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THE JOURNAL OF BIOLOGICAL CHEMISTR YVol. 238, No. 1, January 1963Printed in U.S. A.

The Action of Hydrazine on CollagenI. CHEMICAL MODIFICATION*

ROGER DE LA BURDE,? LEONARD PECKHAM, AND ARTHUR VEIS$From the Armour Leather Company and Northwestern University Medical School, Chicago, Illinois

(Received for publication, July 16, 1962)

The current view o f the collagen fibril (1) pictures it as anordered aggregate of collagen rods, which themselves consist ofthree intertwined peptide chains. The macromolecular mono-mer unit rods, tropocollagen, appear to be packed in the sameway in all collagens regardless of the origin of the collagenoustissue or its age. The variations in the properties of the colla-gens have been ascribed in part to the presence of covalent cross-linkages between tropocollagen units (2-10) (inter-TC).l Thereis also good evidence for the existence of covalent cross-linkagesbetween the individual peptide chains within the tropocollagenmolecule (3, 6, 11, 12) (intra-TC). Some of the current im-portant questions in the chemistry of the maturation of collagenare those of the chemical nature of the various cross-linkages,their number, and their distribution.Several investigators have proposed that the cross-linkages incollagen are ester-like in character (10). The first direct evi-dence came from the work of Grassmann et al. (8), in which itwas shown that lithium borohydride reductively cleaved a smallnumber of covalent bonds in citrate-soluble cal f skin collagen.Konno and Altman (9) reported the isolation of a glycine-carbo-hydrate complex, reducible with lithium borohydride, from ratmuscle collagen. Major support for the presence of ester-likelinkages came from the work of Gallop et al. (7), who reportedthat hydroxylamine and hydrazine reacted with gelatins toproduce protein-bound hydroxamate and hydrazide, respec-tively . These investigators relied on the fact that. both reagentsreact much more rapidly with simple esters than with simpleamides or peptides in proposing that the hydrolyzable bondswere ester linkages. They found that with acid precursor gela-tin,2 or the gelatin produced by the denaturation of calf skin or

* This work was performed in the Central Research Laboratoryof Armour and Company.t That part of the work carried out by R. de la Burde was usedfor the preparation of a dissertation in partial fulfil lment of therequirements for the degree of Dr.-Ing. at Technische HochschuleAachen, at which he had completed other requirements beforecoming to the United States. He wishes to thank the ArmourLeather Company for encouraging this work and permitting pub-lication and Prof. H. Zahn, of Aachen, for acting as his sponsorand assisting in the preparation of the dissertation.$ Inquiries should be addressed to Dr. Arthur Veis.1 The abbreviation used is: TC, tropocollagen.2 Commercial gelatins are manufactured by either o f two pro-cedures. Pretreatment of collagen with acid, followed by neutralor acid extraction, yields the acid-precursor gelatins. Pretreat-ment with a base, usually Ca(OH)z, followed by neutral extrac-tion yields the alkali-precursor gelatins. One essential distin-guishing feature is that the acid-precursor gelatins haveisoelectric points near pH 9 while the alkali-precursor gelatinshave isoelectric points near pH 5. This difference is the result of

ichthyocol TC, 6 moles of hydroxamate were formed per lo5 g ofprotein. The gelatin was degraded to units with molecularweights near 20,000. Since there seemed to be neither a decreasein the number of amide groups in the gelatin, nor any increasein NHz-terminal amino acid residues, it was concluded thatnative collagen and acid-pretreated gelatins contained 6 x 10-cequivalent of weak ester-like linkages per g. Alkali-pretreatedgelatins, from which approximately one-half of the amide groupcontent was removed during pretreatment, formed only approxi-mately 3 X lo+ equivalent per g of bound hydroxamate. How-ever, in this case, the molecular weight was still reduced to avalue near 20,000.The reduction of the molecular weight of the individual or-pep-tide chains, with the concomitant formation of bound hydroxa-mate, led Gallop et al. (7) to postulate that the ester-like link-ages were intrachain bonds, that, is, part o f the gelatin back-bone. From this point of view, the ester linkages could not beinvolved in intra-TC or inter-TC cross-linking. Gallop et al.(13) did, however, suggest that the ester linkages occurred inpairs in the a-chains and that an isomerization and rearrange-ment of bonds could establish ester-like cross-linkages.The hydrasine and hydroxylamine reactions were carried outby Gallop el al. (7) at pH 9 or pH 10 in the range of 1 to 3 Mreagent concentrations. Under these conditions, high concen-trations of urea or thiocyanate were required for the reaction toproceed at an appreciable rat.e at room temperature. The reac-tions could be carried out in the absence of the urea at 40.

Bello (14) attempted to apply these same reactions to fibroussteer corium collagen as well as to an acid precursor gelatin. Inhis experiments, the acid precursor gelatin yielded 0.5 to 1.5 Xlop5 equivalent of bound hydroxamate per g of collagen, andthe insoluble corium collagen, presumably more highly cross-linked, only 2 x 10e5 equivalent of hydroxamate per g. Therewas no way to reconcile his data for the acid precursor gelatinwith that of Gallop et al. (7). Bello found, in addition, that theinsoluble collagen would not react at all with hydroxylamine orhydrazine (pH 8.6) unless the collagen was denatured beforetreatment with the base. Bello suggested that all the boundhydroxamate could be accounted for by the presence o f carbo-hydrate in his collagen preparations. Hormann (15) also ex-amined the reaction o f hydroxylamine with fibrous collagen.He confirmed that the collagen had to be denatured before itwould react. Hijrmann et al. (16) found that, on the average,mature steer corium collagen yielded 11 X lop5 equivalent ofbound hydroxamate per g of collagen. They also treated solu-partial deamidation of the alkali-precursor gelatin stock duringthe pretreatment step.

