454 AMES-COLLAGEN A N D GELA TIN

Speke, Liverpool, for supplies of procaine penicillin and of vitamin B,, concentrates, and to
the The Distillers Company Ltd., Yeast Research Out-Station, Alloa, for riboflavin concentrate.
The Agricultural Chemistry Department
The Queen’s University
Belfast
The Ministry of Agriculture for Northern Ireland
and
The Agricultural Research Institute of Northern Ireland
Hillsborough
Co. Down
Received 7 April, 1952

References
Anderson, G. C. & Hogan, A. G. (1950). J . Nutr.
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Braude, R . & Foot, A. S. (1942). J . agric. Sci.
32, 71.
Braude, R . (1949). Brit. J . Nutr. 3, 293.
Briggs, J . E., Elrod, R. & Beeson, W . M. (1951).
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Brown, W. 0. (1951). Priv. comm.
Colby, R . W. & Ensminger, M. E . (1950). J . Anim.
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Cunha, T . J., Burnside, J . E.. Buschman, D. M.,
Glasscock, R. S., Pearson, A. M. & Shealy, A. L.
(1949). Arch. Biochem. 23, 324.
Cuthbertson, W . F. J . (1952). J . Sci. Food Agric.
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Dunlop, G. (1935). J . ugric. Sci. 25, 445.
Dyer, 1. A,, Krider, J . L. & Carroll, W. E. (1949).
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Edwards, H . M., Cunha, T. J., Meadows, G. B.,
Shawyer, C. B. & Pearson, A. M. (1951). Proc.
Soc. exp. Biol., N . Y . 76, 173.
Guerin, H. B., Hoefer, J . A. & Beeson, W. M. (1950).
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Hogan, A. G. & Anderson, G. C. (1949). Fed. Proc.
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Jukes, T. H., Stokstad, E. L. R., Taylor, R . R.,
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G. B. (1950). Arch. Biochem. 26, 324.
Krider, J. L., Becker, D. E., Van Poucke, R. F. &
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Luecke, R . W., Thorp, F., Newland, H. W. &
McMillen, W.N. (1951). J . Anim. Sci. 10, 538.
McMeekan, C. P . (1940). J . ugric. Sci. 30, 277
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James, M. F. & Johnson, B . C. (1950). J .
Anim. Sci. 9, 83.
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Powick, W . C., Ellis, N. R., Dale, C. N. & Zinober,
M. R . (1951). J . Anim. Sci. 10, 617.
Richardson, D., Catron, D. V., Underkofler, L. A,,
Maddock, H. M. & Friedland, W. C. (1951).
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D. V. (1951). Antzbiot. Chemother. 1 , 41.
Speer, V. C., Vohs, R. L., Catron, D. V., Maddock,
H. M. & Culbertson, C. C. (1950). Arch.
Biochem. 29, 452.
Terrill, S. W. &.Krjder, J . L. (1950). J . Anim. Sci.
9, 670.
Vohs, R . L., Maddock, H. M., Catron. D. V. & Cul-
bertson, C. C. (1951). J . Anim. Sci. 10, 42.
Wishart, J. (1950). Commonwealth Bureau of Plant
Breeding and Genetics : Tech. Bull. No. 15.
Zucker, T. F. & Zucker, L. M. (1950). ‘ Vitamins and
Hormones ’, vol. 8, p. I (New York : Academic
Press).

THE CONVERSION OF COLLAGEN T O GELATIN

AND THEIR MOLECULAR STRUCTURES
By W. M. AMES

A systematic account is given of the conversion of collagen t o gelatin. It is shown

that collagen can be converted into two types of gelatin of differing nitrogen content.
A study of heat degradation has made i t possible t o separate the effects of heat and chemical
treatment in the preparation of one type. The results have been collated by means of
diagrams of molecular structure which lead t o useful deductions and suggestions for further
investigation.

