2I 4 BIOCHIMICA ET BIOPHYSICA ACTA

T H E M E C H A N I S M O F G E L A T I O N O F G E L A T I N

T H E I N F L U E N C E O F pH. CONCENTRATION, T I M E A N D D I L U T E

E L E C T R O L Y T E ON T H E

G E L A T I O N O F G E L A T I N A N D M O D I F I E D G E L A T I N S *

JAKE BELLO**, HELENE RIESE BELLO** AnD JEROME R. VINOGRAD Gates and Crellin Laboratories,

Calilornia Institute o/Technology, Pasadena, Calil. (U.S.A.)

(Received July x9th, x96I)

SUMMARY

The effects of concentration, pH, time and dilute electrolyte on the melting points of gels of gelatin and chemically modified gelatins have been investigated. At I -5 % concentration the melting points of high-isoelectric-point gelatin, low-isoelectric- point gelatin, amino-acetylated gelatin and carboxyl-esterified gelatin are nearly independent of pH. At low concentration (o.6-o. 7 %) deionized gelatins have several melting point maxima and minima. Upon long standing at o ° or upon addit ion of electrolyte the curves become independent of pH from 2-I2. The results are discussed in relation to the conformation and charged groups of gelatin and to present knowledge of the aggregation process.

INTRODUCTION

The gelation of gelatin is a classic example of protein interaction. A bet ter under- standing of this interaction would be of interest also in respect to the association of collagen molecules into multiple strands and of strands into higher order structures. While gelation of gelatin in the cold may be different mechanistically from gelation of collagen at 37 ° , nevertheless knowledge of the former may help clarify the latter.

The melting and gelling points of gelatin gels have been the subject of much in- vestigation. Various aspects of the gelation of gelatin have been reviewed by FERRY 1. MERCK~L ~ reported that the melting point of a IO% gelatin gel is independent of pH from pH i - 4 and from pH 5-9 with a sharp increase of about 3 ° at pH 3-5. GERNGROSS~ reported that the gel strength of xo% gelatin is independent of pH from 4.2-9.5 and drops gradually at either extreme. MEUNmR AND GRIGNARD 4 reported tha t isoelectrie gelatin (with o.5 % ash) had melting point maxima at pH 2.i and 5 with a minimum at pH 2. 9, but no other data were given.

* Contribution No. 2093 from the Gates and Cre!lin Laboratories of Chemistry of the California Institute of Technology, Pasadena, Calif. (U .S.A.).

** Present address: Department of Biophysics, Roswell Park Memorial Institute, Buffalo 3, N.~'. {U.S.A.).

Biochim. Biophys. Acta, 57 (x962) 214-221

MECHANISM OF GELATION OF GELATIN 215

GARREAU a al. 5 reported a minimum gelation temperature and rigidity at the isoelectric point (pH 4.7) for I ~/o gelatin of unspecified purity. GORDON s found that in the presence of o.15 M sodium chloride the melting point is independent of pH in the range from pH 5.2 to 9.2 for I . I ~/o gelatin. KRAEMER AND FANSELOW ~ found that o.5 ~/o gelatin (de-ashed) has a greater gel-forming abili ty at about pH 5 than at other pH values and that the specific rotation is ,~ maximum at pH 5. REIGER AND BACH s reported that gelation is independent of pH from pH 4.o to 7.8 for 3.5% gelatin. PLEASS 9 working with nearly ash-free gelatin (o.oI-o,oz ~/o ash), investigated the mini- mum concentration of gelatin required to form a gel at various temperatures and pH values. If one considers FLEASS' da ta in terms of melting point, there is a minimum melting po;nt at about pH 2.5, a maximum at 1. 3 and near independence from 4 to 11. LLOYD l° determined the minimum concentration of ge~'atin required to set to a gel at 5 ° and obtained results in general agreement with those of PLEASS. These scattered and inconsistent data provide no clear picture of the effect of concentration, electrolyte and pH on the gelation of gelatin.

We hkve investigated the melting points of gelatins of high isoelectric point (IP 9.2), low isoelectric point (IP 4.8 and 5.1), amino-acetylated gelatin and carboxyl- esterified gelatin at various concentrations, pH values and in the presence or absence of sodium chloride.

