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Low-angle X-ray diffraction studies

of reduced and silver-stained
G. A. Wilson*
Textile Physics Laboratory, Department of Textile Industries, The University,
Leeds 2, UK
(Received 31 March 1971 ; revised 11 June 1971)

When the cystine in Lincoln wool fibres is reduced and the fibres subsequently
'stained' with silver nitrate, the low-angle X-ray diffraction pattern becomes
considerably enhanced, and to a good approximation represents the regular
distribution of silver atoms throughout the fibre. This regular distribution of
silver atoms is equivalent to the distribution of cystine, since if the reduced
cystine is Jolocked by alkylation before staining, the silver uptake is low and the
only enhancement which occurs is a very small intensification of the low-angle
equatorial reflections. The precise nature of the enhanced diffraction patterns
varies considerably with the prior reduction treatment and it is possible to
distinguish between matrix and microfibril fractions of cystine. When the
microfibril cystine is labelled the low-angle meridional reflections contract in
spacing and this is accompanied by a similar change in fibre length. There is
no apparent change in the high-angle, helical pattern, however, which leads to
the conclusion that the ordered keratin protein chains are made up of alternating
helical and non-helical sections. A model for the matrix is also proposed,
consisting of short, randomly orientated c~-helical segments, which become
orientated about the fibre axis where they form the surfaces of the microfibrils.
A possible mode of interaction between the matrix and microfibrils is also

INTRODUCTION microfibrils, and hence it has been concluded that the
stain is entering, preferentially, the matrix. The fact that
The low-angle X-ray diffraction of ~-keratin impregnated the observed effects take place after prior reduction of the
with heavy metals after reduction of the disulphide bonds disulphide bonds to thiol groups suggests that the binding
has been reported by several workers 1 4. Both the site for the heavy atom is at a sulphur atom, and thus a
equatorial reflections (at approximate Bragg spacings of cystine-rich matrix is implied.
80/k (el), 45/k (e._,) and 27• (ea))and the meridional The interpretation of the changes in the meridional
reflections (as orders of a macroperiod of 198,~) have X-ray diffraction patterns produced by heavy atoms has
been shown to be enhanced. been made difficult by the fact that previous workers
The equatorial patterns of wool fibres obtained using have not only used different metals but also different
silver nitrate and phenyl mercuric hydroxide as the redmction treatments prior to staining. This paper
heavy metal reagents have been related to the histological examines the effect of difl'erent prior reduction treatments
distribution of the stains throughout the fibre 3. The on the X-ray diffraction of silver-stained keratin.
matrix-microfibril contrast observed in the transmission
electron microscope 3, 6 has suggested a line structure of
'holes' arranged in an electron-dense medium '~ and this EXPERIMENTAL
structure has been related to the spacings and intensities
of el, e2 and e3. The 'holes' have been equated with the Lincoln wool samples were prepared in which (i) 40~o
* Present address: School of Biological Sciences, The University, of the wool cystine was reduced with thioglycollic acid
Bradford 7, Yorkshire, UK. (termed 'most reactive' cystine); (it) the 'most reactive'

POLYMER, 1972, Vol 13, February 63
X-ray diffraction studies o f ~-keratin : G. A. Wilson

