272 BIOCHIMICA ET BI()PHYSICA ACTA

Bt~.\ 35 I~4

St'ONTAN EOUS CROSS-I.INKI NG OF COLI.AGEN :

EVIDENCE FOR METAL-CATALYZED AND OXIDATI\ . 'E REACI ' IONS

J : \ l ( l : BI'H.L() ANI) HELI:.NE R. 1~,I".I.1.()

l)cpartment of Biophysics, l?osaell Park 3h'morial Institute, lhtCfalo, N. Y. (U.S .A, )

(R('ccivcd June 23rd, 1967)

SUMMAI(Y

Incubation ()I aqueous collagen gels at about 37 ' is known to cause insolul)iliza- tion. The clot is slowly soluble at 80-9 o°, but insoluble in strong denaturants; the collagen is probably covalentlv cross-linked. (iross-linking is retarded by several amino acids, especially by histidine, lvsine and arginine, and by F.D'I'A. Removal ot ED'I'A or histidine sh()rtens the inhit)ition period; removal of arginine eliminates in- hibiti()n. Thus, there may be at least two mechanisms ()f inhibition, one invoMng metal ions, the other competition for cross-linking agent(s). Cross-linking is als() re- tarded by borohvdride t reatment and by removal ()f oxygen.

I NTROI)UCTI()N

(]Ross ~ reported that the heating of neutral c()llagen soluti()ns to 37" resulted in the formation of a turbid gel, the matrix of which consisted of collagen fibrils. Cooling from 3 7 ~ caused reversal of the gel to a clear solution of undenatured c.ollagen. Heating to about 50: caused (tenaturation (ff the collagen and dissolution of the gel. llowever, on prolonged incubation at 37", reversibility was gradually lost, and there arose resistance, to dissolution by heat or cokl". (]RANT AND AI.BI'RN a Ioun(l that several polyhydroxyphenols, as well as tyrosinase, r e n d e r the gel resistant to dissolu- tion on cooling, but that EDTA or arginine reverse the effect. They also found that replacement of air by X 2 resulted in greater dispersi()n in ttw presence ()f epinephrine, but not greater in c()ntrols. They sugge.sted that ()xidation processes are important in the stabilization of the fibers t)y polyhydroxybe.nzenes and tyrosinase. They proposed that ('ross-linking is brought al)out by quinones fi)rmed t)n oxidation, but pointed out that evidence for collagen .quinone reactions was not conclusive. GRANT AN I) .'kI.IIURN used incubation periods of i h in most experiments, and up t() 2 4 h in s()mc. \Ve b.ave

been studying the effects (ff amino acids, EDTA, NaBH~ an(l (.)., ()n the cross-linking of collagen tibers, using as the test the dissolution belutvior on healing. Also, we have studied the effect of longer incubation peri()(ls, up to 2 months.

.MF.IHOI)S AN1) MATEIIlAI.5

Collagen was extra('ted at 5 ' with o.5 M acetic acid from tail tendons of Charles River male rats weighing 35o -4oo g, and purified by extensive high-speed centrifuga-

H ioch ira. H ioph.vs..4 eta, 147 I x ()() 7) -' 72 • 279

CROSS-LINKING OF COLI.AGEN 273

t ion, f i l t rat ion th rough W h a t m a n No. I, 2 and 5o papers, and Millipore IO V and 5

filters, and prec ip i ta t ion by dialysis against phospha te (o.oi M NaH2PO ~, o.o23 M Na2HPO4) and water. The collagen was freeze-dried and s tored at - -2o :'. In la ter exper iments , it was found tha t collagen filtered through No. I, 2 and 5o papers, d ia lyzed agains t wate r and freeze-dried gave s imilar results. This was inade the s t anda rd procedure. For the cross-l inking exper iments , the freeze-dried collagen was dissolved in cold o.5 M acetic acid and dia lyzed against phosiflutte (ptI 7, o.o4 M NaH2PO 4, o.o 9 M Na,,HPO4). F ina l concentra t ions (after all man ipu la t ions and addi t ions of reagents) of o.o5 to o.2 % were used.