MATERIALS AND METHODS

Gelatins

Most of the work was done with an acid-extracted pigskin gelatin of isoionic point 9.2 (as determined by turbidi ty maximum, viscosity minimum, and mixed-bed ion-exchange resin xx) obtained from the Wilson Laboratories, Chicago, Illinois, and designated "Wilson U-COP-CO, Special Non-pyrogenic Gelatin". In some experiments there was used a limed ossein gelatin (Charles B. Knox Gelatin Company) of isoelectric point 5.1, or a limed, de-ashed calfskin gelatin (Eastman Kodak Company) of isoelectric point 4.8, and o.o2~/o ash and o.oI~/o albumin content, as reported by the maker.

Concentrations were determined by drying aliquots at lO8 °. Acetylation of aqueous gelatin with acetic anhydride was carried out at pH 9.5-

xo. 5 (see ref. 12). Modified VAN SLYKE amino-nitrogen 13 determination showed that 99~/o of the amino groups had been acetylated. Esterified gelatin was prepared by t reatment with methanol and thionyl chloride 14.

Deiouization was done by the column procedure of JANUS et al. lx, or batchwise, with Amberlite MB- 3 mixed bed, ion-exchange resin.

Melting points

Gelatin solutions were adjusted to the desired pH and placed in I5o × 18 mm test tubes which were stoppered and then warmed at 5 o° for IO rain. Solutions of pH lower than 3 or higher than IO were warmed for 3 min. Warming for Io min caused li t t le difference. The tubes were then stored at o ± o.o5 ° for 2o h. A Neoprene ball (d~ ° 1.17; 0. 5 cm diameter) was inserted under the surface and the tube was warmed at the rate of about 5°]h. The melting point was taken at the temperature at which the ball reached the bottom of the tube. At high concentration the reproducibility was about ~ o.3 ° and at low concentration about 4- o.5 °. In some cases lumps of gel formed; these samples were disregarded.

Biochim. Biophys. Acta, 57 (I962) 2t4-22I

216 J. BELLS, H. R. BELLS, J. R. VINOGRAD

opt ical rotation

R o t a t i o n s were m e a s u r e d in 4 - d m t u b e s u s i n g t h e s o d i u m l a m p . T h e t u b e s w e r e s t o r e d a t 4 ° for t h e r e q u i r e d t i m e .

RESULTS

T h e r e s u l t s for t h e de ion ized g e l a t i n of i soe lec t r ic p o i n t 9.2 (Wilson) a re s h o w n in Fig . I for c o n c e n t r a t i o n s of o .60 t o 5.0 % . A t 5 % a n d 1.2 °/o, t h e m e l t i n g p o i n t is i n d e p e n d e n t of p H ove r t h e r a n g e f r o m 2 -12 . C o n c e n t r a t i o n s lower t h a n 0 . 6 % c o u l d n o t be u s e d

b e c a u s e of t h e f o r m a t i o n of i n h o m o g e n e o u s s y s t e m s c o n s i s t i n g of l u m p s of gel in

o t h e r w i s e f lu id gelaf in~ A t 0. 7 % t h e r e a re m a x i m a a t p H 1.5, 5 a n d a b o v e 12. (Be low p H 2 a n d a b o v e p H 12 t h e m e l t i n g p o i n t fa l l s a t all c o n c e n t r a t i o n s of al l t h e g e l a t i n s s t u d i e d . ) I n o . I or o . o i M s o d i u m ch lo r ide t h e m e l t i n g p o i n t b e c o m e s i n d e p e n d e n t o f p H ove r t h e r a n g e 2 - I 2 e x c e p t for a s m a l l m a x i m u m n e a r t h e i soe lec t r i c p o i n t .

,':i fh k oL, , ~

pu

; - - ~ ~ - - S --~-'-V - ~ - - ] -~-~T-

~ '°i '

a -~ v s s ~ t~

pH

Fig. 1. Melting point of IP 9.z, pigskin gelatin. Fig. 2. Melting point of low-isoelectric-point Deionized: O - - O , 5 % ; O - - - 0 , 1._~%; ~ - - ~ , gelatins. Deionized: t ) - - tO, 5% calfskin; 0 .7%; ~ - - 1 0 , o .6%; ( ) - - @ , o . 6 % w i t h o . x M e - - Q , 5°0'0 ossein; @ - - ~ , 0.57% calfskin, added sodium chloride. Non-deionized: @- -@, (the dotted curve indicates tha t gelation oc-

0.73 %. curred at pH 6, 7 and, weaker, at 8, bu t melting points were not measured, and vertical dotted

lines show the effect of o.1 M sodium chloride]; O - - O , 0 .7% calfskin. Non-deionized: ( I - - ( ) , 0. 7 % ossein.