Introduction
Collagen is readily converted into gelatin, by the apparently simple process of heating
in water. However, as both substances are proteins, the structures of which are unknown
and whose compositions have only recently been established with some degree of accuracy
(Bowes & Kenten, 1948,p. 358), the nature of the transformation cannot yet be completely
J . Sci. Food Agric., 3, October, 1952
 AMES-COLLAGEN A N D GELATIN 455
explained. Various aspects of the preparation and properties of gelatin have been studied
by the author (Ames, 1944, 1945, 1947). The present paper summarizes those parts of the
investigation which can be fitted together to make a coherent picture of the relation of gelatin
to collagen. Inconsistencies and irregularities in earlier figures have been checked, sometimes
by the use of new techniques. Having established the conditions necessary for the conversion,
molecular models have been constructed for collagen and gelatin which satisfy these conditions.
Experimental
The isoelectric point of gelatin.-The investigations, as frequently happens, were started
as a result of chance observations. .In the course of making gelatin, isoelectric dullness was
noticed a t pH values higher than 4.75, which, a t the time, was generally agreed to represent
the isoelectric point. I t is true that the values reported in the literature (Ames, 1944, p. zoo)
differed so much that one might have concluded that the position of the isoelectric point could
vary. I t was also noticed that dried rabbit skin did not conform to the generally accepted
value of pH 4.75 for the isoelectric point of collagen (Ames, 1944. p. 277). The skin was
swollen a t pH 4.75, and shrunken a t pH 8. Such inconsistencies called for elucidation.
In the experiments now to be described, the isoelectric point of gelatin was determined
by finding the pH a t which a dilute jelly showed the maximum opalescence (Ames, 1944,
p. 200). Although this point is generally denoted by means of its pH, there are certain advan-
tages in using a titration method, particularly when the point lies on a portion of the titration
curve nearly vertical to the pH axis. Gelatin can be titrated to a phenolphthalein end-point.
The isoelectric point is then denoted by the amount of standard acid or alkali which has to
be added to neutral gelatin to bring it to its isoelectric point. This method has been used
t o fix the isoelectric points of the commercial gelatins shown in Table I.

Table I
Type of gelatin Isoelectric point
Pig skin
Alkaline process .. .. 0.67
Pig skin
Alkaline process .. .. 0'73
Ossein
Alkaline process .. .. 1.42
Ox sinew
Alkaline process .. .. 1'45
Pig skin
.
Acid process . .. .. - 0.25
Ox sinew
Acid process . . .. .
. - 0.30
Isoelectric points are shown as acidities, C.C.of o.zw-acid/g.

It is evident that the isoelectric points of commercial gelatins do not fall a t a fixed acidity ;
nor do the results give any indication whether the nature of the precursor or the method of
treatment is the cause of the variability.
However, by preparing gelatin from various precursors, using different methods as in
Table 11, it can be shown that the position of the isoelectric point depends upon the pre-
treatment, and not upon the origin of the precursor. Gelatin made from a precursor subjected
to alkaline treatment is isoelectric in the region of 1.5 C.C.of acidity, whereas acid-prepared
gelatin, with the exception of that made from ossein, is isoelectric on the alkaline side of
neutrality to phenolphthalein in the region of pH 8. It has recently been suggested that
the exceptional behaviour of acid-prepared gelatin made from ossein, which is quite definitely
established (Janus, Kenchington & Ward, 1951) is due to the long acid soaking used to remove
the mineral constituents of bone in the preparation of ossein. Gelatin made by heating collagen
with water alone is isoelectric on the acid side of neutrality to phenolphthalein. It might
therefore be possible by varying the length of alkaline treatment, to obtain gelatin isoelectric
at any point on the acidity scale between 0.5 and 1.5, and the use of acid would extend the
range to - 0.25 on the alkaline side.
Alkaline $re-treatment.-By preparing gelatin from collagen soaked for increasing periods
of time in calcium hydroxide, the gradual movement of the isoelectric point can be clearly
demonstrated. The figures in Table I11 range from pH 5.46 to pH 4.75. Careful and repeated
experiments have more recently shown that even after exhaustive alkaline treatment of
the precursor, the isoelectric point of the resulting gelatin does not fall below pH 4.8. I n
J . Sci. Food Agric., 3, October, 1952
456 AMES-COLLAGEN AND GELATIN

Table I1
Treatment Material Time of Jelly Isoelectric
heating strength point
152 0.48
Soaked in water and washed(g%gskin
free of dirt Ox sinew
4 --
159 0.47
0.23
8 115
Ossein 34 I46 0'43
Pig skin I 242 - 0.27
Soaked in 2ojb conc. HCl soln. 255 - 0.27
and washed O x sinew 2 207 - 0.24
Cossein I 21d
? 0.46
Pig skin 14 251 I .48
Soaked in Ca(OH), for 12 weeks 2 63 1.41
and washed Ox sinew I t __
255 1.50
lossein I 255 "45
Time of heating, hr. a t 80"
Jelly strengths, bloom
Isoelectric points, C.C.of 0,2~-acid/g.

conformity with the alteration in the position of the isoelectric point, collagen becomes more
readily convertible into gelatin, the gelatins obtained gradually acquiring the property of
forming stiff jellies. Gelatin appears to reach its maximum jelly strength before the isoelectric
point has reached its minimum pH value. The rate of conversion increases from start to
finish.
Table I11
Time of Rate of Jelly Isoelectric point Formol
soaking, days conversion, strength Zi- ~ TH titration, C.C.of
g./hr. o.IN-NaOH/g.
Nil .. 6.0 86 0'57 5.46 3'23
3 "
8.1 91 0.66 5'27 3.21
6 .. 9'7 106 0.90 5.00 3'46
I0 .. 1.5'9 I10 1.13 444 3.36
14 .. .. 18.3 I20 I .08 4.89 3'44
18 .. .. 14.8 130 1.29 4.8 I 3'41
22 .. .. 22.4 141 I '30 4.80 3'45
29 .. .. 18.3 162 1.42 4.76 3.48
36 .. .. 25.0 '65 1'49 4'76 3.60
43 .. .. 37.1 I82 1.jO 4'75 4.01
Jelly strengths, 6QO/, bloom
Isoelectric points, c c. o.zN-acid/g