Incubat ions were carr ied out at 38.7 °, on o.5-ml samples in small test tubes. Dissolution was accomplished by raising the t empera tu re at a ra te of about lo° /h ; the dissolution t empera tu re was taken as tha t t empera tu re at which almost all of the collagen dissolved.

Exper imen t s with amino acids uti l ized twice as nmch amino acid as c, fllagen by weight. The amino acids were added in pH 7 ph°stf lmte buffer, at a concentra t ion of 2o mg/ml to give a final concentra t ion of 1.8 mg/ml and a collagen concent ra t ion of o. 9 mg/ml. In some exper iments (as noted later) the respect ive concent ra t ions were 2.o and I .o ing/inl.

React ions in the absence of air were carried out e i ther m vacm, or by , t isplacing air by N.,. When the vacuum method was used the tube was sealed with a flame. When the evacua ted tubes were pa r t i a l ly immersed in the bath , wate.r condensed in the narrow upper par t , t he reby changing the collagen concentrat ion. That this was not significant was shown by the s imilar results ob ta ined when the tubes were com- plete ly immersed so tha t no l iquid condensed in the upper par t of the tube. When the N., method was used, N 2 was in t roduced with a small d iamete r hypodermic needle th rough a serum s topper while a second needle allowed gas to escape. Af ter gassing, a second serum s topper was placed over the first. A few exper iments were d, me in the da rk by placing the small test tubes into larger, blackq~ainted tubes ; the test tubes were removed from the b lackened tubes just before heat ing above 39 ~.

Reduct ions wi th N a B H 4 were carr ied out at p l l 8.2 in phospha te cthe same buffer as above, but wi th pH ad jus ted with a very small volulne of s t rong NaOH). The higher pH was used to reduce decomI)ositioil of N a B t l 4 and the a t t e n d a n t foaming. A solution of 5o mg of collagen in I2 ml of buffer was mixed with IO ml of phosphate buffer (o.o3 M in EI)TA, pH 8.2) containing 25 mg of NaBH 4. The solut ion was s t i r red at 4 5 ° for o.5-1 h, t rea ted with 2 ml of acetone (which produces no foaming) to des t roy excess NaB It 4, d ia lyzed against water and against t ,hosphate buffer (pH 7) in a N 2 a tmosphere . The react ion with NaBH~ cause.d some foaming bv l I 2 gas; the foam was discarded. As this may have resulted in sonic loss of collagen, cor~trols were also carried through at collagen concentra t ions of o . i and o.e 5 %.

Ra te of heat gelat ion. Collagen solutions, 2 ing/ml in the usual t)hosphate buffer and at room tempera ture , were added to pre-warmed cuvet tes at 38~ in the. thermo- s t a t t ed cell compar tmen t of a Beckman DB spect ropl lo tometer . The t ransmission was measured at 45o rap. and recorded on logar i thmic char t paper. To avoid bubble format ion in the cuvet te , the samples were. degassed for ~ rain at a pressure of about 2oo toni of mercury. Subsequent operat ions were conducted with exposure to air.

Ul t racent r i fuga t ion was carr ied out on o.2 % solutions in the usual phospha te buffer.

Biochim. 13iophys. Acta, 147 (1967) 272 279

274 j. BEIA.(), H. R. BEI.I.()