TABLE I MELTING POINTS AND ELECTROLYTE CONCENTRATIONS OF INITIALLY DEIONIZED GELATIN

0.7% WILSON GELATIN, IP 9.2

pH Concentration of ions 3[ellDig point* (moles/I) (°C)

2.0 0.022 17. 3 3. I 0.003 No gel 4.1 o.ool 5 I I .o 5.I 0.0006 14.2 6. I 0.0003 No gel 7.0 0.00005 No gel 8.1 o.oooo2 No gel 9.2 o.ooooo** (IP) No gel

xo.o o.ooo 5 No gel I I . I O.OO16 NO gel 12.x o.o1 I3. 5

* The indication "no gel" means the solution flowed freely, and a neoprene ball fell as soon as it was placed in the tube.

*" Assumed, not determined, value.

Biochim. Biophys. Acta, 57 (1962) 214-azt

MECHANISM OF GELATION OF GELATIN 2T 7

The adjustment of pH of the deionized gelatin involves the addition of some electro- lyte, hydrochloric acid or sodium hydroxide, in amounts given in Table I. The 0.6 and 0. 7 % solmions just form uniform gels under the conditions llsed here, and these solutions are greatly affected by small c~langes in composition. When o. 7 % non- deionized gelatin (o.5% ash, corresponding to 6-lO -4 M electrolyte, calculated as sodium chloride) was used, the melting point was again nearly independent of pH ~ron-t 1.5 to i i . This emphasizes the importance of specifying or eliminating the electrolyte content in work at low concentration.

The melting points (Fig. 2) of the 5% solutions of low-IP gelatin were independent of pH from 5 to nearly i i . Between pH 4 and 3, both the ossein and calfskin gelatins exhibit a drop in melting point: This drop was not eliminated by sodium chloride, in agreement with the findings of MERC" :EL 2 for lO% gelatin. In dilute solutions the melting point-pH curves of the two gelatins are complex. Again the minima vanished on addition of o.I M sodium chloride. The dashed portion of the curve in Fig. 2 does not represent measured melting points, but is based on the forma~.'on of firm gels at pH 6 arid 7, and weaker gels at pH 8. It is included to show the approximate shape of the curve r~ther than to show its exact height.The melting point-pH curve for limed ossein gelatin, IP 5.1, was similar to that of the limed calfskin. The non-deionized ossein gelatin (o.7% ash on dry basis) at low concentration showed only a slight minimum a'~ pH 4 and a maximum at pH 5, the isoelectric point.

The isoionic point of the acetyl derivative of the high isoelectric gelatin was 4-7, by ion exchange. The results for several concentrations of gelatin, with and without sodium chlorido, are shown in Fig. 3. Here again, in the dilute solutions there are mini- ma which are eliminated by addition of dilute sodium chloride. At 5 % the melting point curves of the acetylated gelatins do not exhibit the drop at pH 5-3 shown by non-acetylated gelatin at low IP.

In Fig. 4 are shown the data for the high-IP gelatin, having 70% of the carboxyl groups methylated. In dilute solutions the usual minimum at pH 3-4 appears, but the high pH minimum is absent. The maximum at pH 12 shown by the previous materials is also absent, no gelation occurring above pH II. Sodium chloride at o.I M con- centration caused gelation at pH 3 but not at pH II .

~o , t ' ~ , i , i , i ,

p s p H

Fig. 3" Melting point of amino-acetylated Fig. 4. Melting p i n t of carboxyl-esterified gelatins; O--O, 5% acetylated pigskin gelatin; ~ - - ~ , 5%: O--O, o.7%; @, o.7% (original IP 9.2) ; 0 - - 0 , 5 % acetylated with o.I M sodium chloride added. calfskin; ~ - - ~ , o.98% acetyiated pigskin; ID--ID, o.74 % acetylated pigskin. Vertical dotted lines show the effect of added o.t M sodium

chloride.