Another measurable quantity which increases slightly as the alkaline soaking is extended
is the formol titration of the gelatin. The difference is slight, but must be considered signi-
ficant, as there is a complete range of titrations showing a gradual rise.
Nitrogeit conted of daferent gelatins.-The difference in the position of the isoelectric
points of gelatin prepared from an alkaline-treated precursor, and that prepared from an
acid-treated precursor suggests that gelatin prepared from an alkaline-treated precursor is less
basic than the other. There should therefore be less nitrogen in the one than the other. I t
can be seen from Table IV, which embodies results obtained using the precautions suggested
by Chibnall, Rees & Williams (1g43), that the nitrogen content of gelatin made from collagen
pre-treated with alkali is lower than that of gelatin prepared with acid. Commercial gelatin
made by an alkaline process, the precursors of which have not received such energetic alkaline
treatment, has a nitrogen content intermediate between the two types. Gelatin made by the
acid method has the same nitrogen content as the collagen from which it is prepared. By
making use of acidity figures for the position of the isoelectric points of gelatin prepared by
the two methods, the loss of nitrogen can be calculated, and is found to agree reasonably well
with the difference as measured.
The isoelectric point of collagen.-Since the nitrogen content of acid-prepared gelatin is
the same as that of collagen, the loss of nitrogen during the preparation of gelatin by the
alkaline method should take place during the soaking period, and hence the isoelectric point
of collagen should alter during alkaline soaking. By making use of the swelling properties
of collagen, it can be shown that the point of minimum swelling, which coincides with the
isoelectric point, moves from neutrality to phenolphthalein to an acidity of between I, C.C.
J. Sci. Food Agric., 3, October, 1952
 AMES-COLLAGEN A N D GELATIN 457

Table 1V
Nitrogen content
%
Ox sinew prepared by the method of Bowes fi: Kenten (1948, p. 358) .. 18.49
Acid-process gelatin .. .. .. .. .. .. .. .. 18.52
Alkaline-process gelatin . . . . .. .. .. .. .. .. 18.08
Commercial alkaline-process gelatin . . .. . . . . . . .. 18.15
Maximum nitrogen difference . . .. .. .. .. .. .. 0'44
Calculated difference .. .. .. .. .. .. .. .. 0.49

and 2 C.C. of o.zN-acid/g. during an alkaline treatment. Swelling curves for rabbit skin, ox
sinew and ox hide, before and after soaking in calcium hydroxide, are shown in Figs. I, 2
and 3 . The method used, which entails titrating a finely-divided aqueous suspension of collagen,
is not exceptionally accurate, but is more than accurate enough to demonstrate the move in
the point of minimum swelling. It is a t least possible to say that the isoelectTic point of
alkaline-treated collagen falls in the same region as that of the gelatin which can be made
from it. The confusion which existed for long as to the position of the isoelectric point of
collagen is thus resolved : natural collagen is isoelectric between pH 7 and 8, alkaline-treated
collagen at pH 4.8.
There is additional evidence for the validity of these conclusions: If the final position
reached by the isoelectric point of collagen, as a result of the action of alkali, coincides with
that of the gelatin obtained, then little or no ammonia should be lost during heating, provided
the preliminary soaking has been of sufficient length. The results given in Table V demonstrate
this to be the case. Small amounts of ammonia are lost when limed sinew is heated in water,
but the quantity is of the same order as that evolved by heating gelatin alone. When untreated
sinew is heated much larger quantities of ammonia are lost ; the quantity lost is of a different
order of magnitude and is more nearly compar-
able with the amount lost during alkaline soaking,
from which it might be concluded that loss of
ammonia is an essential feature of one method
of converting collagen to gelatin.
Heat degradation of gelatin.-As we have seen,
the conversion of collagen to gelatin entails the
use of chemical treatment and heat. A study of
the effect of heat on gelatin solution might
therefore lead to a better understanding of the
function of heat and chemical treatment in the
preparatory process, I t has long been known
that exposure of gelatin solution to heat results

FIG.I.-Swelling curves for rabbit skis

Unlimed, continuous CUNS ; limed, broken curves

80

70
:6 0
;
f 50
P 40
-
30
ACIDITY,
c.c.-o~0 . 2 ~ - a c i d
iCIDITY,c.c.of 0 . 2 N - a c i d
FIG. 2.-Swelling curves for ox sinew FIG. 3.--Swclli~zg citvues f o v ox hide
Unlimed, continuous curves ; limed, broken curves Unlimed, continuous curves ; limed, broken curves

