222 BIOCHIMICA ET BIOPHYSICA ACTA

T H E F U N C T I O N A L G R O U P S IN T H E G E L A T I O N O F GELATIN*

JAKE BELLO**, HELENE R. BELLO** AND JEROME R. VINOGRAD Gates and Crellin Laboratories, Cali/ornia Institute o/ Technology,

Pasadena, Calif. (U.S.A.)

(Received July x9th, x96x)

SUMMARY

The participation of the side-chain dissociable and polar groups o{ gelatin in the gelation process has been investigated by studying the gelation of gelatins chemically modifit:d in one of the following ways: Carboxyl groups esterified with methanol, amino groups acetylated, hydroxyl groups acetylated or guanidino groups nitrated, the last with simultaneous part ial sulfation of hydroxyl groups. The melting point - intrinsic viscosity relations of the modified gelatins were compared with those of unmodified gelatins of similar molecular weights. I t was found that the amino, carboxyl and guanidino groups are not involved in the equilibrium state of 5 ~/o gels. The hydroxyl groups are not indispensable for gelation, but may contribute. The role ~f the hydroxyl groups is par t ly obscured by a rearrangement that appears to take place in one of the two methods of acetylation.

INTRODUCTION

There has been speculation and some experiment as to which groups in the gelatin molecule are involved in the thermally reversible cross-linking that results in the formation of the gel. Evidence against the participation of carboxyl and amino groups is the fact that the melting point of x - 5 % gels is nearly independent of pH from pH 2-I1 (see ref. I). Also, FERRY has proposed 2 that the insensitivity of the melting point to the presence of sodium chloride is evidence for the non-participation of charged groups. At low gelatin concentrations (o.6--o. 7 °/o ) the rate of gelation, but not the melting point at equilibrium, is dependent on pH and electrolyte t.

GV, ABAR AND MOREL 3 found that when the amino groups were removed by nitrous acid or blocked with dinitrofluorobenzene the gelatin still gelled, indicating that the amino group is not indispensable in gelation. However, quanti tat ive correlation of gelling abil i ty with molecular weight, which might show partial dependence of gelation on amino groups, was not made. The same authors found that destruction of the guanidino group by alkaline hypobremite resulted in reduced gelation accompanied by reduced molecular weight 4. However, similar reductions in rigidity and molecular

Abbreviation: TFA, trifluoroacetic acid. * Contribution No. 23x7 from the Gates and Crellin Laboratories of Chemistry of the Cali-

fornia Institute of Technology. "* Present address: Department of Biophysics, Roswell Park Memorial Institute, Buffalo,

N.Y. (U.S.A.).

Biochim. Biophys. Acta, 57 (I962) 2z2-2z9

FUNCTIONAL GROUPS IN THE GELATION OF GELATIN 2 2 3

weight resulted from ultrasonic treatment, so that no conclusion can be drawn as to Lhe importance of the ~anidino groups. P. DAvls ~ reported that alkaline hypobromite treatment of gelatin prevents gelation, but again no correlation with molecular weight was made. DAvis found that ~,kali alone did not prevent gelation. However, DAVIS' assumption that alkaline hypobromite does not degrade gelatin more than alkali alone is not correct 6. It has been suggested 7 that added guanidinium ions or compounds containing guanidino groups inhibit gelation by competing with the guanidino group of gelatin for available cross-linking sites. JANus 8 has studied the retardation of gelation by guanidinium compounds, but could not conclude that they behave as specific competitors for ~uar Minium receptor sites of the gelatin. JANUS 9 has also reported that the setting time of gelatins with amino groups converted to guanidino groups decreased, but that the rigidity after aging one day at o ° was not affected, i~dicating a "cataiytic" role of the guanidino groups in early stages of gelation. It has been reported t° that sodium in liquid ammo1~ia destroys both guanidino groups and gelling power without reduction of molecular weight. In private conversation with Dr. BERGER he informed us of additional osmotic data from which a molecular weight of about roooo was indicated. At this molecular weight, gelation of chemically un- modified gelatin hardly occurs except weakly at Ligh concentration. That the hydroxyl grou F may be involved in gelation is suggested by the correlation of the shrinkage temperature of collagen with hydroxyproline con~entU.lL Co~n~ TM has suggested that hydroxyproline is important ir~ the structure of gelatin films.