RESULTS

The dissolut ion telnt)eratures shown in the figures are. approx imate . The ex- perinlent det)ends on observing the d i sappearance of tile opaque mass of insoluble collagen cm heat ing at about io"/h. For tim mater ia ls of low dissolution tenlpera ture , this is fair ly easy, as t he collagen ix dena tured , and the tu rb id gel is dissolved s inmltane- ously or soon after, over a small t empera tu re range (about 2 -5:'), and ix conver ted t~, a clear solution of gelat in. However, as cross-l inking procecles dur ing incubat ion, the dissolution t empera tu re becomes a different phenomenon. There is first a syneresis of the gel at about 5 ° '55 :' to produce a clear l iquid and an opaque mass, usual ly cylin- drical, often changing to a sphere. After incubat ion of 2o weeks, the syneresis t empera- lure rises to about 6o ~, p resumably as a result of more perfect in termolecular associa- tiun and/or more extensive cross-linking. Thus, on l~mg incubat ion the syneresis teml)era ture approached the melt ing t empera tu re of nat ive malnmal ian fibers. (;ROSS 2 found tha t on incubat ion for one year the ' shr inkage tempe.rature ' , correspond- ing to our syneresis t empera ture , rose by about the same amount . Dur ing the syneresis the na t ive collagen conformat ion p robab ly is mel ted out. As the t empera tu re is ra ised the opaque m~Lss even tua l ly dissolves, p robab ly by hydrolysis . "['his takes place over a ra ther wide t empera tu re range, and in the cases of collagens t ha t have been incuba ted for some weeks, is not complete itt 85 °, dur ing the usual hea t ing process. The process of dissolut ion becomes increasingly diffuse ms the cross- l inking becomes more extensive., atnd the. e s t ima t ion of the dissolut ion t empera tu re beeonles more diffcult and more subject ive. The es t imate is faith" reliable up to about 7 ° ". In consider ing the d a t a of Figs. z and 2, dissolut ion t empera tu res over 8o" should not be thought of as d is t inc t t)oints, but merely as indica t ions tha t tile collagen was s t rongly cross-l inked and res is tant to dissolut ion except on prohmged heat ing at high t empera tu re . Gm)ss ~ found tha t insolubi l izat ion d id not occur dur ing incubat ion tinles of 4 weeks, but did occur at some t ime (luring an incubat ion per iod of 53 weeks. Gm)ss also used rat ta i l tendon collagen, ex t rac ted with o.I M ace.tic acid, where we used collagen ex t r ac t ed with o.5 bl acid. There are other differences ill the exper iments : types and (probably) age of rat , me thod of purif icat ion, lmffer, ;rod also t empera tu re , 3 7 ' i n ( ;Ross ' ex- pe.riments and 38.7 ' in ours.

The effects of urea, guanid in iu ln chloride and NaCIO v which are effective ( t ena tu ran t s of collagen t, 5 were s tudied. "1"o gels aged 8-IO weeks at 39 :, were. added sufficient IO M urea, IO 3I guanidinun~ chloride or 9 M NaCI() 4 t() make a tinal con- cen t ra t ion of 6 M. These were incubated i6 h at 39", and then heated at the usual rate. These collagen salnples appeared to 1)e its res is tant to dissolut ion as controls to which corresponding amounts ()f water had t)een added.

The effects ()f amino acids are shown in Fig. i. The d a t a of Fig. I represent the major por t ion of the s tudy (Jr the effects of amino acids, being the results ob ta ined from 5 -7 sets ()f experinu,nts on each amin() acid, each yet in t r ipl icate , and all on one ba tch of collagen. At about 6 -8 days it is evident tha t serine is sontewhat inh ib i to ry ; the remainder are. more inh ib i to ry but are too eh)sely grouped to discern any real differences among them. At the end of 3 weeks, the effects are be t te r resolved, and it is seen tha t the h y d r o x y amino acids are, as a gr()up, less inh ib i tory than the others, except for glutamine. Asparagine ix in termedia te , while lysine, arginine and hist idine art: the most effective. At 7 weeks, the inhibit(wv effects have wmished f()r all lint the

Biochim. Bioph>'s..-Iota, 147 (t9~7) 272-279

C R O S S - I . I N K I N G OF C O L I . A ( ; E N 275

last three. The effect of arginine at 7 weeks is not certain as dissolution temperatures above 80 ° are difficult to measure. Only histidine has an effect that we feel confident continues to 7 weeks. (The results of Fig. I were obtained with crystalline amino acids of the Ajinomoto Company of Japan; similar results were obtained with amino acids from several domestic suppliers. The Ajinomoto amino acids were used to insure an independent set of data; different domestic suppliers may provide products of the same manufacturer under their own labels.)