J . Sci. Food Agric., 3, October, 1952

2: **
458 AMES-COLLAGEN AND GELATIN
in rapid degradation. The loss in jelly-forming power and viscosity is illustrated for
alkaline-prepared gelatin in Table VI, which shows that a reIatively short exposure to heat
brings about a marked reduction in jelly strength and viscosity. This deterioration in physical
properties is not accompanied by other major changes, although there are modifications of
a minor nature. One such change is an alteration in pH ; when gelatin is heated on the
acid side of neutrality the pH value increases ; on the alkaline side it diminishes. Reference
to Table VII shows that there are also corresponding changes in the position of the isoelectric
point. Thus the isoelectric point of a definite type of gelatin does not fall a t a particular
pH value, but will depend to a small extent upon the previous history of the sample examined.
The increase in pH on heating gelatin in an acid condition is probably due to loss of carbon
dioxide, which has long been recognized as a product of protein hydrolysis (Thiele, 1567).
In particular it has been shown to be evolved when casein is heated in the presence of acid
(Dunn, 1925). Although there is a measurable loss of carbon dioxide when gelatin is heated
in a faintly acid condition, as some inorganic carbonate may have been present, the loss cannot
with certainty be attributed to the gelatin.
The alteration in pH when gelatin is heated in a faintly alkaline condition can definitely
be ascribed to loss of ammonia. With commercial gelatin, there is a considerable loss of
ammonia, which may be explained by assuming that the alkaline treatment of the precursor
had not been sufficiently long for the evolution of ammonia to reach completion. It should
also be noted that the ammonia comes off rapidly in the early stages, when commercial alkaline-
process gelatin is heated, and thereafter a t a much slower rate, an indication that there may
be two sources. By preparing gelatin from a. precursor which has been exhaustively treated
with alkali (12 weeks in calcium hydroxide, followed by several days in 10% sodium hydroxide
solution), one of the sources of ammonia is removed. On heating in an alkaline condition, gelatin
prepared in this manner still loses ammonia, but a t
a very slow rate. As the precursor, after exhaustive Table VI
alkaline treatment, rapidly goes into Solution in Alkaline-prepared gelatirz ; 2 soltttions
heated f o r 2 hr. at 85'
Table V ~~ PH Jelly Viscosity
Nitrogera loss during the conversion of collagen Before After strength
to gelatin - - 217 74'1
4.20 4.20 98 14.6
Nitrogen loss as a 7'0 4.48 4'49 116 19'4
of the nitrogen in s o h . 4.96 4.46
.. 127 24.0
Untreated ox sinew 5.06 5'15 '43 30'9
heated 7 hr. at 80" .. . . 0'49 5'45 5'64 I59 40'2
Limed ox sinew at pH 8 6.02 6.20 I 66 444
heated 4 hr. at 80" .. . . 0.023 6.59 6.74 I 63 444
heated L hr. at 80" .. . . 0'010 7'40 7'53 170 48.5
0.5% gelatin soln. a t pH 8 8.44 8.56 170 494
heated 7 hr. at 80" . . . . 0.17 9'17 8.90 I57 46.1
496 gelatin soln. at pH 8 9.64 9'45 I 66 43'7
heated 7 hr. a t 80" .. . , 0.081 I 0.08 9.76 I33 36.5
Jelly strength, bloom 63%,
Viscosity, centistokes 2 0 9 ; at

Table VII
Gelatilz solutions heated f o r 7 hr. at 95'
Commercial alkaline-firocess gelatin
PH ~ __ Isoelectric point pH ~

Before After Before After

10'0 9.72 . 5'25 4.8
Ammonia loss, C.C.of o.znr-acid/g. of gelatin after 2.5 hr. .. .. 0.116
I, >,
I , ,I I,,, a further 4.5 hr. . .
I, I, 0.035
Total 0.151
Gelatin wade f r o m collagen after exhaaistive alkaline treatment

