Jan. 20, 1958
AGGREGATION O F ACIDICBOVINESERUM ALBUMIN
treated with 302 mg. of FeClr.6HzO at room temperature in an atmosphere of nitrogen. After a few minutes a brilliant red color was observed which appeared to be stable. After 1 hr., water was added and the solution extracted with ether. The combined ether extracts were washed with water, dried over sodium sulfate and evaporated to dryness. Even after chromatography on Alumina, the red colored oxidation product remained amorphous, m.p. 185-187" (under dec.). Anal. Calcd. for CteHloOs (282.2): C, 68.09; H, 3.54. Found: C, 67.94; H, 3.69. The ultraviolet spectrum revealed absorption a t Xmsx 209, 245 and 282 my. Degradation with Alkali.-Fifty mg. of the original product was fused with 10 pellets of KOH for 1 hr. at 200" in an atmosphere of nitrogen. The brown melt was taken up in water and extracted with ether. From the alkaline ether extract 25 mg. of starting material was recovered in crystal-
381)
line form, m.p. 117-118'. The alkaline aqueous solution was acidified with dilute HCl and continuously extracted with ether. The dried and evaporated ether extracts were mm. in a subjected to high vacuum sublimation a t 1 X Willstatter tube. A t an air-bath temperature of 120125', a colorless crystallized sublimation product was found in the &st receiver. After recrystallization from etherpetroleum ether, 4 mg. of prismatic needles was obtained, m.p. 190-191' (Berl, uncor.). The infrared analysis offered conclusive evidence that the alkaline degradation product was a pon-substituted aromatic carboxylic acid. This is substantiated by the broad absorption extending from 3400 to 2200 crn.-l with submaxima a t 2670 and 2445 cm.-'. The absence of any absorption from 3400-4000 cm. -1 provides evidence that the acid does not contain a phenolic or alcoholic hydroxyl group. PHILADELPHIA, PA.
[CONTRIBUTION NO. 1447 FROM THE STERLING CHEMISTRY LABORATORY, YALE UNIVERSITY]
On the Aggregation of Bovine Serum Albumin in Acid SolutionslJ! BY P. B R O ,S. ~ J. SINGER AND J. M. STURTEVANT RECEIVED JUNE 7, 1957 Bovine serum albumin forms aggregates in 0.10 M NaCl-HCl solutions below PH 3.4. The aggregation reaction appears to be reversible with respect to PH, but the rate of the reaction in both directions is subject to erratic fluctuations. The reaction is inhibited by excess mercuric ion. The aggregates can be converted to monomer on a n ion exchange column in the presence of thioglycolate. The experimental evidence available is consistent with the hypothesis that the reaction is a thiol-disulfide exchange.
Introduction order to answer that question it was necessary to It has been observed by several investigators learn more about the chemical nature of the aggrethat bovine serum albumin (BSA) may aggregate gates and to devise a method for changing the agin acid solutions. Reichmann and C h a r l ~ o o d , ~gregates back to monomer. observed aggregates a t pH 1.9 in the absence of Experimental added salts. In 0.10 M KCI solutions aggregates Crystalline BSA was purchased from Armour and Co. did not appear to form, whereas they did form in G-4302) and from Pentex, Inc. (Lots A-1201 and B0.50 M KC1 solutions. Saroff, Loeb and Scheraga5 (Lot 12016 P). Crystalline bovine mercaptalbumin (BMA) found that aggregates appeared in 0.10 M NaCl was prepared by way of its mercury dimer by the method and sodium acetate solutions of BSA. The aggre- described by Dintzis.8 Analytical grade reagents were gation phenomenon was investigated in some de- used throughout. The investigations were carried out with 1% protein in NaCl-HCl solutions 0.10 M tail by Kronman, Stern and Timasheffa who at- approximately in C1-, unless otherwise stated, a t approximately 25'. tempted to establish the conditions under which The protein concentration of the various solutions was the aggregation reaction occurs. Their findings measured with a Phoenix B-S differential refractometer were inconclusive in that no clearly defined physical calibrated with albumin solutions of known concentration as determined by the micro-Kjeldahl method. The nitroand chemical factors could be isolated which were gen content of the albumin was taken to be 16.07%. The responsible for the formation of aggregates. Ag- experimental results are reported