9O,

I

~ I , , ~ " i so: N~CtO _ 60

ic ACtD

o CONTROL

~-- v EOTI & BH; < 80

• VACUUM BROKEN AFTER SEALING

I-- 70 '- TUBE

z

g - - 0 CONTROL ¢q a s o a s o ' . _ ~ . . . . . . . . . . .

I 2 3 4 5 6 7 8 I 2 3 4 5 6 7 8 9

WEEKS W E E K S

Fig. I. Effects of amino acids on thermal cross-|inking of collagen.

Fig. 2. t-ffects of deoxygenation, EDTA and borohydr ide on thermal cross-linking of collagen.

In Fig. 2 are shown the effects of EDTA and 02 removal. ] 'he data of Fig. 2 are representative, except for the sample in vacuo; this sample became cross-linked more quickly than the others; the other 3 samples gave curves similar to that for EDTA. The.re was some variation from run to run arising from the difficulty in establishing tile dissolution temperature and from variations in different batches of collagen. The abnormal sample is shown to indicate the largest variance that was found With all batches there was a distinct difference between tile control collagens on the one hand and those subjected to NaBH4, EDTA or deoxygenation on the other hand.

Tile possibility that the deoxygenation process may have affected the collagen, making it less susceptible to cross-linking was tested by breaking tile seals on evacuat- ed tubes to admit air and then incubating; these samples showed little or no retarda- tion of cross-linking (Fig. 2).

It is of interest that EDTA-treated collagen gelled at 4 ° during dialysis to re- move the EDTA, while controls remained fluid. This effect presumably arises from removal of metal ions. However, experiments in which added histidine and arginine were removed by dialysis did not result in gelation at 4 °. It is well known from numerous studies and patents that metal ions aggregate gelatin and collagen and tan hide.

Collagen treated with EDTA was subjected to ultracentrifugation, with the results: for control collagen, S4a = 3.82 and 5.31 for ~ and fl peaks; for EI)TA-treated collagen, .%a = 3.80 and 5.19.

The combination of EDTA and deoxygenation resulted in longer retardation of cross-linking than either treatme.nt alone. This is to be e.xpected if the rate of cross- linking depends on both O., and metal concentration.

Experiments were also done with arginine and histidine, in which the amino

I3iochim. Biophys. Acta, I . t7 (1967) 272-279

276 J. BEI.IA), H. R. BFLI.()

acid was removed by dialysis, as in the case of EDTA. The results are shown in Fig. 3. The data shown are averages of 3 ext)e-rimcnts, each in triplicate, all on one batch of collagen. The difference between these amino acids points to there being at least 2 mechanisms of cross-linking iilhibition, The arginine-treated san)pie showed no in- hibition of cross-linking; presumably this amino acid when present during incubation acts by a xnechanism not inw)lving metal binding. Comparison of Fig. x with Fig. 3 shows that solutions from which the histidine has bee.n removed by dialysis required about twice as much time as did the control to reach a dissolution teinperature of 75 ~; while solutions containing histidine required about 8 times as inueh tinw. as (lid the controls. "l'he relatively short period of inhibition in samples which have. had the histidine removed, compared to those in which histidine was not removed, suggests that histidine acts by more than one mechanism.

9 0

8O

~- 70 nr

6o

b- 5 0

O.4-" 0'3 I 0 CONTROL

• His, 2.0 o~

H i s , 0 . 2 L • Arg

o o ', ~ ~ o

WEEKS

,I j~CONTROL I 25 ~ 5 3 75

M I N U T E S

5 0

Fig. 3. f-flects on cross-linking of adding and then removing histidine and argmine. Note longer time for cross-linking of this control batch compared to those of l:igs t and 2.