~
PH - . Isoelectric point pH
~~

Before After Before After

4.83 4'95 4.83 4'97
9'71 9.38 4.83 4'74
Ammonia loss, C.C. of o.zN-acid/g. of gelatin .. .. .. .. 0.04
J. Sci. Food Agric., 3, October, 1952
 AMES-COLLAGEN A N D GELATIN 459
water when the temperature is raised to 40°, and has therefore been subjected only to chemical
treatment, the alterations in pH and the small loss of ammonia are effects due to heat alone.
Collagen can thus be converted to gelatin by alkaline treatment ; when heat has to be applied
to make good deficiencies in pretreatment, as has invariably to he done in practice, degradation
will take place.
Acid-prepared gelatin.-When gelatin, prepared by the acid method, is subjected to heat,
there is a similar fall in certain physical properties, accompanied by small changes in pH,
and in the position of the isoelectric point. The presence of readily available nitrogen, which
has been removed from the other type of gelatin, gives rise to certain small variations in
behaviour which are dealt with in Table VIII. When heated in alkaline solution, acid-prepared
gelatin exhibits a marked diminution of pH, and its isoelectric point moves from neutrality
to a point well on the acid side, changes which are accompanied by a relatively large loss of
ammonia. There can be no doubt that the ammonia which is evolved from the precursor
during the preparation of alkaline-prepared gelatin, and which is still present in acid-prepared
gelatin, tends to be evolved readily when the gelatin is heated. I t might be expected that
the one kind of gelatin could be converted into the other by prolonged heating in faintly alkaline
solution. This, however, is not possible, as the gelatin becomes completely degraded before
the nitrogen content is lowered to that of alkaline-prepared gelatin.
On heating acid-prepared gelatin in solution in an acid condition there is a slight increase
in pH, as happens in the case of the other variety. However, the isoelectric point, which
is determined after the heated solution has been dried off, does not show an increase in pH,
as would be expected. Instead, there is a marked lowering of the pH of the isoelectric point,
suggesting a loss of ammonia sufficiently large to outweigh the small loss of carbon dioxide
which may be assumed to cause the alteration in the pH of the heated solution. When the
isoelectric point of the heated solution is determined by making use of ion-exchange resins
(Janus et al., 1951),it is found to have moved to a higher pH, which is in accordance with
the previously observed change in the pH of the solution. Both types of gelatin thus behave
in the same way when heated in acid solution. From the peculiar behaviour of acid-prepared
gelatin when dried after heating, it may be concluded that it is less stable than the other kind
of gelatin.
Formol titrations.-An estimation in common use for following the process of protein
degradation, where rupture of peptide chains is entailed, is the formol titration. Results for
estimations carried out on dried samples of the heated gelatins are shown in Table IX. There
is little change in the formol titration figures. Alkaline-prepared gelatin shows a slight increase
when heated in alkaline solution, and acid-prepared gelatin shows a slight increase when heated
in acid solution. Estimations carried out on solutions which have been heated show that if
the heating is prolonged, even a t moderately high and low pH values, there is invariably a
small rise in the formol titration in every case. Consequently it would be unwise to attach
undue significance to the differences shown in Table IX.
The rate a t which the formol titration increases is so small, relative to the complete loss
of physical properties brought about by heat, that it raises doubts whether chain rupture
can be one of the major factors.
Structure of collagen and gelatin
Multi-chain models.-The molecular structures of collagen and gelatin are still a matter
of speculation, but any scheme covering the more general properties of proteins will also have

Table IX
Table VIII Gelatzn heated f o r 6 hr. at 85'
Gelatin solutions heated f o r 6 hr. at 85' Formol
A cad-process gelatin titration
PH Isoelectric point p H Ammonia loss, Alkaline gelatin
Before After Before After C.C. of o,ZN-acid/g.
CJnheated .. . . 3'92
of gelatin Heated a t pH 4.75 . . 3-92
Heated at pH 10.40 . . 4'24
10.36 9.09 8.10 5.38 0.3
4.94 5.02 8.10 6.55 - Acid gelatin
4.54 4.68 %08* 825* -.

*
Unheated .. ' . 3'59
Determined in s o h . by the ion-exchange resin Heated a t pH 4'94 . . 3.79
method (Janus et al., 1951). Heated a t pH 10.36 . . 3.57
Formol titration, C.C. of o.IN-NaOH/g.
of gelatin