To determine if any of the polar sid~ ¢b~n groups of gelatin arc important in gelation we have prepared modified gelatins having each of the groups blocked by a small, non-ionizing substituent and have compared the gelation of these modified gelatins with unmodified but partially hydrolyzed gelatins of comparable molecular weight.

EXPERIMENTAL Materials

The gelatin used in this work was Wilson and Co.'s U-COP-CO, Special Non- Pyrogenic Gelatin, of isoelectric point 9.2, prepared by acid extraction of pigskins. Carboxyl-esterified t4, amino-acetylated 1~ and hydroxyl-acetylated t5 gelatins and the melting point method t6 have been described.

Guanidino-nitrated gelatins were prepared as follows: I. Five grams of lyophilized gelatin, vacuum dried for one day before use, was

dissolved in a mixture of 98 ml of concentrated sulfuric acid and 2 ml of 70% nitric acid at o* in a flask fitted with an airtight stirrer. After 45 min the solution was poured into r.51 of ether, pre-cooled in ice. The precipitated gelatin was filtered, washed with 3 × xoo ml of ether and 3 × ioo ml of acetone, suspended in Ioo ml of ice water and neutralized to pH 7 with 2 N sodium hydroxide. On neutralization the solution turned yellow. After week-long dialysis against daily changes of distilled water, the solution was lyophilized. The sulfur content was 3.57 ~/o, correstmnding to xz.7 moles of sulfate introduced/to 4 g of original gelatin, or 75 ~/o sulfation of the hydroxyl groups, corrected for the sulfur content of the original gelatin. Nitration of guanidino groups was 50% complete. The determination of the extent of nitration is given below.

2. In a second preparation, 70 % nitration was achieved using a mixture of 92 ml concentrated sulfuric acid and 8 ml of lOO% nitric acid. The extent of sulfation of hydroxyl groups was 36 ~/o.

Biorhim. Biophys..4aa, 57 (I96-') 222-229

: :4 J. BELLO, H. R. BELLO, J. R. VINOG~AD

The extent of nitration was determined by estimation of residual arginine by the SAKAGUCHI reaction. The variation of FRANKSTON AND ALBANESE 17 gave with un- hydrolyzed, 7o°//o nitrated gelatin only 5-xo~/o of the color intensity given by the original gelatin (corrected for the weight of substituents). When the test was carried out on hydrolyzed gelatins, no red color was obtained. As this may have been due to the formation of by-products that interfere with the color-forming reaction, paper chromatography was used to separate the arginine from the interference. About o.15 or 0.30 ml of solution, containir, g 0.35-o.7 mg of solute, was applied to each half of ao × 3 inch strips of Whatman No. 3 MM paper, folded lengthwise. The part i t ion mixtures were: (a) dicyclohexylamine--n-butanol-water (mixture M of HARDY a al.lS); (b) acetic ac id -e thano l -wa te r (mixture E of HARDY et al.lS); (c) valeric ac id-95 % ethanol-water (4: 2: I, v/v). After I6 h the strips were rinsed with acetone, dried in air, cut in two, and one strip sprayed with hypobromite reagent according to ACHER AND CROCKER 19. After locating the arginine spot on this half of the strip, the corresponding spot on the other half was cut out, cut into small strips and extracted with 5 ml of water at 5 °0 for 1.5 h. After filtration 3-ml aliquots were taken and the color developed with the SAKAGUCm reagent. The extent of nitration produced in the 70% nitrated gelatin as obtained with the three part i t ion solvents were 68, 70 and 72%, respectively. The 50% nitration product was parti t ioned with mixture M.

