellow to similar extents.
It is interesting that conversion of the free amino groups of glycine
and alanine to acetyl goups accelerates yellowing. 'Ibis tends to
support other evidence that the presence of a peptide bond enhances
yellowing, e.g. serine and serylglycine yellow at vastly different rates.
 104.

TABLE IIIVI

The Effect of Heating Selected Compounds at 130°c in Air for up

to 2J+ hours.

Canpound Effect of Heat

Colour Fluorescence
Bnission (run)
glycine no colour change nil

acetylglycine yellows slowly 410

alanine no colour change nil

acetylalanine yellows 415

acetamide volatile - no apparent
colour change nil
ac:rylamide no colour change nil
~ acetimidoac:rylic acid yellows uo
glycylglycine discoloured nil
g1.ycylserine yellows 415

The only conmon factor among the compounds in Table llVI 'Which yellowed
is the presence of the p\ptide bond. However, it would appear that
there is no s~le relationship between the compound's structure and its
tendency to yellow. In fact, there is no definite evidence that the

same chranophore is involved in all cases.

 105.

Chapter IV:

A review of the literature concerned with the effect of dry heat on

proteins showed that considerable controversy existed as to which amino
acids were affected, the importance of oxidation and the causes of
yellowing. In the investigation reported here a variety of proteins was

examined in attempts to relate amino acid canposition and degradation

with yellowing. However, it soon became apparent that a great many
reactions occurred, only sane of which contributed to yellowing, and
furthermore, the extent of each reaction depended on both amino acid
canposition and peptide configuration. In many cases it is impossible

to gauge the extent of a particular reaction by measures of physical

properties such as viscosity since a number of reactions have conflicting
effects. Thus peptide breakdown is compensated for by peptide formation
at salt linkages.
The reactivity of the peptide bond has been shown to be important in
the heat degradation of proteins. Both oxidative and hydrolytic break-
down appear to occur and there is evidence that the peptide is involved
in several condensation reactions with amino acid residues.
'!he heat induced decanposition of protein bound cystine is a complex
process, in which the nature of the final products is influenced by the
presence of OJcy"gen. The major degradation product isolated from cystine
in wool heated in air at 130°c for 24 hours was cysteic acid. However
the need for hydrolysis of the protein to isolate degradation products
canplicates the situation am a variety of cystine oxidatioo products are
possible.
 106.

Protein-bound tryptophan is also degraded by heating at 130°c but

the extent of degradation depends on the particular protein as well as
the presence or absence of oxygen. At least two mechanisms of
tryptophan degradation exist: one oxidative, the second probablY'
involving condensation with the peptide to fo:nn a product which apparently
contributes to the yellowing. Wool appears to have a configuration W1ich
f avcnu, such condensation since wool-bound tryptophan is degraded far more

extensively than that in silk or l.ysozyme. Sane of the oxidation

products of tryptophan may also contrirute to the discolouration.
Amino acid analyses revealed that heating at 130°c for 24 hours also
caused significant degi-adation of serine, threonine and tyrosine.
Dehydration appears to be the most probable decanposition reaction of
serine and threonine and there is little or no evidence that this

si@'lificantly effects yellowing. Tyrosine ma_y be oxidised to quinones

or may condense with the peptide: either reaction would probablY'
contribute to the heat discolouration of the protein.
While no one amino acid was found to be the sole cause of yellowing,
the presence of free amino groups is a requirement for one of the major
yellowing reactions. Proteins rich in lysine yellowed most and the
blocking of the free amino groups caused a significant reduction in the
heat yellowing of wool. &sterification of free carboxyl groups also
reduced heat yellowing and since blocking either carboxyl or amino groups
was as effective as blocking both, a condensation between the two appeared
to be the initial step in yellowing.
 lCfl.

Since the yellow material is chsnically bound to the protein and

is destroyed by acid eydrolysis and since only a very snall amount of
yellow material is necessary to cause si~ificant colour changes, its
isolation and identification is extremely difficult. Studies of
fluorescence spectra and colour reactions of fractions obtained from
heated wool revealed that substituted pyrroles were formed in proteins
heated in air. The protein configuration is most important to facilitate
the formation of such compounds since the free amino and free carbaxyl
groups must be adjacent for the formation of the peptide bond and a
second free amino group must then be in the right position to condense
to form the pyrrole structure. Such coofigurations apparently occur
in gelatin and casein as well as in wool since all. gave positive tests
for pyrroles after extensive heating.
'!he results obtained during this investigation have emphasised
the importance of the amino acid sequence and the spatial configuration
of the protein as factors influencing· heat degradation. This means that
generalisations from one protein to another may- not be valid and the use
of results obtained from model compounds is of doubtful value. The
degradation of proteins by dry heat is an extremely complex process
involving a considera'ble number of interactions. While sane progress
in understanding these reactions has been achieved, there is still need
for further detailed investigations.
 108.

APPBNDII.

A.. The Determination of Primary Amino Groups in Proteins6 5.