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190 Action of Hydrazine on Collagen. I Vol. 238, No. 1TABLE 1

Characterization of purified and limed collagenAnalysis

1. Content of solu-ble compo-nentsNeutral salt-solubleAcid-soluble2. SwellingpH 2.5pH 5.33. Total nitrogen4. Amide nitrogen

5. Arginine 8.43-8.55 8.47-8.526. Citrulline Negative Negative7. Hydroxyproline 12.9 12.98. Shrinkage tem-perature 62-64 58-59

Purified Liedcollagen collagen%

0.040.59

415 515310 32518.08 17.820.66 0.42

Method

Weight and hy-droxyproline(Neuman and Lo-gan (22))

Solution uptake(Bowes (23))Kjeldahl analysisNH3 in partial hy-drolysate (Casseland Kanagy (24))Rosenberg et al. (25)Fearon test (26)Neuman-Logan (22)

ble TC with hydroxylamine and found 6.8 X 10e5 equivalentof bound hydroxamate per g. They therefore concluded thatthere were 4 moles of ester-like linkages per 10-S g of insolublecollagen which participated in inter-TC linkages. Followingthe hydroxamate formation, carried out with 0.75 M NHQOH in4 M LiCl at 37 and pH 9.55 for 10 hours, the collagen was com-pletely dissolved and degraded, presumably to an average mo-lecular weight near 20,000. Both the collagen and TC con-tained a total of approximately 4 X IO-+ mole of glucose or glu-cose and galactose per g. Hormann (17) concluded that thecross-links in collagen consisted of hexoses whose two hydroxylgroups formed ester links between the different peptide chainswhile the hexoses in tropocollagen were bound by only a singleester group. The reducing group of the hexose was supposed tobe attached to a hydroxyl side chain of one of the backbone pep-tide units by a glucoside-like oxygen bridge.

All o f these data provide substantial evidence for the presenceof hydroxylamine-sensitive, ester-like linkages in collagen butdo not provide conclusive proof that the ester linkages partici-pate in inter-TC unit bonding. The dissolution of the maturecollagen fibers after hydroxamate formation could conceivablyoccur merely due to the extensive degradation of the individualbackbone peptide chains and not because of the cleavage of in-terchain bonds. A clarification of this question would appearto require the selective cleavage o f the inter-TC bonds whileleaving intact the intra-TC bonds of weak ester-like character.If one accepts the premise that the inter-TC bonds are esterbonds as Hormann proposes (15-17), it is diff icul t to see whythese groups would not be readily accessible for reaction withhydroxylamine or hydrazine.

Veis and Cohen (2, 18) and Veis et al. (3, 11) showed thatcollagen fibers could be selectively degraded in acid or at neutralpH to yield soluble fractions with nongaussian molecular weightdistributions and containing substantial numbers of moleculeswith both the peptide backbone chains and the intra-TC cross-

linkages in tact. These molecules could be reversib ly denaturedand renatured. Courts (19) showed that treatment of maturecorium collagen with acid in the cold, followed by prolongedsoaking in Ca(OH)2 in the presence of salts that reduced swell-ing, also appeared to lead to the selective rupture of inter-TCbonds. Collagen fibers treated in this way were soluble in colddilute acetic acid to give high viscos ity solutions very similar toTC in behavior. Fibrous precipitates of this soluble collagencould be prepared by dialysis (20). These data illustrate theaccessibi lity of the inter-TC bonds to both acid and alkalinehydrolysis but do not indicate their chemical nature.

In a search for a system in which the inter-TC bonds could becleaved in fibrous collagen without denaturing the collagen struc-ture, we found that hydrazine, in contrast to hydroxylamine,would react with native collagen under appropriate conditions.We therefore investigated the chemical and physical changesoccurring in purified native steer corium collagen, and Ca(OH)2-soaked, but otherwise undegraded steer corium collagen follow-ing reaction with hydrazine. This report describes the chemicalaspects of the collagen-hydrazine reaction over an extendedrange of hydrazine concentration.

EXPERIMENTAL PROCEDUREPreparation of Collagens-Purified steer corium collagen was