Molecular weights Weight average molecular weights were determined by the approach to equili-

brium method ~°. The gelatin sample was dissolved in, and dialyzed against, 2 M potassium thiocyanate before use. The concentration of gelatin was determined refractometrically using a specific refractive index increment of o.oo168 at z5 ° (see ref. 21).

The apparent part ial specific volume, l?apv, was determined at 25 ° at a concen- tration of about i°/~) gelatin in 2 M potassium thiocyanate; values of o.7o5-o.7II were found for both modified and unmodified gelatins, except for o.66 for the 7o% nitrated gelatin. Other reported values of part ial specific volume are o.68 for a lime process gelatin a2, o.695 for a soluble collagen of carp swim bladder m and ~.7o for acid pigskin gelatin ~4. The value calculated by COHN ANY EDSALL 25 from the IJ's of the amino acid residues of gelatin is o.7I.

A $pinco Model E analytical ultracentrifuge was used for the sedimentation studies. The optical arrangements and cells used were essentially as described by KLA~ER AND KEGELES ~. About 0.o2 ml of carbon tetrachloride was added to the cell when it was desired to photograph the bottom surface of the solution. The plates were evaluated from enlarged tracings. An average value for the molecular weight was obtained from data at the top of the cell from 2 or 4 photographs taken over a period ot xo--6o rain of centrifugation, depending on the speed. No drift in the results with time was observed. Data from the bottom of the cell were used in one instance only, which is referred to below.

Details of the molecular weight determination of the parent gelatin are given here. A series of runs was made at a constant speed of 8363 rev./min at a8 °. The results of these runs are represented by plus signs (+) in Fig. I where I/M is plotted as a function of concentration. One run was made without the temperature regulator, but allowing the machine to warm up for a period of two hours before the run. The

Biochim. Biophys. Acta, 57 (I96z) aaa-zz9

FUNCTIONAL GROUPS IN THE GELATION OF GELATIN 22~

molecular weight so obtained is indicated as a circled plus sign (®) in Fig. I. It is seen that there is no significant difference with and without the temperature control. This run also establishes the reproducibility of the results. Runs were also made at 12590 rev./min and 162oo rev./min to see whether a lower value for the molecular weight is obtained because of the fractionation of the solute through differential

1.5.

1,3,

I,I.

0.9-

O7-

0.5-

O.3-

O.I-

+

CONCENTRATtON, WEIGHT PERCENT

Fig. I. Ultracentrifuge molecular weight of parent gelatin. See EXPERIMENTAL for details.

sedimentation. These results are indicated by full circles (1259 ° rev./min) and triangles (162oo rev./min) in Fig. I. Since these points appear to fit the straight line obtained from the results at lower speed, it was concluded that at these speeds fracfionation did not affect the results significantly. In one run, the data at the bottom of the cell after 4o-12o rain of centrifugation at 8363 rev./min were also used to calcu- late the molecular weights. These values agreed with the molecular weights obtained at the top of the cell in the same photographs within the experimental error,, which is estimated to be i 5 % in the results reported here.

The plot of I / M vs. concentration (weight per cent) in Fig. I is seen to be linear. From an extrapolation of the plot to zero concentration the molecular weight was found to be 22oooo. The slope of the straight line was 6.1. IO -e.

Viscosities were measured at 4 °0 in an Ostwald type viscometer having a buffer flow time of about 24o sec for 5 ml of solution. The buffer used was a pH 5 acetate buffer containing o.i M sodium acetate and o. t5 M sodium chloride. Intrinsic viscosi- ties were obtained by e x t ~ o l a t i o n of double plots of ~]sp/c vs. C and log ~]sp vs. C.