cystine was reduced as above, alkylated with ethylene Table 1 Uptakes of silver after reduction of cystine in Lincoln
dibromide, and the remaining 'least reactive' cystine wool
reduced with tetra(hydroxy methyl) phosphonium
Thiol content Silver uptake
chloride (THPC); (iii) 80~o of the cystine was reduced Reduction treatment (Fmol/g wool) (t~mol/g wool)
with T H P C ; and (iv) all the cystine was reduced and
alkylated with ethylene dibromide by two reduction- 0.45M thioglycollic acid (pH 6) 340 1220
alkylation cycles (using thioglycollic acid and THPC 0.2M THPC (pH 5.4) 700 2120
0.45M thioglycollic acid (pH 6)
as the two respective reducing agents).
alkylation with (CH2)2Br2
The samples were all stained in solutions of silver further reduction with 0.2M
nitrate, and X-ray photographs were taken of the THPC (pH 5.4) 580 1470
washed and dried samples. Complete reduction and
Parallel experiments were carried out on single fibres alkylation 0 310
and tests were made for fibre contraction at each stage
of the reaction sequence.
Lincoln wool was prepared for analysis by Soxhlet
extraction followed by a dilute (6 × 10-4 N) hydrochloric
acid wash to remove any metal ions and reduce the ash Table 2 Intensity distribution of the low-angle equatorial reflec-
content of the wool 7, and finally washing in conductivity tions el, e2, e3 at approximate respective Bragg spacings 80 ~.,
42 ~, 27 ~ in silver-stained Lincoln wool
water to the isoelectric point of wool (pH = 5.1).
Low-angle X-ray photographs were taken using a Approximate
'pin-hole' camera designed in this laboratory s. Nickel enhancement
filtered CuK~ (,~= 1.54A) radiation was used and was Intensity ratio (intensity ratio
generated from a Hilger Y-25 100t~m microfocus tube Treatment lel : le~ : le3 le2 : I~.s~)*
operating at about 35 kV and 2.5mA. Untreated sample 10 : 0.75 : 1 0-4 : 1
High-angle photographs using CuK~ radiation were 'Most reactive' cystine silver
taken on simple glass-capillary collimated cameras, also stained 14 : 3.75 : 1 50 : 1
designed in this laboratory 9. The source of X-rays was a 'Least reactive' cystine silver
Hilger and Watts Y-90 X-ray generator, working at stained 3 : 1.9 : 1 50 : 1
80% of cystine silver stained 14.5 : 3 : 1 50 : 1
34kV and 14mA. A nickel filter was employed as before, All of cystine reduced and
and silver foil was inserted in the centre of each sample alkylated before silver staining 14 : 1.2 : 1 1 :1
in order to measure the exact reflection spacings.
The X-ray photographs were analysed on a Joyce * Intensity of e2 compared with that of the main equatorial high-
Loebl Automatic Recording Microdensitometer Model angle helical reflection at a Bragg spacing of 9.8 ~,
MkIIIC. All traces were corrected for background
scatter by comparison with a trace at an angle of 45 °
to the equator.
The reduction procedure with 0"45M thiogtycollic far too high to be accounted for by a stoichiometric 1 : l
acid followed by a standard wash to yield samples of reaction with thiols. Most of the reaction appears to take
known constant thiol content (equivalent to 4 0 ~ place with these groups, however, as the introduction
reduction), and the silver nitrate staining procedure at of an alkylation step between the reduction and staining
pH 5-4, were both developed in this laboratory 3. Uptakes reactions considerably decreases the metal uptake.
of silver were measured gravimetrically against blank The low-angle X-ray diffraction patterns of the
samples. samples, with the exception of that of the reduced and
Reduction with 4 ~ (w/v) THPC at pH 5.410 yielded a alkylated sample, become considerably intensified with
sample with 80~o of the cystine converted to thiols. respect to the high-angle coiled coil diffraction after
In the case where the sample had already been reduced staining (Table 2). To a good approximation, therefore,
by thioglycollic acid and subsequently alkylated, all t h e they describe the distributions of silver atoms in the
remaining cystine was reduced. Before further reaction different samples. The low-angle diffraction pattern of
THPC reduced samples were quickly washed in successive the reduced and alkylated sample, however, shows no
changes of conductivity water for 10 rain. enhancement along the meridian after staining (dens-
The alkylation procedure with 0.05 M ethylene di- itometer traces showed the meridional pattern to be
bromide at pH 8 has been described elsewhere al. identical to that obtained from untreated Lincoln wool),
The thiol contents of all the samples were measured and very little along the equator (Table 2). Thus the
by gravimetric uptake of phenyl mercuric hydroxide3, enhanced diffraction patterns may be assumed to be
and by measurement of the remaining cystine after due to silver atoms located at cystine residues, and hence
alkylation of the thiols, by the Shin O'Hara method 12. describe the distribution of cystine throughout Lincoln
This was compared with the cystine content of untreated wool fibres.
wool, which was found to be 860 t~mol/g, in agreement
with the analyses of other workers ~, 13.
Low-angle X-ray diffraction of the reduced and stained
RESULTS AND DISCUSSION The low-angle X-ray diffraction pho.tograph of the
sample in which the 'most reactive' cystine has been
Specificity of silver nitrate for reduced cystine reduced and labelled with silver is shown in Figure 1, and
The silver uptakes for all the samples considered are that of the sample in which the 'least reactive' cystine
shown in Table 1. It should be noted that the uptakes are has been reduced and labelled is shown in Figure 2.