prepared from fresh steer hide by the method of Veis, Anesey,and Cohen (3). The limed collagen was obtained from a simi-lar hide subjected to a 5-day lime treatment. This material waspurified by the method of Bowes and Kenten (21). Both col-lagens were lyophilized for storage. A number of determinationswere made to characterize the collagens. These data are sum-marized in Table I. The short liming process did not decreasethe arginine content of the collagen, and there was no indicationof citrulline formation. The principal result of the liming wasto reduce the amide content by 36.4%.Preparation of Hydra&e-Hydrazine (Olin Mathieson Chemi-cal Corporation, 95 + %) was redistilled and used as a standardfor the preparation of hydrazine solutions of different concentra-tions. The concentration of the standard was determined colori-metrically by the method of Pesez and Petit (27) and was keptin the range o f 96 to 98%. Dilutions of this stock solutionwere made in an acetone-Dry Ice bath to avoid overheating.Since aqueous hydrazine easi ly undergoes autoxidation, whichcan be accelerated by dilution, by the addition of NaOH, or bythe presence of trace metals (2830), the change in concentra-tion of 10 and 70% aqueous hydrazine on standing in closedglass containers at room temperature was determined. Therewas no significant decrease in hydrazine concentrat ions within 6days.Reaction of Collagen with Hydra&e-Lyophilized collagen wastreated with aqueous hydrazine at 25 =t 2 for 30 hours. Aratio of 1 weight of collagen to 10 volumes of hydrazine solutionwas used. The moisture content of the collagen (-9%) did notcause an increase in the solution temperature on mixing. Thesereaction mixtures were shaken in glass-stoppered Erlenmeyerflasks, When solutions above 58.8% hydrazine were used, awax coating was applied to the stoppers.At the end of the reaction period the collagen was washedwith many changes of cold distilled water (-7) for 3 to 4 days.The washings were monitored for the presence of hydrazine asfollows. After each 20 hours of washing, 2 g of collagen werewashed separately for 3 hours in 50 ml of distilled water. This

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January 1963 R. de la Burde, L. Peckham, and A. Veis 191washing solution was tested qualitatively with salicylaldehyde(31) for the presence of hydrazine. After the first negative test,the protein was washed for another 30 hours and then lyophil-ized. The hydrazide content of the collagen was determinedby the method described by Seifter e t al. (32). Crystalline hy-drazine sulfate, the reaction product of hydrazine hydrate andexcess sulfuric acid, was used for the preparation of the hydra-zine standards.It was noticed that the amount of protein-bound hydrazideappeared to decrease with prolonged washing. To determinethe extent of this loss, samples of collagen treated with 40y0 and70 $Zo ydrazine were extracted with repeated changes of absoluteethanol for 40 hours to remove the free hydrazine. The spot testwith salicylaldehyde indicated the absence of free hydrazine inthe final rinse. The extracted collagens were rinsed with waterto remove the ethanol, lyophilized, and then subjected to theusual aqueous wash procedure. The hydrazine content was de-termined at intervals during a 70-hour wash. These data areshown in Fig. 1 and indicate a gradual decrease in protein-boundhydraaide that could decrease the values reported subsequentlyby5tolO%.Arginine Detemination-Half-gram portions of collagen werehydrolyzed under reflux for 16 hours in 10 ml of 20.5% HCl.The hydrolysates were analyzed by the method of Rosenberget al. (25). The presence of hydrazine in the amounts expectedfrom the hydrolysis of the bound hydrazides (up to 90 x 10m5equivalents of NHfNHz per g) did not affec t the arginine analysis.Citrulline Analysis-The hydrazine-treated collagens wereexamined for the presence of citrulline, one of the possible break-down products of arginine. The method described by Fearon(26) was used, and no citrulline was found.Amide Analysis-The amide content of the collagens wasdetermined as the amount of ammonia distilled from partial acidhydrolysates in the presence o f excess sodium and calcium hy-droxide (24). A comparable standard hydrazine solution wasprepared for each of the distillates, according to the hydrazidecontent. The steam distillates of these solutions were titratedas in the amide analyses. In each case, the amount of steam-distilled hydrazine that could have accompanied ammonia in thecollagen distil lates was negligible and could not be detected bytitration.

Amino Acid Analyses-Purified collagen and the residual andsoluble fractions resulting from 40 and 70 7O hydrazine treatmentwere hydrolyzed and examined for their amino acid content by aquant itative paper chromatographic technique (33). Particularattention was given to the ornithine content. The standardamino acid mixtures contained ornithine as well as the aminoacids present in collagen. The presence of ornithine increasedthe color yield of the lysine. Correction factors were computedfor various concentrations of ornithine. These were obtained byrunning the standard amino acid mixture with ornithine con-centrations ranging from 0 to 6.02%.

NHz-terminal Amino Acids-Purified native collagen treatedwith 50% hydrazine was dinitrophenylated under the conditionsdescribed by Lev y (34). The dinitrophenyl derivati ves wereidentified qualitatively by paper chromatography.

RESULTS

The Hydrazine SystemThe reaction systems examined ranged from 0 to 50% hydra-zine, corresponding to 0 to 21.8 M. The basicity of the solutions

rGs5z 40, I I I I I I I I II I 8 16 24 32 40 48 56 64 72

DURATION OF WATER WASH, HOURSFI G. 1. Decrease in protein-bound hydraaide during the wash-ing procedure. A---A, collagen treated in 7070 hydrazine;

O-O, collagen treated in 40% hydrazine. The upper figureson the ordinate refer to the 707, and the lower to the 4070 hydra-zine-treated collagens.varies continuously over this range. Deno (35) has shown thatthe Hammett function, H- is a linear function of the hydrazineconcentration. H- increases from a value of 10.75 for an in-fini tely dilute hydrazine solution to 15.93 at 18.72 M (60%).In the overlapping range up to pH 14, the H- and pH functionshave identical values and, therefore, the pH is also a directlinear function of the hydrazine concentration. In the ensuingdiscussion, all the data could have been presented in terms ofH-, or pH, but are usually presented in terms of the hydrazineweight concentration since this is a more easi ly appreciated unitand was the experimental variable.