in terms of the PH of the gregates also were observed by the present authors,' solutions as measured with a Beckmen model G pHmeter who reported on the sedimentation behavior, and a t 2 5 O , the PH-meter being standardized with a 0.10 M strength acetate buffer having a PH of 4.65.O made the observation that the aggregate appeared ionic The extent of the aggregation reaction was measured with to undergo a reversible swelling similar to that ob- a Spinco Model E ultracentrifuge operated a t 59,780 r.p.m. Ultracentrifuge cells with plastic centerpieces were used. served with monomeric BSA. During a series of calorimetric studies on serum I n analyzing the sedimentation patterns, i t was assumed that the refractive increments of the various aggregates are albumin, the question arose as to the effects which the same as that of the monomer, and the areas under the the aggregation reaction might have on the ther- partially resolved peaks of the sedimentation patterns were mochemical behavior of the protein a t low pH. In divided symmetrically. The ratios of the areas were taken
as the ratios of the concentrations of the various compo( 1 ) From a dissertation submitted by P . Bro in partial fulfillment nents. The usual correction for the dilution effect was o f t he requirements for the Ph.D. degree, June, 1956. introduced. The errors in the observed areas due to the (2) This research was aided by grants from the National Science Ogston- Johnstonlo effect were within the experimental Foundation (G 179) and the United States Public Health Service error. On the basis of the analysis of 24 different sedimen(RG 3996 C). tation patterns, the standard deviation in the determination (3) General Electric Co. Fellow, 1954-1956. of the aggregate to monomer ratio was found to be 0.04 and (4) M. E. Reichmann and P. A. Charlwood, Can. J . Chcm., Sa, 1092 to be independent of the value of the ratio. (1954). ( 5 ) H. A. Saroff, G. I. Loeb and H. A. Scheraga, THIS JOURNAL, The aggregrate under investigation here, al77, 2908 (1955). (6) M. J. Kronman, M. D . Stern and S. N. Timasheff, J . Phys. (8) H. XI. Dintzis, Ph.D. Thesis, Harvard University, 1952. Chcm., 60, 829 (1956). (9) R. G . Bates, Chcm. Reus., 41, 1 (1948). 77, (7) P. Bro, S. J. Singer and J. M. Sturtevant, THISJOURNAL, (10) J. P. Johnston and A. G. Ogston, Tranr. Faraday Soc., 11, 789 4924 (1955). (1946).
P. BRO,s. J. SINGER .4ND J. R f .
390
though apparently dimeric in character, is not to be confused with the mercury dimer'l formed from mercaptalbumin, nor with the disulfide dimer of mercaptalbumin formed by oxidation.12 Several differences between the low pH dimer and the others will appear in the results to be discussed below. Results The albumin samples as purchased contained approximately 5% of a heavy component (Fig. la). The low PH aggregation under study gave much higher concentrations of heavy components (Fig. l b ) , ranging as high as 65y0with the Armour BSX and 87y0with BMX. Although a t least two heavy components were observed in acid BSX solutions, our attention was concentrated on the smallest of these since it was more readily available for quantitative studies. The comparison of sedimentation constants of the various aggregates with that of the mercury dimer of BMA, given in Table I, indicates that the smallest aggregate is a dimer. TSBLE 1 SEDIMENTATION CONSTASTSAT pH 3.0 OF BSA AND AGGREGATES AND OF THE MERCURY DIMEROF BMA
ITS
Substance
Sm,zo
Substance
SW,20
BSA First aggregate
3.06 3.91
Second aggregate Hg dimer of BMA
5.12 3.97
The $H Dependence of the Aggregation and its Reversal.-Solutions of BSA were adjusted to several different PH values and examined in the ultracentrifuge after varying intervals a t room temperature. The results, summarized in Table 11, indicate that a significant increase in the concentration of dimer occurs a t p H values below 3.40, a conclusion substantiated by numerous additional experiments. This observation is in disagreement with the report by Reichmann and Charlwood4that no aggregation takes place in 0.10 M KCI solutions a t low 9H. The data in the table illustrate the erratic rate with which the reaction takes place. Experiments performed with exclusion of oxygen showed that oxygen has no significant effect on the rate.