Biochim. Biophys. Acta, 57 (1962) 2z4-22I

218 J. BELLO, H. R. BELLO, J. R. VINOGRAD

The melting points described above are for gels stored at o ° for 2o h. This period of time was chosen because previous investigators have generally used times of one day or less and because, in earlier work with 5 ~ gels, we found that longer storage (up to one month) caused almost no change in the melting point at pH 5 (see ref. 15). It was thought desirable to determine the effect of time at low concentration. In Fig. 5 are shown the melting points of o.6%, IP 9.2, gelatin solutions aged at o ° for different times. I t it apparent that the low melting points at pH 3-4 and 9 in time are raised to about the same level as the maxima. The minima are therefore the result of a decrease in the rate of formation of the gel. The modified gelatins we have studied also gelled at pH 3-4 and pH 9 on long standing at o °, although melting points were not determined. On long storage of esterified gelatin at pH 11.6 and o °, gelation also took place. However, as it was found that under these conditions hydrolysis of ester groups occurred, gelation may have been due to reversion to non-esterified gelatin.

, i , i , i r - . T .- i - - r - T ]

i:!I .... .240 t o ~ , o 2 4 6 8 i o i;~

pH pH

Fig. 5. Eitect of time of storage at o ~ on melting point of 0.6°/0, IP 9.2 pigskin gelatin: O--O, 2o h; O--O, 4 days; O--O, 7 days; ©--~ ,

Ii days; @--~, 4I days.

Fig. 6. Specific rotation as a function of pH: 0 - - 0 , 0.7% IP 9.2 gelatin after 20 h at 4°; O--O, same after xo days at 4 ° ; O--O, o.7 %

esterified gelatin after 2o h at ¢%

The specific rotations of o. 7 °/o deionized, high-isoelectric-point gelatin and. of its methyl ester at 4 ° and various pH values are shown in Fig. 6. Throughout the entire range the specific rotation of the non-esterified gelatin is greater than w 25o ° and from pH 5 to 12 is about - - 3oo% values in agreement with those reported for gelatin at low temperature and about double those reported 1~ for gelatin above 35 °, where no aggregation occurs 17. There is a 2O~/o reduction in specific rotation at pH 4 where, at the concentration studied, gelation is retarded. The results are in substantial agree- ment with those of CARPENTER et al. 16 who found minima at pH 3 and perhaps 5 (where turbidity made measurements uncertain). KRAEMER AND FANSELOW ~, working with 0.5 % gelatin, found a maximum rotation slightly below pH 5 at 25 ° or less. CARPENTER eL al. reported that after three days the specific rotation rose slightly at all pH values but somewat more at the pH 3 minimum than at other points. By using gelatin of high isoelectric point we have been able to obtain measurements near pH 5 without turbidity difficulties (there was turbidity at pH 9, but readings were not hampered) and found no minimum. In Io days an increase was found at the minimum but not at pH 5. This result parallels the observations on the effect of time on melting point. In both sets of data, the minima tend to disappear with long storage at low temperature, although in the case of melting points the curve as a whole rises. The specific rotation of the esterified gelation was nearly constant from pH 7-11.6

Biochim. Biophys. Acta, 57 (I962) 2x4-221

MECHANISM OF GELATION OF GELATIN 2I 9

but iell at pH 4. The lower rotation of the esterified gelatin is probably due to its lower molecular weight.

DISCUSSION

In other work TM, we have shown that blocking of the carboxyl, amino, hydroxyl and guanidino groups has li t t le effect on gelation at 5~/o concentration. At the low con- centrations studied here, it is apparent from the pH and salt dependence that the charged groups are of importance in the early stages o5 gelation, but as inhibitors rather than as required cross-linking groups. At these borderline concentrations, charge effects tha t would be insignificant at high concentration are of great importance. I t has been suggested that a c¢oss-link in gelation consists of multiple hydrogen bonds TM, 2o. While the major contribution to gelation is probably made by multiple-bonding ~ites, some contribution, especially in the early stages at low concentrations, may be made by single hydrogen bonds, salt links or interactions between non-polar groups.