Reactions during HydrazinolysisFormation o f Protein-bound Hydrazide-Hydrasine was found to

react with the undenatured fibrous collagens to yield protein-bound hydraaide over the entire hydrazine concentration range.The amount of protein-bound hydrazide in the native and limedcollagen is shown in Fig. 2 as a function of the hydrazine solu-tion concentration. The two striking features of these data arefirs t, that the native collagen reacted with substantially morehydrazine than did the limed collagen at. each hydrazine con-centration, and second, that in the 40 to 50% hydrazine concen-tration range (H-, 14 to 15) there was an abrupt rise in theamount of hydrazide bound to the native collagen, whereas thisdid not take place with the limed collagen.

The collagen fibers did not appear to be denatured by theirtreatment and reaction with hydrazine. Electron micrographsof the limed fibers, the native hydrazine-treated fibers, and thelimed, hydrazine-treated fibers showed all of the cross-striationsand periodicity of structure characteristic of native collagen aslong as the hydrazine reaction solution concentration was < 55 %.The original limed collagen, however, showed some evidence fordecreased structural integrity in having a shrinkage temperaturerange of 58-59 as compared with the native collagen 62-64Orange (Table I). Amorphous, nonstriated, treated collagenfibers appeared at hydrazine concentration > 60%. This didnot coincide with the 40 to 50% hydrazine concentration rangein which the native collagen showed its enhanced formation ofbound hydrazides.

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192 Action of Hydraxine on Collagen. I Vol. 238, No. 1

0 IO 20 30 40 50 60 70HYDRAZINE CONCENTRATION, %

FIG. 2. The formation of bound hydrazide by collagen as afunction of hydraeine concentration. O-O, native purifiedcollagen ; O-0, limed collagen.

3632262420

1 t I I I I I0 IO 20 30 40 50 60 70

HYDRAZINE CONCENTRATION, %FIG. 3. The amide group content of hydrazine-treated collagen .

O--O, native purified collagen ; O-0, limed collagen .

Deamidation-The amide analyses (Fig. 3) showed that theamide contents of both the native and limed collagens were de-creased during hydrazinolysis and that the decreases weredirectly proportional to the hydrazine concentration. An un-expected resul t, however, was that the treatment of the nativecollagen with 60% hydrazine removed only an amount of amideequivalent to that removed by the 5-day liming. The residualamide in the limed collagen was not very reactive. These dataclearly suggest that two groups of amides exist in collagen andthat approximately 640/, of the amides are situated in such away as to be sterically inaccessible to the hydrazine, or are heldby particularly strong bonds. The correspondence between theextent o f deamidation and the basic ity of the reaction mediumprobably indicates that the amide removal is a general base-catalyzed reaction and is not governed by any specif ic reactiv ityof amide bonds with hydrazine. However, in the presence o f anucleophilic reagent such as hydrazine, every amide hydrolyzed

I I I I I I ! I0 IO 20 30 40 50 60 70

HYDRAZINE CONCENTRATION, %FIG. 4. The arginine content, o f hydrazine-treated collagen .

O-O, native purified collagen ; a-0, limed collagen .TABLE II

Arginine and ornithine content of native collagen afterreaction with hydrazine

Hydrazineconcentration Collagen fraction Arginine* Ornithinet% moles/l0~ g

0 Native fibers 48 040 Insoluble residue 45 2.570 Insoluble residue 23 3340 Soluble, nondialyz- 23 33

able70 Soluble, nondialyz- 8 53

able* Determined by direct analysis, method of Rosenberg et al.(25).t Determined by paper chromatography (33). This method i ssubject to a larger experimental error than the arginine analysis.These data seem to be consistent.ly high by approximately 157,.

will react to form a protein-bound hydrazide except at very lowhydrazine concentrations.

Deguanidination-Analyses of the arginine content of the twocollagens, illustrated in Fig. 4, showed that at high hydrazinesolution concentrations (hydrazine > SO%, H- > 15) the ar-ginine content was decreased. The reaction was that of con-version o f the arginine to ornithine and, as shown in Table II,there was reasonably good quant itative correlation between thedecrease in number of residues of arginine and the number ofresidues of ornithine created. There was no indication of citrul-line formation, and in accord with the work of Hamilton andAnderson (36)) it is safe to conclude that the direct deguanidina-tion of arginine was the principal reaction in the presence ofhydrazine. The deguanidination reaction also resulted in thefolmation of several amino-substituted guanidines and of sym-diaminotetrazine. This interesting aspect of the chemistry ofthe reaction system will be described elsewhere.3

The deguanidination reaction does not involve the formationof protein-bound hydraaide, as does the cleavage of an amidebond. Therefore, although the deguanidination reaction began

3 R. de la Burde and A. Veis, in preparation.

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January 1963 R. de la Burde, L. Peckham, and A. VeisI t I I t I

ZO-

0 10 20 30 40 50 60

WDRAZINE coNcENmA TioN , %

193

FIG. 5. The number of peptide bonds cleaved in the residue fibers of hydrazine-treated collagen. O--O, native collagen; O--Olimed collagen.to be signi ficant in the same hydrazine concentration range as thevery rapid rise in bound hydrazide in the native collagen, thesetwo reactions were not directly related.