PH Age of s o h , days Dimer, yo
Irol. 80
STURTEVANT
THE REVERSALOF
TABLE 111 AGGREGATIOS REACTIONAT HIGH pH
THE
fiH of, aggregation
.4ge, hr.
Aggregate, %
p H of reversal
Age, hr.
2.97 2.97 2.9; 2.9; 3.11
26 49
31 34 34 3-1
7.15 3.61 3.31
2 2
3
0
31 28 59
69 74 ,.
fG
(11) (a) W. L. Hughes, Jr., TEIS JOURNAL, 69, 1836 (1947); (b) W. L. Hughes, Jr., Cold Spring Harbor Symposia Quant. B i d , 14, 70 (1950). (12) R. Straessle, THISJOURNAL, 76, 3138 (1954).
5
Y
3.41 5.1;
3 24
Comparison of the Aggregate with the Mercury Dimer of BMA.-A sxnple of Pentex crystalline BSA was analyzed spectrographically and found to contain the following metals13: copper, 20 =t 3 p.p.m.; tin, less than 2 p.p.m.; zinc, 20 =I= 3 p.p.m.; lead, less than 2 p.p.m. X sample of BSA solution containing 30y0 of the dimeric aggregate was found to contain very nearly the same metallic contamination on a dry protein basis. These amounts of metallic ions, if fully utilized in forming dimers analogous to the mercury dimer of BMA, could account for no more than 970 of dimer. It is therefore evident that the dimer present was largely of some other type. Further evidence for this conclusion was obtained from a coniparison of various properties of the mercury dimer of B X I with those of the aggregate. Previous study of the mercury dimer of inercaptalbumin8~1'~14 has been largely confined to PH values above 5.0. I t was therefore necessary t o investigate the stability at low pH of the mercury dimer formed a t high pH and the stability at high pH of the mercury dimer formed at low PH. After this investigation was in progress, Kay and Edsall15 published their yuan titative studies which demonstrated the stability of the mercury dimer of BMA a t low pH Our qualitative results confirm their findings. B X - 1 prepared by the method of Dintzis* was used in the experiments summarized in Table I V , a t molar proportions of HgC12:BMX of 1: 2. It is apparent that with respect to stability a t high PH, even after preparation a t low pH, the mercury dimer differs significantly from the
TABLE I1 THEAPPEARANCEOF DIMERICAGGREGATE OF RSA AS A FVNCTIOS OF p H 2.70 2.81 2.07 2.48 2.90 2.90 2.99 3.30 1 1 1 1 1 5 1 1 9 40 15 18 24 31 47 48
To test the possibility that the absence of aggregates a t pH values above 3.40 might be due to rate control rather than to equilibrium control, solutions in which aggregate had formed a t a low pH were adjusted to a higher pH and after various lengths of time subjected to ultracentrifugation. The first four experiments listed in Table I11 indicate that the aggregation can be reversed by raising the pH, so that the reaction can properly be described as a low pH phenomenon. The last experiment in the table indicates that the reversal of aggregation, like the aggregation reaction itself, is erratic in nature.
Aggregate, 70
3.41) >
i
10
3.68
-> )
low pH aggregate. Xs required by the dimerization eq~ilibriall,'~ 2BMA-SH + HgCI? J ' (BX1-S)ZHg + 2HC1 ( l j ( a ) (BMA-S)2Hg
+ HgC12 JJ 2BXIA4-SHgC1
(b)
the mercury dimer was found to disappear rapidly on the addition of an excess of HgC12. The series of experiments in Table LT demon. strates that the mercury dimerization of BMA takes place also in the presence of BSA a t low QH. (13) Analyses performed by Ledoux and Co., Teaneck, E.J. I n preparing t h e sample for spectrographic analysis, the elements H g and As probably are lost. (14) H. Edelhoch, E. Katchalski, li. H. Maybury. W. L. Hughes, Jr., and J. T. Edsall, THISJ O U R N A L , 7 5 , 5058 (1053). (15) C. M. K a y and J. T. Edsall, Arch. Biochem. Biophys., 65, 354 ( I 956).