Fig..I . Effect on gel formation of adding and then removing histidine. Curves are drawn through points showing averages of 3 experiments. Vertical bars show total range of values of 3 cxperimmlts. Bars are offset Iroln points for clarity.

Since histidine, even when removed by dialysis, inhibited cross-linking, it was of interest to learn if similar addition and removal of histidine would affect gel forma- tion. The results are shown in Fig. 4- The experimental da ta were represented as smooth curves made with a pe.n-re.corder. The data of Fig. 4 were. taken at intervals of o.5 recorder time unit. The histidine-treated collagen lagged in absorbance at first, but then speeded up and reached the same tinal value as the control. Par t of the early lag in both cases arises from the time required for temperature equilibration. But, in the case of the histidine sample the lag portion of the curve is quite horizontal for 30 sec, while in the control it has a small, but positive slope.

Collagen was also treated with NaI~[t t, to reduce aldehydes to alcohols and to reduce oxidizing agents. NaBI-I~ reactions were carried out it) the presence of EI)TA; it was the control experiment which showed that inhibition of cross-linking occurred even after removal of EDTA by dialysis. Fig. e shows that NaBtt~ t reatment inhibits insolubilization for a longer period than does EDTA; therefore, it is probable that Nal3lt~ t reatment itself inhibits insolubilization. Since NaBtI~ t reatment may result in sonic loss of collagen by foaming, controls also were run at one-half and one-fourth of the usual concentration. The results were tile same a.s for the s tandard controls.

htiochim. Hi.phys..qcta, 147 (t967) "7-"- '79

CROSS-LINKIN(; OF COLLAGEN 277

Experiments in black-painted test tubes gave results similar to those in which the collagen gels were exposed to light, indicating that the oxidation process is not catalyzed by light.

DISCUSSION

GRANT AND ALBURN suggested that tile collagen is cross-linked, but left open other possibilities of aggregation. Their experimental observation was reduced solubility on cooling; this technique leaves open the possibility that more efficient orientation, rather than covalent cross-linking, might lead to loss of solubility in the cold. "l'axzv.i{, MoxRo]~. AND GROG.":, 6 stated that the insoluble gel i~" cross-linked. The fact that the long-aged gel does not dissolve readily at 8o-85 ° , but does so only slowly, points to a cross-linked material that dissolves as a result of hydrolysis. This is also supported by the observation that 6 M NaCIOa, urea, or guanidinium chloride did not dissolve the clot at lower temperatures than controls.

At this stage of the investigation of the in vitro cross-linking definite mecha- nisms of action are not known, but we may consider several possible explanations for the effects of amino acids in inhibiting cross-linking.

The most extensively explored theory of cross-linking of collagen in zivo is that of reaction with aldehyde. (For recent pertinent work see refs. 6-8.) The results re- ported here are consistent with reactions inw~lving aldehyde. NaBH, can reduce aldehydes to alcohols; several of the amino acids can react with aldehydes. The ira- hit)itorv effect of lysine suggests that e-amino groups are involved; but the lesser effect of some of the other amino acids suggests that c~-alnino groups are less effective. The latter, of course, are rare in collagen.

All of the amino acids were used at the same concentration. Since the various side chains are present in collagen to different extents, the ratios of added amino acids to amino acid residues in collagen are not tire same. These ratios vary from about 2o: z to about 5o:x, except for histidine at 2oo: z. If competition for cr~,ss-linking agent ix the cause or retardation, the coinpetition should be greatest in the case of histidine. The eventual cross-linking of collagen from interchange reactions between the amino acid-cross-linker product with collagen; from slow reaction between the cross-linker and collagen functional groups which are intimately associated, but not accessible to the inhibitory amino acid ; from gradual exposure of reactive groups as a result of slow hydrolysis of blocked cross-linking sites; from oxidation of saccharides or amino acids to reactive cross-linkers.