Hydrazinolysis of Peptide Bonds-Bradbury (37) determinedthe kinetics of the hydrazinolysis of simple peptides and foundthat the rate o f cleavage of peptide bonds was finite in anhydroushydrazine even at 2, the freezing point of hydrazine. Rees andSinger (38) also reported that there was a continuous decreasewith time of the specific viscos ity of a sample of y-globulin inanhydrous hydrazine at 25 and that this was due to peptidebond hydraz inolysis. We therefore examined the hydrazine-treated collagen for evidence of peptide bond hydrolysis andchose the 50 To hydrazine-treated native collagen for this analysis.At this hydrazine concentration, the number of bound hydra-aides was almost at its maximal value of 40 X 10m5 mole boundper g, whereas only approximately 0.3 of the arginine residueshad been deguanidinated and there had been no dramatic in-crease in amide removal. Paper chromatography of hydroly-sates of the fluorodinitrophenylated, 50% hydrazine-treatedcollagen showed the presence o f glycine, aspartic acid, andglutamic acid as new NHn-terminal residues. Semiquantita-tively , the NH2-terminal glycine was present in largest amountand glutamic acid in the smallest amount. These data are inaccord with those of Bradbury (37, 39) who found that peptidebonds containing glycine were particularly susceptible to hy-drazinolysis, and that, in addition, bonds involving serine, glu-tamic acid, and aspartic acid were also especially labile in insulin,Iysozyme, and wool.The number of peptide bonds cleaved by the hydrazinolysiscan be determined by subtracting the number of moles of boundhydrazide formed b y amide replacement from the total hydraaidecontent. Fig. 5 shows the results o f such a calculation for boththe native and limed collagen. At the maximum, approximately22 x lOA mole of peptide bonds are cleaved per g of nativecollagen. At the same hydrazine concentration, only 3 x lop5mole of peptide bonds per g were converted to the hydrazide in

the limed collagen. These data indicate that approximately19 X 1O-6 mole of peptide bonds per g collagen were hydrolyzedduring the liming process and that the same bonds were appar-ently involved in both liming and hydrazinolysis. Bowes (23)found evidence for an increase in both basic groups and fluoro-dinitrobenzine-reactive groups in limed collagen. She ascribeda portion of these to newly created residues from peptide bondhydrolysis and the remainder to the presence of ornithine result-ing from the deguanidination of arginine. In this study , thenative and limed collagens had the same arginine content andall of the increase in reactive groups appears to be related to thehydrazinolysis of particularly labile peptide bonds. The ideathat a set of such bonds exists arises because of the clear-cutmaximum in hydrazide formation at 55% hydraaine (Fig. 2).

Dissolution during HydrazinolysisAs the hydrazinolysis reaction proceeds, a number of physical

changes indicate a decrease in the structural integrity of thecollagen fibers. The fiber tensile strength decreases, the swell-ing increases, and part o f the collagen dissolves. Each of thesealterations will be discussed in detail in a subsequent report.For the present purpose, we can take the extent of dissolution asa measure of the degree of rupture of cross-linkages as well as ofbackbone peptide chain hydraz inolysis. The jilled circles inFig. 6 represent the solubility of the native collagen as a functionof hydrazine treatment. The limed collagen (open circles) wassomewhat more readily dissolved at low hydrazine concentra-tions, but in both cases the amount of collagen dissolved in-creased most. rapidly at hydrazine concentration greater than70%. The rapid increase in ease of dissolution, in other words,did not coincide with the abrupt increase in content of boundhydrazide at 40 to 50% hydrazine (Fig. 2) but followed the samecourse as the deguanidination reaction, As shown in Table II,the soluble fractions had a much lower arginine content than theinsoluble fibrous residues and a correspondingly higher amount o fornithine. This observation suggests that the arginine residues

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Action of Hydrazine on Collagen. I Vol. 238, No. 1

0 to 20 30 40 50 60 70 00 so 100HYDRAZINE CONCENTRATION. %

FIG. G. The disso lution of collagen during reaction with hy-drazine. O-O, native purified collagen; O----O, limed col-lagen.52

1264

0 IO 20 30 40 50 60 70HYDRAZlNE CONCENTRATION, %

FIG. 7. A summary of the modification of native purified colla-gen. All points except the open circles refer to the Zeft-hand ordi-nate. l ----0, moles of bound hydrazide; O-U, moles ofamide removed; V-V, moles of arginine destroyed; O--O,dissolution of the collagen.may play an important role in maintaining the insolubility of theintact collagen structure.

DISCUSSIOli AND COiTCLU SIONSThe results reported in the foregoing are fai rly clear-cut andunequivocal. The objective of this discussion is 2-fold: 17-ewish

firs t to correlate the state of intermolecular bonding in nativesteer corium collagen with its hydrazine react ivity and chemicalmodification; second, we wish to relate our data to the studies ofothers on the hydroxylamine and hydrazine reactivi ty of systemscontaining denatured fibrous collagens or gelatins. Because ofpossible chemical differences in collagens from different sources,and because of the differences in reaction conditions, this latter

comparison may be considered more speculative in nature. Theensuing discussion is, therefore, somewhat artific ially divided intotwo sectionsReaction of Native Fibrous Collagen with Hydra&e

Evidently, three reactions take place when fibrous undena-tured collagen is treated with aqueous hydrazine: deamidation,peptide bond hydrazinolysis, and deguanidination of the argi-nine. The firs t two reactions result in the formation of protein-bound hydrazide, while the deguanidination reaction does not.The extent of deamidation is directly proportional to the hy-drazine solution concentration over the entire range examined.The peptide bond hydrazinolysis becomes important only whenH- = 14 and is complete, at room temperature, when H-reaches 15. The deguanidination reaction becomes significantwhen H- > 15. As the hydrazinolysis reactions proceed, thefibers begin to dissolve, and they pass rapidly into solution atH-. > 16. The course of each of these reactions is summarizedin Figs. 7 and 8 for the native and limed collagens, respect ively.