64 POLYMER, 1972, Vol 13, F e b r u a r y ,
X-ray diffraction studies of =-keratin: G. A, Wilson

lower the intensity of the external interference giving rise
8th to el. By drawing an analogy with untreated Lincoln
wool, where le2 : l e a ~ l : 1, the observed lowered
intensity ratio from 4 : 1 to 2 1 may be similarly
explained. The theory explaining the low-angle equatorial
ii7 ~ii!!i~ diffraction of untreated Lincoln wool is based on the
scatter from regularly distributed protofibrils (3-strand
coiled coils) inside the microfibri114. In the usual stained
specimens as typified by Figure 1, the electron density
difference between the matrix and microfibrils is so large
that the arrangement of protofibrils inside the micro-
fibrils becomes unimportant and may be neglected.
However, if the stain enters the microfibrils in large
quantities, the density difference between the microfibrils
and matrix will become equalized, and essentially the
Figure 1 Low-angle X-ray diffraction photograph of Lincoln wool same diffraction conditions as those for untreated Lincoln
that has been silver stained after reduction of the 'most reactive' wool will occur, i.e. the low-angle equatorial diffraction
will be based largely on the arrangement of protofibrils.
The effect of this in the stained sample will presumably
be to lower the intensity ratio lee : le3.
these results suggest, tneretore, that the 'most reactive'
cystine is located mainly in the matrix, and the 'least
- ~th reactive" cystine in the microfibril.
The meridional diffraction. When the 'most reactive'
cystine is labelled the 8th order meridional reflection
alone appears to be notably enhanced (Figure 1). When,
however, the 'least reactive' cystine is labelled the 3rd,
5th a n d 8th orders are enhanced, with the 3rd order
particularly strong (Figure 2).
This last feature of the meridional difl'raction confirms
the results of workers using mercury compounds as the
stains ~,4 and provides a simple explanation for the
non-correlation of their results with those of workers
Figure 2 Low-angle X-ray diffraction photograph of Lincoln wool using silver compounds. Evidently the prior reduction
that has been silver stained after the 'most reactive' cystine has
been blocked and the 'least reactive' cystine reduced
treatment is extremely important in determining which
reflections are enhanced on staining. The 5th order
enhancement has also been reported previously, but this
has been associated with a lysine repeaO ~', HL It is quite
possible that this repeat is present in addition to that
Important differences are apparent, both along the attributed to cystine in the present paper.
equator and the meridian. These are discussed in detail The intensity ratios of the meridional reflections of all
below. three enhanced diffraction patterns are shown in Table 3.
It will be noted that when enhanced, the intensity ratio
The equatorial diffraction. The intensities of the low- of the 3rd and 5th orders is constant, but the relative
angle equatorial reflections are summarized in Table 2. intensity of the 8th order varies with the exact chemical
The equatorial diffraction of the sample in which the treatment, being strongest when the 'most reactive'
'most reactive' cystine has been reduced prior to staining cystine has been labelled, and weakest when the 'least
is identical to that found by other workers ~, a This type reactive' cystine has been labelled. It would appear that
of pattelm, taken in conjunction with the observation in the intensity of the 8th order is associated with 'most
such samples of matrix-microfibril contrast in the reactive' (or matrix) cystine, and the 3rd and 5th orders
transmission electron microscope, is generally interpreted with 'least reactive' (or microfibril) cystine.
as being produced by scatter fi'om a system of 'holes'
(microfibrils) embedded in an electron dense (i.e. silver
dense) medium (the matrix). This type of scatter is
characterized by a high intensity of el due to external Table 3 Relative intensities of enhanced meridional reflections
interference between microfibrils, and e,, and ea repre- in silver-stained Lincoln wool
senting the scatter from a single cylinder, radius 37'5A,
Intensity ratio
having an intensity ratio le,, : l e a ~ 4 : 1. When, however, Treatment lard : /5th : /Sth
the 'least reactive' cystine is labelled, the relative intensity
of et becomes much lower, and le., : lea ~ 2 : 1. 'Least reactive' cystine silver stained 10:2:1.5
These differences may be explained, qualitatively at 80% cystine silver stained 10:2:4
'Most reactive' cystine silver stained 10" : - - : >10
least, by postulating that when the 'least reactive" cystine
is reduced and stained, considerable quantities of silver * I n this sample the 5th order is very weak and has not been
enter the microfibril. The effect of this will be to random- measured. Also there appears to be very little enhancement of
ize the histological distribution of silver, and hence the 3rd order

POLYMER, 1972, Vol 13, F e b r u a r y 65
X-ray diffraction studies of =-keratin: G. A. Wilson