Unless otherwise noted, the following discussion refers only to the low-concen- tr'ation data. Since it was found that, at the lowest concentrations, the non-gelation at pH 3-4 and 6-11 (8-11 for limed gelatin) was in reality a retardation of gelation, the explanation of the minima in the curves must be sought in terms of factors affecting the rate of gelation. The retardation may be due to one or more of the following general causes: The inhibition of molecular conformations required for the formation of cross- linking sites; the formation of aggregates having insufficient exposed cross-linking sites as a result or burying of sites in the interior; or the adsorption of molecules of low functionality to the aggregate surfaces, such molecules acting as terminators ~z of t~ross- linking. Molecules of low functionality would be more probably found on the surface of an aggregate than in the interior because as chain terminators they would inhibit the accretion of additional molecules, and being less firmly held to the aggregate (because fewer bonds need be broken to detach them) they would be dissociated from the aggregate and their place taken by polyfunctional molecules until most of the poly- functional molecules are in the aggregates, The appearance of gels after extended cold storage may be regarded as resulting from the chan~e of molecules of conformations of low functionality to conformations, of higher functionality, from the reorganization of aggregates to expose more cross-linking sites or to dissociation of low functional molecules, thereby permitting aggregates to coalesce.

I t has been shown that at concentrations too low for gelation, aggregation of gelatin molecules occurs 2°.21 and that dissociation of molecules from the aggregates is slow but measurable 2°.

The various minima and maxima in the melting point curves are obviously due to associations or repulsions of the charged groups since they are dependent on pH and on electrolyte. The distribution of charge appears to be more important than net charge. This is shown by comparison of the high-IP pigskin gelatin with its low-IP acetyl derivative; their melting point curves are the same, and one gels at its IP while the other does not. There are two apparent points of correlation between net charge and gelation: the high charges at pH 2 correspond to high melting points; the high charges at pH 12 for three gelatins correspond to high melting points, and the low charge at pH i i for the fourth corresponds to its non-gelation. However, at these extreme pH values, the electrolyte concentration is sufncient to screen the charged groups except, perhaps, in the case of tile esterified gelatin at pH xx (near its iso-

Biochim. Biophys. Acta, 57 (x962) 214-22t

220 J. BELLO, H. R. BELLO, J. R. VINOGRAD

electric point). I t is probable that the easy gelation at pH 2 and 12 is due to electrolyte. A similar efiect appears in the viscosity-pH date of STAI~SBY ~3 for a low-IP gelatin, the ~iscosity rising to a maximum at pH 3 (our gelation minimum) as a result of electrostatic repulsions, and falling at lower pH as a result of screening of charge by electrolyte. Additional support is given by the effect of added sodium chloride, or of the original ash, in eliminating the minima.

At very low pH, below I, gelation is reduced, and when the acid concentration is in the 1-3 M range (depending on the acid) gelation is completely prevented, without extensive hydrolysis iv. This may be due to protonation of peptide groups, thereby preventing intraxnolecular hydrogen bonding required to form a cross-linking site, or intermolecular hydrogen bonding between sites.

Fig. 3 shows that the amino groups are not involved. The shape of the gelation curve for esterified gelatin suggests that the carboxyl

groups are involved. The inhibition of gelation of non-esterified gelatin at pH 6-11 (or 8-I I ) is probably due to inter- or intramolecular repulsions between carboxylate groups, or association between carboxylate groups and other groups. Since there are fewer carboxylate groups at pH 3-4 than at higher pH, and since the amino-acetylated gelatin is similar in its behavior to its parent gelatin, it is possible that the guanidininm groups are involved in this pH range. This possibility cannot now be tested by studying the deguanidinated gelatin as the available deguanidination procedures cause extensive reduction of molecular weight. The report 24 that treatment of gelatin with alkaline hypobromite does not cause much degradation of the gelatin is inccrrect ~. I t has been reported by BERGER et al. that sodium in liquid ammonia destroys the guanidinium groups of gelatin without degradation ~. Dr. BERGER has informed us that the molecular weight had been reduced to IOOOO, as determined by osmometry. Low- molecular weight gelatin does not gel at o.7%, and at concentrations high enough for geiation, there is no pH effect. The gelation of high-IP gelatin on going from pH 4 to pH 5-6 may be due to elimination of guanidinium repulsions by formation of com- ponsating carboxylate groups. The similarity of the high-IP pigskin gelatin to its 7O~/o-esterified derivative at pH 5 m a y be due to the remaining free carboxyl groups.

The cause of the difference between the curves for high-IP and low-IP gelatins probably lies in different distributions of charged groups. High-IP gelatin has more amide groups and correspondingly fewer carboxyl groups than low-IP gelatin ~. Differences between these two types of gelatin in viscosity characteristics are known ~.