AGGREGATION OF ACIDICBOVINE SERUM ALBUMIN
Jan. 20, 1958
TABLEIV THE STABILITY OF THE MERCURY D ~ VARIOUSVALUESOF pH 9HofPrePn.ofdimcr 5.13 5.13 5.13 $Ii of *ging 4.57 4.02 3.77 Age. dnyr 2 2 2 Dimer. % 35 75 79
391
OFR BMA AT
5.13 3.0 3.0 3.0 3.48 3.35 3.60 4.82 3 2 2 2 66 80 66 64
The protein used contained 57% BMA, so that maximum dimer formation should be observed with a mole ratio of Hg++ to BSA of 0.29. TABLE V
Hg++/BSA mole ratio Age, days Dimer, %
lb
la
D1mn OF BMA PRESENCE OF BSA AT p H 2.93
FoR&l.4T10N OF
IN
0 0.22 0.54 0.75 1.60 2.70 2 1 2 1 2 2 5 3 4 14 8 6 5
Fig. l.-Ultracentrifuge diagrams of crystalline BSA; sedimentation proceeds to the left: (a) sample that did not aggregate. in 0.1 M NaCl-HC1. p H 2.91, after 126 minUtes at 59.780 r.p.m.; (b) sample containing 59% dimer, in 0.1 A4 NaCl-HC1, pH 3.11. after 88 minutes at same speed.
a BSA preparation whose sulfhydryl groups were completely blocked by reaction with a derivative of iodoacetamide, N-(p-benzenearsonic acid)-iodoacetamide. This preparation, labeled BSA-S-RI, has been described in other studies.1B Analyses indicated that one atom of arsenic was attached per SH group originally present in the BSA sample, and TABLE VI all SH was reacted. In five experiments with ACTION OF EXCESS HgC4 ON THE Low PH AGGREGATE BSA+Rl aged from 3 to 5 days a t p H 2.9, no agHg++/BSA mole gregates were observed, which result implicates ratio 0.00 0.52 0.52 1.57 1.57 thefreesulfbydrylgroupof BMAintheaggregatiofl 5.2 5.2 3.0 3.0 3.0 reaction. PH
Whereas excess HgClz leads to complete and rapid replacement of the mercury dimer by BMASHgCl, only a very slow disappearance of the low PH aggregate is caused by excess HgClz. This is illustrated by the data in Table VI.
Age, days Dimer,
1 31
1
2
37
23
3 21
8 13
The small apparent increase in dimer at p H 5.2 may be due to the formation of some mercury dimer. It may be noted that the lack of p H reversal in the first 2 samples in the table is similar to what was observed with the last sample in Table 111. A series of experiments was performed to see whether the low pH aggregation is inhibited by a large excess of Hg++. These experiments were, performed about a year later than the other experiments described in this paper, and utilized different samples of proteins. With BSA and BMA at p H 3.0 in the absence of HgClZ, with aging periods of 1 to 5 days, amounts of aggregate ranging up to 22% were observed. On the other hand, with Hg++: SH ratios from 5 to 10, no aggregate formation was observed in any case. Although the experiments with no HgClz present illustrated again the erratic character of the reaction, i t may be concluded that excess HgC12 completely inhibits the low PH aggregation. It is evident from the experiments detailed above that the low pH aggregate is of a type quite different from the mercury dimer of BMA. Nevertheless, there appears to be a direct connection between the presence of BMA and the low $H aggregation phenomenon. Samples of BSA which contain approximately 60% of BMA as shown by snlfhydryl titrations* have yielded a maximum of 65% of dimeric aggregate. On the other hand, a sohtion of BMA behaved as shown in Table VII, giving eventually nearly complete dimerization. No aggregation of BMA was observed at p H values above
3.4. Furthermore, experiments were performed with
TABLE VI1 AGGR~GATION OF BMA Age, hr. 1 4 Dimer. % 11 21
AT
pH 3.0 7 29
10
87
The Effect of Reducing Agents on the Aggregate.+traesslel2 has shown that the disulfide dimer of human mercaptalbumin, formed by oxidation of the mercury dimer by iodine, can be converted to the monomer by the reducing action of cysteine. It was therefore of interest to investigate the effect of reducing agents on the low $H aggregate of BSA. Treatment of an aggregated sample in 0.10 M NaCl a t the isoelectric pH with cysteine (4.4 moles per mole of BSA) for 2 days, or with potassium thioglycolate (1.0 mole per mole of BSA) for 1day, caused no decrease in the extent of aggregation. It was found that a reduction in aggregate content can be achieved by passing a solution containing the aggregate through an ion exchange column containing adsorbed thioglycolate. Columns were prepared using Rohm and X a a s resins IR 120 and IRA 400, thoroughly washed with distilled water before use, and having the composition listed in Table VIII. TABLE VI11 COMPOSIT~ON OF GLYCOLATE COLUMNS ~ w e r
E;;m'. En,+
1 (top)
z ~s,Ii,Coo-
4 (mixed bed) 3 20 30 30 CH,COOOA.