At hydrazinc concentrations 400/& and the concomitant appearance of new

32 1 I I 4 I II -IGO

HYDRAZlNE CONCENTRATION , %

FIG. 8. A summary of the modification of limed collagen. Al lpoints except open circles refer to left-hand ordinate. O--O,moles of bound hydrazide, t-W, moles of amide removed;V-V, moles of arginine destroyed; O--O, dissolu tion ofthe col lagen.

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NH*-terminal residues, indicates the presence of some particu- formed on the reaction of aldehydes with proteins. Landucci,larly labile peptide bonds in native collagen. There must be a Pouradier, and Durante (46) claim that aldehydes occur in alllimited number of such bonds since the enhanced hydraeide collagens and gelatins. The major portion of these aldehydesformation levels of f at hydrazine concentrations > 55% and are thought to be bonded to the peptide chains and in alkali-since the limed collagen, in which these bonds have presumably labile cross-linkages. The existence of very stable inter-TC andalready been cleaved, shows no such increase in bound hydrazide. intra-TC cross-linkages also fi ts in well with the data of CourtsThe peptide bond hydrazinolysis occurs at an H- o f 14 to 15 and Stainsby (47) and Veis, Anesey, and Cohen (11,48). Theseand that the basici ty of the liming solutions approximates 14. workers have each presented evidence that both alkali- and acid-It is possible that the enhanced hydrazide binding includes precursor gelatins of high molecular weight can be obtainedhydrasides formed from the cleavage of ester linkages. The under fa irly drastic conditions and that these high molecularNH*-terminal amino acid residue content was not determined weight gelatins are cross-linked multichain structures. Somequantitative ly or directly. However, the hydrazinolysis reac- peptide bonds were cleaved during the preparation of these gela-tions in the H- 14 to 15 range do not lead to the marked solu- tins (47), but the chain cross-linkages were still present.bilization of the native collagen which would be expected if ester In summary, the data presented here suggest that the inter-cross-linkages were severed. Correspondingly, the limed colla- TC cross-linkages are rather strong bonds, more stable than atgen did not become markedly more soluble in this hydrazine least one set of weak peptide bonds in the collagen backboneconcentration range, and the lack of solubility of the limed chains. The resistance o f these interchain cross-linkages tocollagen provides evidence that the cross-linkages in the limed base-catalyzed hydrolysis or to hydrazinolysis at high basici tycollagen were intact. This is in spite of the fac t that approxi- argues against their being considered as of weak ester-likemately 20 X 10v5 mole o f peptide bonds per g had been cleaved nature. On the other hand, a portion of the amide groups inin both collagens at this point. It is diffi cult to imagine that native collagen are readily reactive with hydrazine to formweak ester cross-linkages between TC units would be less ac- protein-bound hydrazide.cessible to general basic hydrolysis or hydrazinolysis than would Alkali-pretreated collagen contains many severed peptidepeptide bonds within the organized framework of the TC-back- chains in the polar regions of the molecule. This hydrolysisbone chains. does not lead to dissolution of the collagen unless some further,The peptide chains in collagen contain segments alternately more stable interchain bonds are hydrolyzed. These bonds arerich in the nonpolar and imino acid residues and in the polar equivalent in resistance to hydrazine to that holding the guani-amino acids. The appearance o f NH&erminal aspartic and dino group to arginine. Nat ive collagen is more react ive withglutamic acids on hydrazinolysis or basic hydrolysis and the hydrazine than limed collagen only insofar as the weakest amidelack of appearance of NHz-terminal proline or hydroxyproline groups are removed and the weakest peptide bonds are severed.following exhaustive liming of collagen (40) provides evidence These reactions reduce native collagen to the state of limedthat the polar regions are most susceptible to basic hydrolysis. collagen and do not render it any more soluble than the limedThe imino acid-rich gelatin chain segments, on the other hand, collagen, until at H- > 16 the deguanidination reaction begins.are stable to basic hydrolysis and, in addition, give to collagen One possibility for a strong but alkali-labile bond is that of aand gelatin gels their particular chain folding and organization. bond joining an c-amino group of lysine to a neighboring amideGallop et al. (13) have suggested that ester linkages are located group or guanidino group by condensation of an aldehyde onin the polar regions o f the gelatin molecule. Since, in fibrous the e-amino group to form an aminomethylol compound thatcollagen, these regions are the most readily deformed and swollen subsequently condenses to hydrolyze out water and join the twoand the most susceptible to general basic hydrolysis , it is diffi cult groups with a methylene or substituted methylene bridge.to see why ester linkages in these regions would not have been Intrachain cross-linkages of this type have been introduced intoattacked by hydrazine. Hence, we consider the presence of native ichthyocol tropocollagen showing that in the nativeinter-TC ester linkages as being rather unlikely. structure the appropriate cross-linking groups are sterically in