RESULTS AND DISCUSSION

In order to determine whether blocking of a functional group affected the gelling power, it was necessary to compare the gelation of the modified gelatin with chemically unmodified gelatin of similar molecular weight, as gelation is known to be a function of molecular weight ~, m and as chemical modifications were accompanied by reduction

Biockim. Biophys. Acta, 57 (x902) 222-229

Z26 J. BELLO, H. Ro BELLO, J. R. VINOGRAD

in molecular weight. For the sake of convenience, it was desired to use the intrinsic viscosity as a measure of the molecular weights of the many samples prepared, rather than the more time consuming absolute measurements. Since the modified gelatins were chemically different from the original gelatin, it was necessary to determine whether the same viscoslty-molecular weight relation existed for both modified and unmodified gelatins. For this purpose the intrinsic viscosities and weight-average molecular weights of four unmodified gelatins of various molecular weights (prepared by hydro- lysis at pH 5 and xoo °) and of one specimen of each of the four modified gelatins were measured. The results are showh in Fig. z. The points, with the exception of the nitrated gelatin, fall close to a straight line, fitting the equation [7] ---= 4 .8. xo-4 M°'~e. These constants may be compared with those of 2. 9. Io -4 and o.62, (equilibrium ultraeentri- fugation) of WILLIAMS e~ al. 81 for low isoelectric point, limed calfskin gelatin.

i i i

°°r ,~o t_ - ~

• oo?

5o t Q

20 l F I I I

0.15 0.25 0.35 0.46

[~] , dl#11

Fig. 2. Molecular weight - viscosity relations: O, standard gelatins: 0 , 7o % of guanidino groups nitrated; ~, O-acetylated by HCIO 4 method ~ O, amino-acetylated ; ~), 8o % carboxyl-methylated.

It having been shown that the intrinsic viscosity may be used as a measure of molecular weight, the viscosities of the modified gelatins were plotted against melting points of the gels. The gels were stored at o ° for 2o h before determining melting point. At 5~/o concentration, little change in melting point occurs after longer storage 2s. The results are shown in Fig, 3. The viscosity-melting point relations of chemically unmodified gelatins oi different molecular weights were taken as standards repre- senting normal gelling behavior.

The carboxyl-esteHfied and amino-acetylated gelatins clearly have melting points equal to those of unmodified gelatins of similar molecular weight. These groups are, therefore, of little or no importance in 2o-h gelation of 5~/o gels.

The hydroxyl-acetylated gelatins fall into two groups: those acetylated with a mixture of acetic acid, acetic anhydride and perchloric acid and having normal or

~ " • I • (in one case) above normal melting points; and those acetylated with acetic an~iydride and TFA and having lower than*normal melting points. The gelatins ot both groups are 96 -xoo~ acetylated, except for the perchloric product of highest viscosity which is 8 7 ~ acetylated. The differences in viscosity between gelatins acetylated to the

Biochim. Biophys. Acta. 57 (I96",) 2-,2-229

FUNCTIONAL GROUPS IN THE GELATION OF GELATIN 227

same degree by the same reagent arise from differences in the time of reaction and amount of anhydride. The TFA products form a curve parallel to the normal curve. The difference in gelation behavior of the two types of O-aeetylated gelatins may be the result of a rearrangement in one of them. A rearrangement might interfere to some extent with formation of a conformation required for gelation. It is, therefore, more likely that the rearrangement, if any, has taken place in theTFA products.This anom- aly is being investigated by deacetylatiort with dilute alkali Some preliminary data are shown in Table I. Comparison of the melting point-viscosity data of Table I with Fig. 3 shows that deacetylation restores tlie TFA product to the main line, suggesting a reversal of a rearrangement.

TABLE l D I d A C F , T Y L A T / O N O N t l Y D R O X Y L - A C E T ' I ' L A T E D GELATIN

•

.~;dl :d Of Per ee:lt Melting point acetylation acetylated ," ~11 before dllg a]teP"

before after

TFA Ioo 0.'7 o.~5 15~4 r5.4 HCIO4 87 0.35 0.25 27.o 24.1

The low melting points of the sulfated gelatins are probably due to the intro- duction of sulfate groups or to acyl shifts rather than to blocking of hydroxyls. That the nature of the group introduced may affect gelation is evident in that acetylation of amino groups does not affect the melting point, while succinylation of the same groups lowers the melting point, perhaps because of charge or bulk.