J. Sci. Food Agric., 3, October, 1952

460 AMES-COLLAGEN A N D GELATIN
to be capable of explaining the relation of collagen and gelatin. I t is possible to construct
molecular models accounting for the results obtained in the experiments described above, and
thus establish conditions which must be satisfied in the final plan of the molecule. By general
agreement, collagen consists of polypeptide chains which may be bonded together by cross-
links of various kinds ; but there is also the possibility that collagen may consist of a long
single chain, coiled in a random fashion, and linked to itself. For this reason a model of each
kind has been worked out.
The conversion of collagen to gelatin by either method shows little evidence of chain
breakage. If the conversion is assumed to entail breakage of cross-links, all three types of
cross-links need to be considered, namely, hydrogen bonds, salt links, and covalent linkages.
As rather energetic chemical treatment or heat has to be used in the preparation of gelatin,
breakage of hydrogen bonds or salt links would seem to be ruled out. The loss of ammonia
which appears to be an essential accompaniment of the conversion unless acid is used, suggests
the breakage of a covalent cross-link formed by two dicarboxylic acid residues sharing an
amide group between them. Such a linkage would have the structure CO-NH-CO and on
hydrolysis ammonia would split off, leaving the carboxyl groups free. I n the multi-chain
models, breakage of this amide link is considered to be responsible for the formation of alkaline
gelatin.
To account for the formation of acid-prepared gelatin, it is necessary to postulate a similar
type of covalent cross-link capable of being broken by acid and heat. There is no evidence
to suggest what this cross-link can be, but a type of bond which is possible would be formed
by condensing an end-NH, group of lysine with an end-CO,H group of a dibasic amino-acid,
forming a CO-NH linkage. Such a bond would not be the same as the polypeptide links
in the main chain, in that there are hydrocarbon links on either side. As breakage of this
bond makes possible acid-process gelatin, it is referred to as the acid bond.
A model of collagen with six chains, making use of amide and acid cross-links is shown
in Fig. 4, and the two types of gelatin in Figs. 5 and 6. When collagen is converted into alka-
line-prepared gelatin the amide links are broken. There is also some breakage of peptide
linkages, and it may be assumed these are broken centrally between the pairs of acid cross-
links. If the molecular weight of collagen is taken as 24, the average molecular weight of
alkaline-prepared gelatin is 4. Its formol titration is 12. When acid-prepared gelatin is
made, all the acid links are broken, and there is some breakage of peptide links which is assumed
to take place on the outer chains where cross-links have been broken. The average molecular
weight of acid-prepared gelatin is then 3, and its formol titration 10. Acid-prepared gelatin
has a lower viscosity than alkaline-prepared gelatin, and hence a lower molecular weight ;
it also has a lower formol titration. The models satisfy these conditions.
When acid-prepared gelatin is heated in alkaline solution, there is a large loss of ammonia,
with only a slight increase in formol titration, pointing to a preferential attack on amide cross-
links. Reference to the model shows that this would cause a major breakdown, with a con-
.sequent deterioration in physical properties. When heated in acid solution, the cross-links
remain intact, but there is a slight increase in formol titration. This would also result in
poorer physical properties, though to a less marked degree. When alkaline-prepared gelatin
is heated in acid solution, as there is a negligible increase in formol titration, by analogy with
the behaviour of acid-prepared gelatin it may be assumed that the acid cross-links are broken.
This would give rise to a severe degradation. Breakage of peptide links is the only possible
source of deterioration when alkaline-prepared gelatin is heated in the presence of alkali.
The multi-chain models give a satisfactory picture of the relation of collagen and gelatin,
and the behaviour of gelatin on heating, except in one respect. The heat-degradation of
alkaline-prepared gelatin in the presence of alkali, and of acid-prepared gelatin in the presence
of acid, is explained by polypeptide chain breakage, and there is no practical evidence to show
that this is more than slight. There are other objections to multi-chain models with covalent
cross-links. By making an analysis of the titration curve of collagen and limed collagen (Bowes
& Kenten, p. 365), it can be shown that the number of carboxyl groups set free by the alkali
is almost exactly equivalent to the amide ammonia lost. For a covalent cross-link it should
be twice the amount.
Single-chain models.-These objections can be met by constructing a single-chain model,
which, however, introduces other difficulties. The loss of amide nitrogen, which appears to
be intimately associated with the conversion of collagen to gelatin must now be regarded as
adventitious. The formation of gelatin has to be explained by chain breakage. As the trans-
formation is unaccompanied by large changes in formol titration the assumption is made that
there are links in the chain, the rupture of which gives rise to end groups incapable of reacting
J . Sci. Food Agric., 3, October, 1952
 AMES-COLLAGEN A N D GELATIN 46 1

‘
.XID
IACID
I I

ACID
I I
ACID
I _I

I
ACID ACID
I I I
ACID ACID

FIG.4 .- Collngew FIG.5.-Alkalzne-prepared gelntzn

with formaldehyde. These groups may be pro-

line and hydroxyproline, which will not react
with formaldehyde in the same manner as amino-
acids having two hydrogen atoms attached to the
AM: DE nitrogen atom. Collagen and gelatin may be
differentiated from other proteins by their large
content of these amino-acids, proline and hydroxy-
proline being present to the extent of one in every
four residues. Suppose that half the proline links
A M DEI are readily split by acid, and the remainder by
alkali, and that these types alternate along the
chain. For the purpose in view it is unnecessary
to consider the unknown causes which result in
~~

one bond being stable to alkali and the other to

FIG. tj.--Acid-prepared gelatzn acid. The hydroxyl group in hydroxyproline may
be included, or the differentiation may depend
on the nature of the acid residues on either side. A single-chain model of collagen, consisting
of 44 residues with the bonds vulnerable to acid and alkali marked Pa and Pb respectively,
is shown in Fig. 7 beside the two types of gelatin.
Pa Pb Pa Pb Pa Pb Pa Pb Pa Pb Collagen