All of the cross-linking may not be dependent on pre-existing aldehyde as in- dicated by the eventual cross-linking of NaBH4-treated collagen. Also, collagen treated with thiosemiearbazide to block aldehydes, shows retarded cross-linking ~. In the case of NaBtt 4, reoxidation of reduced aldehyde or oxidation of other groups to aldehyde perhaps occurs. In the case of thiosemicarbazide oxidation may occur or the thiosemicarbazone may slowly react.

Another mechanism of inhibition is the inactivation of catalytically active metal ions by complexation. This is supported by the fact that EDTA and histidine retarded cross-linking even when they and their complexed metal ions were dialyzed out before incubation. "l'.axzE~, MoxRoI,: AxD GRoss found that adding thiosemicarbazide immediately before incubation (incubation conditions are not suitable for reaction of

Biochim. 13iophys..4eta, I47 (1967) 272-279

278 J. BELLO, H. R. BEI.I.O

thioselnicarbazide wi th aldehydes) does not cause retardation of cross-linking even at th iosemicarbazide levels comparable to our ED'I 'A levels. Since th iosemicarbazide (:an chelate some metal ions, its re.tarding action appears to be the result of tlfi(~semi- carbazone formation. That different amino acids may opera te bv different mechanisms, or tha t one amino acid n lay operate, by more than one mechanism, is suggested by the diffi 'rence between arginine and hist idine, the former more like.ly ac t ing its a com- pe t i tor for cross-l inking agent, and the la t te r both its a metal eomt)le.xer and as a compet i tor . The metal ions tha t are known as ox idan ts or as ox ida t ion cata lys ts , e.g.

Ire ( I I I ) , ( 'u (II), etc. form insoluble phosphates . If such ions are involved in cross- l inking collagen they must e i ther be s t rongly bound to react ive sites, or are act ive its the 1)hosl~hates.

()ANI)LISH AND "]'I(I.'-;'I'RAM 'a repor ted on the effects of several amino acids on the rates of fiber fl~rmation and redispersion of the fibers by urea or KI. Their results are not comparab le to ours as they incubated collagen for I5 h, and they used about one- ten th the amount of amino m'id. They flmnd tha t serine aided redist)ersion and tha t o ther amino acids had small effects. ( ;m)ss AXD KIm~ m s tudied the effects of amino acids on fibril format ion and flmnd large effects, especial ly s t rong r e t a r d a t i , n by arginine and lysine. However, their work was on fibril formation at ear ly stages (minutes to a few hours) while our work is on long incubat ion. Perhaps the r e t a rda t ion of fibril formation ~" by sonle amino acids m a y also result in a hmg re ta rda t ion of subt le conformat ional or aggregat ional changes which are required for effective cross- linking. In the case. in which histidine, was removed by diah 's is the gelat ion exper iment showed tha t the ra te of gelat ion was near ly normal, except for a slight lag at first, and tha t the extent of gelat ion was normal, as indicate.d by the overall change in abs~wb- ante. The small difference m a y arise from removal of metal ions, whose role in fibril format ion requires s tudy. "l'he various levels of organizat ion probal) ly are ge.lmrally s imilar to those of the. control, and the inhibi t ion of cross-l inking is not the result of a difference in organizati~m. This is not certain, of course, its there may l)e an impor t an t difference tha t is not ( tetectable by the gelal i ,m rate exper iment .

The re ta rda t ion of cross- l inking in vacuo or under N,, is con t r a ry to the report of GRANT AND :\I.I~URN :~. I t al.)l)ears tha t the cross- l inking we are deal ing with is different from theirs ; t ha t O., is n - t involved in ear ly cross- l inking s tudied by ( im\xT axD AI.Bt:RN, in the absence of po lyhydr ic phenols, but is involved in the slower cross- l inking we have studied. In the ear ly cross-l inking metal ions m a y act d i rec t ly its oxidants . In the vat 'uunl exper iments , cross-l inking occurs af ter some weeks. The solutions were. not exhaus t ive ly evacuated , lint only evacua ted for about 2- 3 lllin at about I mm of pressure. The slow cross- l inking of deoxygena ted collagen mav arise front residual O~, o ther oxidants , or from prefl~rmed cross-l inking agents, such a aldehydes. The hmger re ta rda t ion of EI ) ' l 'A- t rea ted , deoxygena ted collage.n is ex- pected for a me ta l - ca ta lyzed oxida t ion .