The most evident correlation in Figs. 7 and 8 is that the de- the proper configuration for reaction (49).guanidination of arginine is paralleled by an increase in thesolubility of both the limed and native collagens. Arginine has Reaction of Denatured Collagen and Gelatin with Hydrazine.been shown to be of importance in the ordering of tropocollagen Presence of Ester Linkages in Collagensunits to form the native structure (41) and in the hydrogen bond Quantitatively, the data presented previously for the forma-stabilization of the ordered fibr ils (42). Similarly, Grabar and tion of bound hydraaides at 2 to 3 M hydrazine concentrationsMorel (43) and Janus (44) have shown the importance of the are in good agreement with the value reported by Bello (14) forguanidino group in the setting and gelation of gelatin. The re- the number of collagen-bound hydroxamic acids following theciprocal relationship between solubility and arginine content reaction of denatured fibrous collagen with hydroxylamine, butleads one to consider the possibility that, in addition to their are lower than the values reported by Hormann (15) and Gallopparticipation in electrostatic chain interact ions, the guanidino et al. (7). Both Gallop et al. (7, 13) and Hijrmann et al. (15-17)groups in collagen may be involved in the covalent cross-linkages used more vigorous reaction conditions to secure reaction ofthat stabilize mature collagen. At least, the covalent cross- their gelatins or denatured collagens with the hydroxylamine.linkages must have approximately the chemical stability of the Hormann, Reidel, Altenschopfer, and Klenk (16); for example,bond linking the guanidino group into the arginine molecule. heated their reaction mixtures at 37 and pH 9.55 for 10 hours.A suitable cross-link might be that joining an e-amino group to Under these conditions, even in the absence of hydroxylamine,an amide or to a guanidino group via a methylene bridge result- the isoelectric pH of acid precursor gelatin or native collagen ising from an aldehyde condensation reaction. Fraenkel-Conrat shifted to lower values indicating the loss of amide nitrogen.and Olcott (45) have shown that such cross-linkages are readily Pentide bond hvdrolvsis is also enhanced at nH 9.55. the firstI I

January 1963 R. de la Burde, L. Peckham, and A. Veis 195

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196 Action of Hydra&e on Collagen. I Vol. 238, No. 1order rate constant for gelatin peptide bond hydrolysis being number of ester bonds in denatured collagens and gelatins bysome 8 times greater at pH 9.5 than at pH 7.1, at which the rate hydroxylamine or hydrazine reactivi ty.constant has its minimal value (50). It is difficult to imaginethat some amide loss and peptide bond hydrolys is did not occur REFERENCESin the presence of the hydroxylamine at elevated temperature 1. HARRINGTON, W. F., AND VON HIPPEL, P. H., in C. B. ANFIN-and high pH. In addition, our experiments indicate the ready SEN, JR., M. L. ANSON, K. BAILEY, AND J. T. EDSALL (Edi-reactivity of approximately one-half the amide groups in native tors), Advances in protein chemistry, Vol. 16, AcademicPress, Inc., New York, 1961, p. 1.collagen even at room temperature. Therefore, the results re- 2. VEIS, A., AND COHEN, J., J. Am. Chem. Sot., 76, 6238 (1956);ported here, -2 x lop5 equivalent of bound hydrazide per g of J. Phys. Chem ., 62,459 (1958).collagen at 3.0 M hydrazine, must be considered as the base line 3. VEIS, A., ANESEY, J., AND COHEN, J., J. Am. Leather Chem.for chemical modification during hydrazinolysis, excluding carbo- Assoc., 55, 548 (1960).4. MECHANIC, J., AND LEVY, M., J. Am. Chem. Sot.. 81, 1889hydrate or ester reactions. Gallop el al. (7) reported that an (1959). Ialkali precursor gelatin that had lost 50% of its amide groups 5. JOSEPH, K., AND BOSE, S., Bull. Central Leather Researchduring pretreatment also formed 50% less bound hydrazide. Inst., 7, 97 (1960).The agreement of this finding with our results seems more than 6. PIEZ, I?., AND GROSS, J., J. Biol. Chem., 235,995 (1960).coincidental. 7. GALLOP, P., SEIFTER, S., AND MEILMAN, E., Nature, 183, 1659(1959).Direct chemical evidence for the occurrence of ester linkages in 8. GRASSMANN, W., ENDRES, H., AND STEBER, A., 2. Natur-collagens has been given by Grassmann et al. (8) and Konno forsch., 9b, 513 (1954).and Altman (9). Recently, Gallop et al. (13) described the 9. KONNO, K., AND ALTMAN, K. I., Nature, 181,994 (1958).isolation of amino alcohols following lithium borohydride reduc- 10. GUSTAVSON, K. H., The chemistry and reactivity of collagen ,tion of gelatin. These amino alcohols could only have arisen Academic Press, Inc., New York, 1956, p. 167.11. VEIS, A., ANESEY, J., AND COHEN, J., Arch. Biochem . Bio-from (Y- and ,&aspartyl ester bonds. Quantitatively, they were phys., 94, 20 (1961).able to recover only 30% of the amount expected on the basis of 12. GRASSMANN, W., HANNIG, K., AND ENGEL, J., 2. Physiol.their hydroxylamine assay , i.e. they recovered only -2 x 10e5 Chem., 324, 284 (1961).equivalent per g of ester reaction products, which would corre- 13. GALLOP, P. M., BLUMENFELD , O., FRANZBLAU , C., AND SEIF-TER, S., Abstr. Meeting of Am. Chem. S ot., 1962,~. 9C.spond to the glucose and galactose analyses of Hormann (17). 14. BELLO, J., Nature, 184, 241 (1960).Thus, while there seems to be little doubt that ester-linkages are 15. H~RMANN, H., Das Leder, 11, 173 (1960).present in collagen, the number of such linkages is. probably on 16. H~RMANN, H., RIEDEL, A., ALTEN&HO&ER, T., AND KLENK,the order of 2 x 10W6 mole per g and the higher values reported M.. Das Led er. 12. 175 (1961).for the hydroxylamine-sensitive bonds in denatured collagens and 17. H~R~ANN, H., ~bst&cts,Seve~th Congress of the InternationalUnion of Leather Chem ists Socie ties, Was hington, D. C.,gelatins probably include some bound hydroxamate resulting 1961, p. 16.from the cleavage of weak amide and peptide bonds. 18. VEIS, A., AND COHEN, J., Nature, 186, 720 (1960).19. COURTS, A., Nature, 191, 1097 (1961); Biochem . J., 74, 238