Pa Pb Pb Pa Pb Pb Pb Acid-prepared gelatin

- Pb Pb Pb Pb Pb Acid-prepared gelatin
heated in acid solution
Pa -- Pa ----- Acid-prepared gelatin
heated in alkaline solution

Pa Pb Pa Pb Pa Pb Pa Pb Pa Pb Collagen

Pa Pb Pa Pa Pb Pa Pa Pb Alkaline-prepared gelatin

Pa Pa Pa Pa Pa - Alkaline-prepared gelatin
heated in alkaline solution
- Pb -- Pb -- Pb Alkaline-prepared gelatin
heated in acid solution
FIG. 7

When acid-prepared gelatin is made, the breakdown of collagen is controlled, so that

only sufficient Pa links are broken to make collagen soluble. In the diagram three have been
broken, giving an average molecular weight of 11. On heating acid-prepared gelatin in an
acid condition, the remaining Pa links are broken, the resulting reduction in molecular size
accounting for the loss in physical properties. When acid-prepared gelatin is heated in alkaline
solution, the chain is reduced to relatively small fragments by the breakage of all the Pb links,
so that loss of viscosity and jelly-forming power would be expected.
Similar models are shown for alkaline-prepared gelatin. Under the conditions of
our experiments we have found alkaline-prepared gelatin to have a higher viscosity than
J . Sci. Food Agric., 3, October, 1952
462 AMES-COLLAGEN AND GELATIN
acid-prepared gelatin, so that the alkaline-prepared gelatin is assumed to have the higher molecular
weight. Accordingly, in passing from collagen to alkaline-prepared gelatin only two Pb links
are broken, resulting in an average molecular weight'of 15 compared with 11 for acid-prepared
gelatin. Heat degradation can be explained by the breaking of the remaining Pb links by
alkali, and all the Pa links by acid. I t should be noted that in the single-chain models any
small increments in formol titration are disregarded. Breakage of a small number of links
other than those specifically attacked by acid and alkali can, however, be fitted into the frame-
work of the models.
In the suggested molecular construction, no attempt has been made to explain the small
changes in pH exhibited by both types of gelatin when heated in solution. These are con-
sidered to be due to causes not necessarily resulting in the breakage of bonds, and are assumed
to play a minor part in molecular degradation. In the case of alkaline heating, the loss of
ammonia may be attributed to the breakdown of the guanidine group of arginine, which is
known to occur in alkaline solution, and in particular, when collagen is exposed to the action
of alkali (Highberger & Stecker, 1941 ; Warner, 1942). When gelatin solution is heated in
the presence of acid the small change in pH is probably due to a loss of carbon dioxide, though
this has not been proved, and it is uncertain which amino-acid residue is responsible.
The nature of the deductions which can be made from either the single-chain or multi-
chain models illustrate their usefulness in collating the results of a large and varied body of
experimental work. These may be summarized as follows:
I . By gentle alkaline hydrolysis of gelatin prepared by the alkaline method continued
over a sufficient length of time, the combination of amino-acids responsible for the acid cross-
link may be found in the hydrolytic products. Or if the molecule is a single chain, the P b acid
will be found a t the ends of chains, provided the mixture of peptides is not unduly complicated
by the rupture of other peptide linkages.
2. Similarly, by gentle acid hydrolysis of acid-prepared gelatin, the amino-acids responsible
for the amide link may be identified, or for a single-chain molecule the Pa acid which will be
at the ends of chains.
3 . Acid-prepared gelatin cannot be converted into alkaline-prepared gelatin, because they
differ in details of molecular structure and also because it would not be possible to remove
the amide ammonia without bringing about a complete breakdown. In the one model, breakage
of the amide cross-links and, in the other, removal of P b bonds by alkali, gives rise to degrada-
tion products which are not the same as alkaline-prepared gelatin. This is in accordance with
experimental results (Ames, 1947).
4. I t will not be possible to make alkaline-prepared gelatin by long treatment with acid,
as has been claimed by Rousselot (1944). Preliminary experiments show that it is, in fact,
not possible to make alkaline-prepared gelatin from a precursor treated with acid.
5. Alkaline-prepared gelatin is less susceptible to heat degradation in alkaline solution,
and acid-prepared gelatin is less susceptible in acid solution. Heating experiments have
been carried out on the two types, the results of which do not conflict with this deduction,
but are somewhat inconclusive (Ames, 1947).
6. Collagen which has been treated with alkali will yield gelatin of better physical pro-
perties when heated in alkaline solution. This deduction, based on theoretical grounds, is
in accord with manufacturing tradition, which asserts that lime-treated skin should be heated
at pH 7-8 when making gelatin.
7. Ossein, which has been obtained from bone by the action of acid, should be heated
in acid solution to obtain the best gelatin.
I t is evident that the suggested molecular structures do explain the limited field they
are intended to cover, namely the relationship between collagen and gelatin. They clearly
show the importance of investigating the end groups set free as a result of the gentle hydrolysis
of both types of gelatin. Some attention might also be given to the significance of the high
proportion of proline and hydroxyproline in gelatin.
Acknowledgments
The author wishes to thank the Director and staff of the British Gelatine and Glue Research
Association for advice and criticism, and J. Br G. Cox Ltd., in whose laboratory these investiga-
tions were carried out, for permission to publish the results.
J. & G. Cox Ltd.
Gorgie Mills
Received ZI March, 1952
Edinburgh, I I
J. Sci. Food Agric., 3, October, 1952
 CALL-DETERMINATION
References
Ames, W. M. (1944). J . SOC.chem. I n d . 63, zoo,
234. 277. 303.
Ames, W. M. (1945). J . SOC.chem. I n d . 64, 242.
Ames, W. M. (1947). J . SOC.chem. I n d . 66, 279.
Bowes, J . A. & K$nten, R. H. (1945). Bzochewz. J .
43, 356, 365.
Chibnall, A . C., Rees, M. W. RC Williams, E. F. (1943).
Biorhena. J . 37, 354.
Dunn, M. S. (1925). J . Amer. chem. SOL.47, 2564.
OF M E T H Y L BROMIDE, etc. 463
Highberger, J . H. & Steclter, H. C. (1941). J . Anzer.
Leath. Chem. Ass. 36, 368.
Janus, J. W., Kenchington, A. W. LE Ward, A. G.
(1951). Research 4, 247.
Rousselot, A . (1944). C . R . Acad. Sci., Paris 219,
62.
Thiele, J. (1867). Chern. Zbl. 12 (new series),
385.
Warner, R. C. (1942). J . biol. Cheni. 142, 705.