The inhibi t ion of cross-l inking l)y Nal-',H 4 t r ea tmen t may also affe.ct it nmtal ion-ca ta lyzed reaction, through reduct ion of metal ions to lower ox ida t ion states, to metal or metal borides. Howcve.r, at this s tage we do not know enougll about the act ion of N a B l l a on collagen to make any firm s ta tements . F~I.UMENH'21.1) AND (]AI.I.(IP s

have found tha t dur ing NaBH a reduct ion (~f a ldehydes in collagen, there aptmars t(~ be a small amount of pept ide clc'avage at a lanine residues. That inhibi t ion of cross- l inking by combined t r ea tmen t with N a B H z (with EI)TA) and deoxygcna t ion resul ted

Biuchim. IHophys. ,lcta, I47 (~967) 272 279

CROSS-LINKING OF COLLAGEN 279

in the longest inhibition of cross-linking is compatible with a greatly diminished capacity (lack of Oz and metals) to reoxidize reduced aldehyde.

It is not entirely clear whether all of the metal ions are present in the tissue or if some originate from the solutions used in the experiments. However, it appears that much of the metal content was present in the original tissue. This is based on the fact that after the EDTA or histidine treatment, the collagen solution was dialyz,.'d against the same batch of phosphate buffer as were controls without EDTA or histidine. This suggests that at least some of the metals involved are present in the original tissue from which the collagen wa.s extracted, and were not introduced during man ipulations subsequent to the original extraction. Some metal ion may have been introduced in the acetic acid extracting medium, but not in the phosphate dialysis n~e(tium as in some experiments the same stock solutions of phosphate troffer were used :o dialyze out the acetic acid and the EDTA.

The processes described here may be related to natural maturation or aging of collagenous tissue, but this is not proved. Aging is slower, but would be altected by intermolecular orientation and by enzymes, inhibitors and reversing agent.-.

A('KNOWLEI)GE.MENTS

We are grateful to Mr. FRANKI.YN C. ~VISSI.ER for the ultracentrifuge runs, anti to Miss HELEN B. PATRZYC for preparation of the collagen.

This work was supported by Grant GM-o9826 from the Institute of General .Medical Sciences, National Institutes of Health and Grant DRG-7oI from tt:e Damon Runyon Memorial Fund.

I(t,FEI(I:.N('I,~S

I J. GROSS, J. Exptl. Med., io8 (I958 ) 215. 2 J. (;ROSS, Science, 143 (1964) 900. 3 N. H. (;RANT AND H. E. AI.PIURN, Biochemistry, 4 (1965) I271. 4 l~. N. VON HIPPEI. ANI) Ix. ¥. X, gONG, Biochemistry, 2 (i963) r387. 5 J. |~EI.LO, H. C. A. ]{IESE AND J. 1{. VINOGRAD, J. Chem. Phys., 60 (r956) 818. b J. L. TANZER, I_). 3IONROE AND J. GRoss, Biochemistry, 5 (1966) I9X9. 7 P' BORNSTEIN AND K. A. ]JIEZ, Biochemistry, 5 (1966) 346o. 8 (). Ih.UME.~FELX) AND P. M. GALLOP, Proc. Natl. Acad. Sci. U.S., 56 (I966) 126o. 9 J. K. ('.,,NI)LISH ANt) G. R. TRISTR.'~r,t, Biochim. I~iophys. Acta, 88 (1964) 553.

lO j . (;ROSS AND D. KIRK, J. Biol. Chem., 233 (1958) 355.

Biochim. Biophys. Acta, 147 (196;') 272-279