It was suggested at the beginning of the DXSCUSSIO~ that changes in the con- formation of individual gelatin molecules may be responsible for the delayed gelation at certain pH values. In this regard the optical data are suggestive but not conclusive. The lower rotation (absolute value), increasing with extended cold storage, at pH 3-4 corresponds to the melting point curve. Thus, the delayed gelation may be due to unfavorable conformations. There is an alternate possibility that the lower rotation is caused by differences in the structure of the aggregates. The shape of the optical rotation curve at higher pH values does not point to conformational factors, although these might be subtle or mutually balancing.

While we have been able to suggest explanations for the various maxima and minima, greater certainty must await more detailed information on distribution of charged groups, intimate details of the aggregation process and of the nature of the

Biochim. Biophys..4eta, 57 (I962) 2x4-~2x

MECHANISM OF GELATION OF GELATIN 22T

aggrega tes , a n d the deve lopmen t c,f non-deg~adati, , 'e m e t h o d s of b locking the guan id ino

(and p e r h a p s hydroxy l ) g roups .

ACKNOWLEDGEMENT

"[his w o r k was s u p p o r t e d b y con t r ac t No. DA-oo7-MD-298 wi th the Office of t h e S u r g e o n General , D e p a r t m e n t of t he A r m y .

REFERENCES

I j. D. FERRY, Advances in Protein Chem., Vol 4, Academic Press, In~., New York, z948, p. t. 2 j . H. G. MERCKEL, Kolloid Z., 78 (I937) 339" 30 . GERNGROSS, Kolloid Z., 4 ° (19.,6) 279. 4 L. ~'~/IEUNIER AND R. GRIGNARD, 14me. Congr. chim. ind.. Paris, Oct. 1934. 5 y . GARREAU, P. GIRARD AND N. MARINESCO, Comp. rend. soc. biol., Io 3 (I93O) 551. 6 R. S. GORDON, Jr., in J. D. FERRY, ,4dvances in Protein Chem., Vot. 4, Academic Press, Inc.,

New York, I948, p. 26. 7'E. O. KRAEMER AND J. R. FANSELOW, f . Phy:. Chem., 29 (1925) II6~). s R. REIGER AND S. BACH, KclloidZ. , 76 (I~J36~ 82. 9 W. B. PLEASS, Proc. Roy. Soc. (London) .4, I26 (193o) 406.

I0 D. J. LLOYD, Biochem. J. , I6 (1922) 530. n j . W . JANUS, A. W. KENCHINGTON AND A. G. ~*VARD, Research, 4 (I95I) 247. ta j . ~3ELLO AND J. R. VINOGRAD, f . . 4 m . Clwm. Sot., 78 (I956) 1369. ta D. J. DOHERTY AND C. L. OGG, Ind. Eng. Chem. Anal. Ed., 15 (I943) 73I. :4 j . BELLO, Biochim. Biophys. Acta, 22 (1956) 426. 15 j . BELLO, H. RIESE AND J. R. VINOGRAD, J. Phys. Chem., 6o (I956) I290. 18 D. C. CARPENTER, A. C. DAHLBEEG AND j . c. HENNING, Ind. Eng. Chem,, 20 (I928) 397. 17 E. O. KRAEMER, o.i Phys. Chem., 45 (194 I) 66o; 46 (I942) I77. 18 j . BELLO, H. R. BELLO AND J. R. VINOGRAO, Biochim. Biophys..-Icta, 57 (x962) 222. 19 j . E. ELDRIDGE AND J. D. FERRY, f . Phys. Chem., 58 (I954) 992. 20 H. BEODTKER AND P. DOT',', J. Phys. Chem., 58 (1954) 968. 21 G. BEYER, J. Phys. Chem., 58 (1954) Io5o. 2~ R. J. GOLDBERG f . Phys. Chem., 2o (I952) i8~6. a3 G. STAINSBY, Nature, I69 (1952) 662. at p. DAVIS, Trans. Faraday Soc., 53 (I957) 139o. -°5 J. BELLO, Trans. Faraday Soc., 55 (1959) 213o. ~9 A. BERGEn, J. KURTZ AND J. NOGUCHI, in G. STAINSBY, Recent .-Idvances in Gelatin and Glue

Research, Pergamon Press, New York, I958, p. 271. av ~,V. M. AMES, f . Sci. FoodAgric., 3 (1952) 579. as A. VEIS A~D J. COHEN, J. Polymer Sci., 26 (t937) x 13.

Biochim. Biophys. Acta, 57 (I961) 214-221