Table 4 Bragg spacings of the meridional reflections enhanced helical regions of the fibre. This, in turn, leads to the
when the 'least reactive' cystine in Lincoln wool is silver stained deduction that the contraction is taking place in non-
Bragg spacing (~) helical regions which alternate in series with the coiled
coil (or other s-helical regions). If this is so, it may
Untreated 'Least reactive' reasonably be assumed that the non-helical regions are
Order Lincoln wool cystine silver stained stabilized to a large extent by disulphide bonds, and the
cleavage of these bonds by reduction, and the subsequent
3rd 65.8 61.65
5th 39* 36.80 reaction with silver nitrate, cause these regions to
8th 24.4 23.20 contract.
Macroperiod (,&,) 198 185 The very great intensity of the 3rd order reflection
suggests that the major repeat of one helical plus one
* A very weak reflection which is only occasionally observed non-helical region is 66A. This agrees with analyses of
in untreated specimens s,~-~
other workers studying the 3rd order reflection in
untreated keratin s, 25, who suggest that it arises from a
major discontinuity at this spacing. This discontinuity
has been linked by these workers with the anti-helical
The shapes of the 3rd and 5th order reflections are amino acids 25 or, more specifically, cystine residues 8.
different (Figure 2). The 3rd order, like the 8th, is a The present results are in full accord with the latter
sharp meridional arc, but the 5th order splits into three postulate.
distinct reflections which form a rudimentary layer line. The much weaker 5th order reflection (suggesting a
In the light of work on collagen iv, and more recently 39.5A repeat) is not considered to be associated with
on untreated a-keratin 8, this would suggest that the helical-non-helical discontinuities. However, as it always
microfibril cystine itself is subdivided into cystine in two has the same comparative intensity as the 3rd order,
different structural positions (although of similar it appears to .represent a repeat which is part of the
reactivity to reducing agents). same chain.
When only the 8th order reflecUon is enhanced, its It is difficult to envisage how regular contractions at
Bragg spacing is the same as in untreated specimens at 66A intervals could bring about a regular contraction of
24.4A. When the 3rd, 5th and 8th orders are enhanced, the 39.5A spacing. One possibility is that in untreated
however, the spacings all contract by 6.5~ and the specimens this repeat is slightly irregular. The reflection
macroperiod falls from 198 to 185/k (Table 4). is extremely weak and does not index satisfactorily on a
This change in low-angle spacing is accompanied by a macroperiod of 198A, its actual Bragg spacing being
contraction in fibre length of 6.3 % (average of five single recorded as 39A s,2~. These characteristics would be
fibres) which occurs only at the final silver staining stage. expected if the cystine residues giving rise to the repeat
No contraction is observed for samples which do not were occasionally spaced at intervals shorter than the
show spacing changes. Thus there is a direct correlation theoretical 39-5 A. The contraction of non-helical regions
between change in X-ray spacing and change in fibre every 66A could then be envisaged as making the spacing
length. more regular, as well as decreasing its overall value.
As the contraction in length occurs at the final silver This explanation would also account for the fact that
staining reaction, it would suggest that the contraction other workers who have recorded intensification of the
takes place in the parts of the protein chain where the 3rd order after labelling cystine with mercury com-
silver is being attached, rather than continuously along pounds but no change in spacing have not reported any
its length. This contention is supported by a study of intensification of the 5th order 2,4.
the high-angle pattern. The high-angle X-ray diffraction
of keratin is generally accepted as being that given by a
2- or 3-strand coiled coiP 8, xg. (Recently, other a-helical
forms have been shown to be possibilities 2°-23, but, this CONCLUSIONS
is irrelevant to the following argument.) In particular,
the strong meridional ~eflection at 5.15A represents the The structure of the matrix
pitch of an a-helix which has been twisted to form one The broad halo (Bragg spacing=25.~) observed in
strand of a coiled coil. A regular contraction would X-ray diffraction studies of silver-stained fibres and the
presumably cause a decrease in the spacing of the sharp diffraction ring (Bragg spacing= 21 ,~) observed in
reflection (for example, compare the fact that when X-ray diffraction patterns of the silver-stained kerato-
wool is extended by 20 ~, the 5.15A reflection increases genbus zone of developing fibres have been interpreted as
in spacing by 2 ~ 24) but accurate measurement showed indicating matrix order2V, 28. In the latter studies 2s
that the reaction sequence had caused no change. electron micrographs revealed the presence of silver
However, further considerahon of the high-angle globules of the same order of size, between the micro-
pattern, and this reflection in particular, suggests that fibrils, and it was also reported that after keratinization
the implied irregular coiled coil contraction is not taking but before dehydration the diffraction ring became
place either. There is no sign of any disorder which broader. These results were interpreted in terms of the
would arise from an irregular contraction of the coiled matrix originally being a globular entity which becomes
coil. Although to establish the fact completely it would be distorted on keratinization owing to oxidation of
necessary to measure quantitatively intensity changes in cysteine to cystine.
the 5"15/~ reflection which result from silver staining-- More recently, very wide angle studies have suggested
a very difficult task owing to the presence of the silver that the matrix consists of short, randomly orientated
swamping the natural diffraction pattern--the results s-helical segments 3, s, and the 25,~ halo has been related
do suggest that there is no contraction in the coiled coil, to the leflgth of a segment 8. A closer examination of this