SUMIllARY (1960).20. WARD, A. G., Nature, 190, 1960.1. Nat ive fibrous collagen was treated at 25 with aqueous 21. BOWES, J. H., AND KENTEN , J., J. Sot. Leather Trades Chem.,hydrazine solutions ranging in concentration from 5 to 70% 33, 365 (1949).

hydrazine (-1.5 to 19 M). The insoluble fibrous residues were 22. NEUMAN, R., AND LOGAN, M., J. Biol. Chem., 184, 299 (1950).freed of excess hydrazine, and the hydrazide content was deter- 23. BOWES, J. H., J. Sot. Leather Trades Chem., 33,176 (1949).24. CASSEL, J., AND KANAGY, J., J. Research Natl. Bur. Standards,mined. Similar studies were made with limed collagen. 42, 557 (1949).

2. Analyses were performed to determine other possible reac- 25. ROSENBERG, H., ENNOR, A. H., AND MORRISON, J. F., Bio-tions, particularly amide loss, peptide bond hydrolysis, and them. J., 63, 153 (1956).conversion of arginine to ornithine. The solubility of the chemi- 26. FEARON, W. R., Biochem . J., 33, 902 (1933).cally modified fibers was also determined. 27. PESEZ, M., AND PETIT , A., Bull. Sot. Chem. France, 37, 122(1947).

3. In the low hydrazine concentration range (< 407,), the 28. CUY, E., AND BRAY, W., J. Am. Chem. Sot., 46, 1786 (1924).amide loss was found to be slightly greater than hydrazide for- 29. GILBERT, E., J. Am. Chem. Sot., 51, 2744 (1929).mation in the native collagen. Limed collagen formed bound

30. AUDRIETH, L., AND OGG, B., The chemistry of hydrazine, Johnhydrazide and lost amide nitrogen in exactly equivalent amounts. Wiley and Sons, Inc., New York, 1951, p. 141.31. FEIGL, F., Qualitative analysis by spot test, Ed. 3, ElsevierThe bound hydrazide content of the limed collagen was one-half Publishin g Company, New York, 1946, p. 186.that of the native collagen in this range. At hydrazine concen- 32. SEIFTER, S., GALLOP, P., MICHAELS, S., AND MEILMAN, E.,trations in the range from 40 to SO%, peptide bonds were cleaved J. Biol. Chem., 235, 2619 (1960).in the native collagen, yielding approximately 19 x 10-b equiva- 33. HIMES, J., AND METCALFE, L., A&. Chem., 31, 1192 (1959).34. LEVY. A. L.. Nature. 174, 126 (1954).lent. of bound hydrazide. This reaction did not occur with the 35. DEN&, N. C:, J. Am: Chem. Xoc., 74, 2039 (1952).limed collagen, indicating that these peptide bonds were also 36. HAMILTON, P. B., AND ANDERSON, R. A., J. Biol. Chem., 211,alkali-labile. At higher hydrazine concentrations, arginine was 95 (1954).converted quantitatively to ornithine. Dissolution of the fibers 37. BRADBURY, J. H., Biochem . J., 68,475 (1958).appeared to accompany thr deguanidination reaction. 38. REES, E. D., AND SINGER, S. J., Arch. Biochem . Biophys., 63,144 (1956).4. The lack of collagen dissolution, under conditions in which 39. BRADBURY, J. H., Biochem. J., 68,482 (1958).amide and peptide bond hydrazinolysis occur, suggests that the 40. COURTS, A:, Biochem . J., 58, $4 (1954):intermolecular cross-linkages in mature collagens are not ester-

41. BENSUSAN. H. B.. Biochem istru. 1. 215 (1962).like in character. The ready removal of amide further suggests 42. RICH, A., AAD CR&K, F. H. C., 7. MoZecuZar L&ok, 3, 483 (1961).that amide loss should be taken into account in assessing the 43. GRABAR, P., AND MOREL, J., Bull. Xoc. Chim. Biol., 32, 643,..-..(1950).

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