Preparation of Ninhydrin Reagent: o.8 €PlS of stannous chloride were

dissolved in 500 mls of citrate buffer pH5. To this solution were

added 20 sns of ninl\Ydrin dissolved in 500 mls of methyl cellosolve.
The reagent was then transferred to a reservoir bottle and stored under
an atmosphere of nitrogen.
Method: Triplicate samples of protein (5-15 mg) were placed in 6" x t"
test tubes and 5 .ml.s of 10.C butanol-water added. To each tube were
then added 5 mls of 10.C aqueous pyridine (free frcm :impurities containing
amino gi-oups) and 10 .ml.s of ninhydrin reagent. The reagents were mixed
and the tubes stoppered before heating in a vigorously boiling water bath
for 4.5 minutes. The tubes were allowed to cool and the contents of each
tube diluted to 250 mls after decanting the coloured solution frcm the
protein and washing the remaining colour from the wool with 1:1 ethanol-
water. The solutions were well mixed and within 1 hour the optical
density read at 'J"IO nm against a reagent blank. Solutions of alanine
in 10.C butanol-water were used as a standard and, from the colour yield
per mg amino-N and the optical densities of the solutions obtained from
samples of known weight, the amino-N content of the various wool samples
were detennined.

B. The Detennination of Tyrosine in Proteins66 •

Preparation of Solutian: 0.1 8J1S of a.-nitros~-naphthol were dissolved

 109.

in 100 ml.s of 0.l.N sodium hydroxide. The solution was heated at 100°c
to complete dissolution, filtered and stored.
Method: Triplicate samples of approximately 10 J11€P1S of protein were
weighed accurately in 25 ml flasks to which 11 mls of 18N sulphuric acid
were added. The nasks were stoppered and heated at ?OOC for 3 hours.
After heating the solution was diluted to 250 mls and duplicate aliquots
of 5 ml.s taken. To each aliquot was added 1 ml of the a.-nitroso-~
naphthol solution, 0.5 mls of 2.5N nitric acid and 1.5 mls of 100
eydrochloric acid. The mixture was well shaken then heated in a boiling
water bath for exactly two minutes, then cooled in a beaker of ice.
The developed red colour was read against a blank treated in the same
manner at 510 nm. Solutions of 1 to 20 g/ml of tyrosine were used for
calibration.

c. The Determination of Tryptophan in Proteins67.

Triplicate samples of 50 mg of protein were weighed into 50 ml

volumetric flasks and 2 mls of 18N H2s:>4 followed by 1 ml of~ P-
dimeteylaminobenzaldehyde in 10.C HzS04 were added. The mixture was
shaken and a further 11 mls of 18N H2so4 were added. After shaking
again the flasks were stoppered, heated at 70°c for 2 hours, 2 mls of
o.OlM sodium nitrite solution were added and the mixture made up to
volume with distilled water and heated for a further 2 hours at 6o0 c.
The solution was filtered and the optical density read at 585 nm using
distilled water for a blank. Solutions of lysozyme treated similarly
were used for calibration.
 no.

o. The Bstimation of Thiol Groups in Intact woo168.

Principle: The determination is based on the reaction

- SH + Me Hg I ~ S Hg Me + H I

Procedure: Two stock solutions were stored and mixed together as

required, viz. tris buffer solution (100 mls; 0.1 Mat pK7 containing
0.1 M potassium chloride) and methyl mercuric iodide (500 mls; 5 x 1o-'tM,
diluted from a 0.1 M solution in pure dimethyl fonnami.de using aqueous
0 .1 M potassium chloride) • '!he methyl mercuric iodide solutions were
stored in the dark.
Wool samples were conditioned and five 25 mg samples weighed out.
Two were used for determining the moisture content and then discarded.

The other three were transferred to 10 ml tubes, aliquots (5 mls) of

bufferred methyl mercuric iodide added by pipette to each flask and the
wool samples thoroughly wet out by suction. The tubes were stoppered
and left agitating gently at room temperature for at least four hours.
A sample of each solution was decanted into clean, dry flasks and the
mercury concentration of the solution determined by atomic absorption
spectroscopy using a Techtron Model AA 4 atomic absorption spectrophoto-
meter. Before aspirating the solution, the aspiration unit was flushed
with stannous chloride to ensure that all the mercury was in the reduced
form. Calibration curves were prepared using methyl mercuric iodide in
the buf ferred solution over a range of 1 x 10-5,( to 5 x 1o-'tM. From the
amount of mercury remaining in the solution the amount absorbed and
hence the thiol content was detennined.
 lll.

1. Detennination of Thiol plus Disulphide Groups in Intact woo168.

Principle: This detennination is based on the fol.lowing reactions

Nt.z S03
- s- s- _ _,,,.,., - S Na + - S so3 Na
and 2 (- S Na) -t Hg Clz ~ - S Hg S - + 2 Na Cl•

Procedure: The stock solution contained amnonium chloride (0.16 M),

amnonium hydroxide ( 0 .04 M), urea ( 8 M) , potassium chloride ( 0. 5 M)
and sodium sulphite ( 0 .2 M) • Mercuric chloride ( 5 x 1o-'tM) was added
and the solution stored in the dark.
Wool samples were equilibrated at constant humidity, and in addition
to the triplicate 7.5 mg samples weighed out into 10 ml tubes, two
separate samples were weighed for moisture determinations and later
discarded. Aliquots ( 5 mls) of the stock solution containing 5 x 10~
mercuric chloride were added by pipette and the samples were wet out by
suction. The tubes were then sealed and agitated gently at room
temperature for 18 - 24. hours. An aliquot (1 ml) of each solution was
pipetted into a 10 ml volumetric nask and the solution made up to
volume with distilled water and its mercury concentration detennined by
atanic absorption spectroscopy. Calibration curves were prepared by
adding lmown quantities of mercuric chloride to the stock solution.
From the calibration curve the amount of mercury in the solution was

calculated, and hence the amount absorbed by the wool. Cystine content
was calculated as total thiol + disulphide content - thiol content.
 1]2.

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