In the case of the guanidino-nitrated gelatin, there are two preparations: one 5O~'o nitrated and 75~/o hydroxyl-sulfated and the other 70% nitrated and 36% hydroxyl-sulfated. The gelling power of the 5o~,, nitrated, 75 ~,, hydroxyl-sulfated gelatin is less than that of normal gelatin, but is, however, the same as that of the non-nitrated gelatin quantitatively sulfated by the method of REITZ et al. a°. More important, the 70°//0 nitrated, 36% hydroxyl-sulfated gelatin has normal gelling power based on its viscosity. Fig. 2 suggests that the 70% nitrated gelatin has a low viscosity for its molecular weight. Therefore, the gelling ability of this material may be somewhat lower than indicated by Fig. 3. This may be due wholly or in part to the sulfate groups. I t appears that the guanidino groups are of relatively little importance in the equilibrium gel, although they may play a part ~ in the early stages. The. re- maining guanidino groups are probably of little importance in gelatio,.; since they are difficult to nitrate, they are probably inaccessible to complementary cross-linking sites.

In a previous paper ~ we reported that the effects of ions on the m~lting points of gels of gelatin and modified gelatins is not due to binding at the polar and charged side-chain groups. From the latter and the result.q reperted here, we conclude that none of the ionizing or polar side-chain groups is of importance in tl:e gelation of gelatin at equilibrium. The other groups to be considered are the non-polar side chains and the peptide groups. There is no evidence concerning the non-polar groups in gelation. While the number of unquestionably non-polar side chains is known (alanine, valine, leueine and proline), the side chains of the hydroxyamino acid, dicarboxylic acids, diamino acids and arginine may act as polar groups or may be arranged, at least

Biochim. Biophys. ,4cta, 57 (t96z) 2:2-229

228 J. BELLO, H. R. BELLO, J. R. VINOGRAD

in the cross-linking sites, so that their polar portions are hidden and the non-polar sections are exposed and arranged in ways appropriate for intermolecular associations.

Tile chief remaining functional group, and present in the largest proportion, is the peptide group. Evidence for the participation of the peptide group in gelation has recently been briefly presented in the demonstration that blocking of these groups by biuret-type complex formation with copper (II) ion completely prevents gelation3L

I I I I I

• .Q-@50~o

- - olz I I I • , o.~ 0 . 4 0 . ,~

[ ,7 l , d~,'~

I I I

I o . o t 0 . 0 2 o , o s

[ c u ] m o l * ~ i l * ,

Fig. 4. Melting points of gelatin-copper com- plexes.

Fig. 3. Melting point-viscosity relations; O, standard gelatins; O, guanidino nitrated; m, hydroxyl groups quantitatively sulfated; 0D, hydroxyl-aeetylated by perchloric method; t), hydroxyl-acetylated by TFA method; Lq, aminoacetylated; A, carboxyl-methylated with

methanol-hydrochloric acid; &, carboxyl-methylated with methanol-thionyl chloride.

Breaking the complex by mild acidification or ion exchange resin allows gelation to take place. The effect of copper (bound as the peptide complex) at p H I I on the melting point of 5~)/o gelatin gels is shown in Fig. 4. Nickel has a similar effect. The mechanism of inhibition of gelation by copper may be one of the following: (a) preventing inter- or intra-molecular hydrogen bonding between peptide groups by occupying the hy- drogen bonding sites; (b) changes in chain geometry resulting in steric interference with chain conformation or inter-molecular association. The shape of the curve of melting point vs. copper concentration conforms to the concept of a required con- formation in gelation because at about o.o2 M copper (II) the melting point begins to decrease very rapidly, as though the conformation could no longer be maintained after a critical amount of derangement had occurred. An alternate possibility is that gelation ceases when specific peptide groups have been occupied.