APPARATUS FOR T H E DETERMINATION O F

LOW CONCENTRATIONS OF METHYL BROMIDE
AND OTHER GASES
By F. CALL

A method is described for the determination of low concentrations (0.01-1.0 mg./l.) of

methyl bromide, based on the length of the colour stain produced by the reaction of the
bromine in the combustion products with fluorescein paper. The length of the stain is
directly proportional to the concentration, provided certain .factors are controlled. The
effect of these factors is discussed and an apparatus is described which automatically
controls them. The method has been shown to be applicable to hydrogen cyanide and
carbon tetrachloride, and it is believed to be suitable for any gas for which a suitable test
paper exists.

Introduction
There are in existence several forms of apparatus for the detection of very low concentra-
tions of gases, which depend on the colour produced by reaction of the gas with paper impreg-
nated with a suitable reagent. Many of these methods can be used to determine approximately
the concentration of the gas, but have the disadvantage that it is necessary to compare the
intensities of colour stains with a set of standards, thus introducing a subjective factor that
may lead to considerable errors. It would be a considerable improvement if concentrations
could be assessed in terms of some quantity more easily measured. The apparatus which will
be described has been designed so that a colour stain is produced whose length is directly
proportional to the concentration of gas under test. The apparatus has also been made semi-
automatic, thus standardizing the conditions of test, with a corresponding increase in reliability
and ease of manipulation, making it suitable for use by relatively unskilled workers.

Experimental technique
I n the present apparatus, the gas stream, after catalytic combustion if necessary, instead
of being allowed to impinge on a fixed area of the test paper, is led at a carefully controlled
flow rate over the paper surface along a groove or channel cut in a polished brass block. The
test paper is clamped firmly and held flat over the groove by means of a second polished plate,
sufficient pressure being applied to prevent leakage of air from outside into the groove. The
gas reacts with the test reagent to give a visible stain of uniform intensity whose length varies
as the concentration of the gas provided certain conditions are controlled.
The chief factors which determine the length of the stain produced in such a method are :
(a) concentration of gas, ( b ) rate of flow, (c) volume of sample, ( d ) concentration of fluorescein
on the paper, (e) dimensions of the groove, (f)type and surface of the paper. These factors
have been investigated by varying one at a time while keeping the remainder constant.
As the apparatus was required primarily for use in fumigation work, the greatest interest
centred on halogenated hydrocarbons, particularly methyl bromide, which is coming into
increasing use as a fumigant. Lubatti (1945)has briefly described a method for the detection
and determination of low concentrations of methyl bromide based on catalytic combustion of
the gas by a glowing platinum filament, and reaction of the bromine thus formed with a paper
impregnated with fluorescein solution to give a red stain of eosin. As with most other methods,
assessment of the concentration is by comparison of the intensity of the stain with a set of
standards. Since tests showed the reaction to be very sensitive and capable of detecting
0.2 pg. of bromine, it was adopted for this investigation, and most of the development work

J. Sci. Food Agric., 3, October, 1952