66 P O L Y M E R , 1972, Vol 13, February
X-ray diffraction studies of a-keratin : G, A. Wilson

200 associated in the present work with a regular matrix
d:D repeat, could arise from complete orientation of the
matrix segments about the fibre axis. This would most
I likely occur at the surfaces of the microfibrils (a possibility
suggested by early workers on the X-ray diffraction of
C silver-stained keratin1), to enable maximum disulphide
C crosslinking between the matrix and microfibrils.

The structure of the protofibrils
Correlation of the 'least reactive' cystine with micro-
fibril cystine and co-ordination of the data obtained
from the diffraction pattern of the sample in which this
fraction has been labelled with silver lead to a general
model for the microfibril sub-units or protofibrils
o l/ (Figure 3b). The helical regions in this Figure are shown
as 3-strand coiled coils, and the non-helical regions are
shown to have a finite length, so that they contain some
cystine residues which form part of the 39A repeat.
This has been done because it is well known that cystine
I00o< is not readily incorporated into a-helical structures.
it is possible that the non-helical regions tbrm a fairly
large proportion of the protofibrils. Optical transform
results show that 607k lengths of coiled coil will yield
well-ordered helical reflections, although if the length is
reduced to 27A they become diffuse e'5.
The idea of alternating helical and non-helical regions
has been the basis of various mechanical models e9, a0,
and also some recent models derived from chemical
50 studies al-aa. It represents a significant departure,
X-X X - however, from recent X-ray diffraction models such as
the segmented rope model, where the coiled coil is
continuous and the coiling takes place at specific
residues2a, ~5, and the straight ~-helix models ~°-'~.
These models have been derived because the experimental
9 data from diffraction patterns do not fit exactly the
theoretical continuous coiled coil diffraction. Models
x ~/ similar to that presented in this paper may explain the
O discrepancies equally well.
tb- ~ b ~
The unified structure
Figure 3 The molecular configuration of c~-keratin. (a) Proposed If the possible disulphide bonds are drawn between the
structure for the matrix, consisting of short units of helical plus matrix and microfibril to give the unified model (Figure 3),
non-helical material, about 25 ~, long. Cystine (half-cystine repre- then it is immediately apparent that only a few may be
sented by © ) i s located in the non-helical segments and the so formed. Presumably the other cystine residues of the
disulphide bonds between the units produce a 'rigid' structure.
The units are orientated randomly except where they form the matrix and microfibrils are used for the internal cross-
surface of the microfibril. Here they become orientated along the linking and stabilization of their respective structures.
fibre axis. (b) Shows one protofibril on the periphery of a micro- Thus some parts of the protofibrils are stiffened by
fibril. It consists of a 3-strand coiled coil, interspersed at regular covalent crosslinks with the matrix, and others are not.
intervals by non-helical sections. The basic unit of one helical
plus one non-helical segment is 66,~ long. The non-helical
This type of model has also been deduced from a study
sections all contain cystine and there is a 66 ~ cystine (half- of the mechanical properties of reduced and methylated
cystine represented by ©) repeat. The other cystine repeat fibres34
(half-cystinerepresented by x ) is shown as 39.5~ but may be The proposed structure is attractive in that it incor-
irregular. This cystine may be located either in the coiled coil or porates the features of three different mechanical models
non-helical segments. Possible disulphide links with the matrix
to form 'rigid' parts of the structure are shown mentioned above. It is also consistent with current
chemical evidence, and the previous apparently contra-
dictory reports from X-ray diffraction observations
concerning the regular distribution of cystine in a-keratin
halo has revealed it to be more intense and ol slightly are easily explained.
greater spacing along the meridian s , suggesting the
segments to be partly orientated about, and extended ACKNOWLEDGEMENTS
along the fibre axis. These deductions are in full agree-
ment with a recently derived mechanical model for the The above work was carried out under a Research
matrix 29. Studentship sponsored by the Science Research Council
In th~s type of model (Figure 3a) it is easy to envisage to whom the author is indebted for their financial
how the 25A meridional reflection, which has been support. The author would also like to acknowledge the

P O L Y M E R , 1972, V o l 13, F e b r u a r y 67
X-ray diffraction studies of ~-keratin : G. A. Wilson

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68 POLYMER, 1972, Vol 13, February