ACKNOWLEDGEMENTS

We wish to thank Dr, R. SRINIVASAN and Mr. M. CURLEY for the determinations of molecular weights. This research was supported under contract No, DA-49-oo 7- MD-z98 with the Office of the Surgeon General, Department of the Army.

Biochira. Biophys. Acta, 57 (196")) a22-2a9

FUNCTIONAL GROUPS IN THE GELATION OF GELATIN 2 2 9

R E F E R E N C E S

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1956, p. r37, 8 j . W. JANus, in G. STAINSBY, Recent Advances in Geiatin and Glue Research, 4~ergamon Press,

New York, I958, p. 27-'. g J, W. JANus, in G. STAINSBY, Recent Advances in Gelatin and Glue Research, Pergamon Press,

New York, 1958, p. 2I 4. 1o A. BERGER, J. KURTZ AND J. Nooucn t , in G. STAINSBY', Recent Advances in Gelatin and (Hue

Research, Pergamon P re~ , New York, 1958, p. z7t . 11 T. TAKAHASHI AND T. TRAVAEA, Bull. japan Soc. Sci.,Fisheries, 19 (i953) 6o3. 1,, K, H, GUSTAVSON, Acta Chem. Scand., 8 (19.55) iz~8. is C. CONES, J. Biophys. Biochem. Cytol., t (t955) 2o3. 14 j . BELLO, Bioehim. 13iophys. Acta, 2". (1956) 426. 15 j . BELLO AND J. R. VtNOtIRAO, J. Am. Chem. Soc., 78 (1956) t369. 18 J. BELLO, H. RII~SE AND J. R. VINOGRAD, J. Phys. Chem., 60 (1956) 1229. l? A. A. AI.BANF, SE AND J. B. E. FRANKS'tON, J . Biol. Ctwm., I59 (1945) 185. 1~ T. L. HARDY, D. O. HOLLAND AND J. l-l. NAYL~R, Anal. Chem., 27 (1055) 971. to H. ACHER AND C. CROCKER, Biochim. Biophys. Acta, 9 {1942) 7o4. ,0 S. M. KLAINER, G. KEGELES AND W. J. SALE. t ' , J. Phys. Chem., 6x (1957) t786. ~t j . w . WILLIAMS, W. M. SAUNDERS AND J. S. CICIRELLI, J. Phys. Chem., 58 (19541 774. v, E. O. KRAEMER, J. Phys. Chem., 45 (1948} 66o. '~l H. BOEDTKER AND P. DOTY, J. Am. Chem. So¢., 78 (I956) 4.267. ~,t p. A. Ct-IARLEW'OOD, J. Am. Chem. Soc., 79 [1957) 776. 2.~ E. J. CoRN AND J . T . EDSALL, Proteins, Amino Acids and Peptides as Ions and Dipolar Ions,

Reinhold Publ i sh ing Corp., New York, I943, p. 375. a8 S. M. KI:AINER AND G. KEGELES, Arch. Biochim. Biophys., 63 (1956) 247. 27 j . E. ELDRIDGE AND J. D. FERRY, J. Phys. Chem., 58 (1954) 99z. is j . POURADtER AND A. M. VENE'r, J. Chim. Phys., 47 (195 o) 391. n j . BELLO, H. RIESR AND J, R. VINOGRAD, J. Phys. Chem., 69 {1956) I;'99. ao t t . C. REITZ, R. E. FERREL, H. FRAENKEL-CONRAT AND H. S. OLCOTT, J. .4nL Chem. Soc.,

68 (1946) to24. at j . BELLO AND J. R. V[NOGRAD, Nature, 18[ (1958) 273.

Bioehim. Biophyo. Acta, 57 (196-') zzz-2zg,