5 10, H
The contact time of the protein solution with the thioglycolate section of the column, as estimated from the flow rate of the solution and the void vol(18) F, pew
s. J. s i n e r .
T ~ J SO ~ ~ ~i sS, 4583 , (19%).
P. BRO,S.J. SINGER AND J. M. STURTEVANT
392
unie of the section, was found to be an important variable, as illustrated by the data in Fig. 2. It would appear that a contact time of several hours would be required to achieve complete conversion to the monomer. It was demonstrated by material balances that the apparent decrease in aggregate content was not the result of adsorption on the column. Thus in the experiment shown in Fig. 2,
BMAIesH
[
BMa or BSA
-S-SBMA]
HS-
[
BbZA or BSA
(2)
Thiol-disulfide exchanges have been observed or hypothesized in several systems. Ryle and SanR-S-S-R
3s
+ S-S-1
Vol. 80
d
+ R'-S-S-R'
IJ 2R-S-S-R'
(3)
ger" studied reactions of the type with cystine and various of its substituted derivatives. They found the reaction to be fairly rapid in 10 N HC1 and very rapid in alkaline solutions, but to have a vanishingly low rate in weakly acid solutions. Since the alkaline reaction was strongly catalyzed by mercaptide ions, for example from cysteine, they concluded that it proceeds in the absence of added mercaptide ions by way of the formation of a small amount of such ion by the reaction R-S-S-R
+ OH-
R-S-
+ R-SOII
(4)
The RS- ion then attacks R'SSR' in a thioldisulfide interchange. The reaction in strongly 0 5 IO 15 20 acid solution is inhibited by the addition of thiols, so that a different mechanism certainly applies in Contact time on thioglycolate resin, min. Fig. 2.-The conversion of the low PH aggregate of BSA this case. Fava, Iliceto and Cameral8 employed to the monomer form on contact with a resin containing ad- S35to follow the reaction between various mercaptans and the corresponding disulfides, and consorbed thioglpcolate. cluded from their kinetic data that the reaction is 96% of the protein was recovered, whereas had the first order in RSSR and RS-. Huggins, Tapley and JensenIg studied the formaindicated reduction of aggregate content been due to adsorption, the recovery would have been only 79%. tion of gels by BSA and human serum albumin in concentrated solutions in the presence of urea, and Discussion presented convincing evidence that thiol-disulfide The results which we have obtained clearly dis- interchange is involved. Kauzmann and Douglas2o tinguish the low p H aggregation of BSA from the have interpreted their data on the denaturation of mercury dimerization of mercaptalbumin," and BSA by urea in terms of the Huggins-Tapleyfrom the oxidative disulfide dimerization of mer- Jensen exchange reaction. captalbumin.12 The salient differences include : Rapid thiol-disulfide exchange reactions have (a) stability with respect to PH, the low ,bH dimer been observed only in neutral or alkaline solutions, being unstable above pH 3.4, while the others are whereas the exchange we are proposing as an exstable a t neutral pH; (b) behavior toward excess planation for the aggregation of B S 4 occurs a t PH Hg++, which reverts the low p H dimer to monomer values below 3.4. The non-occurrence of the reaconly very slowly, reverts the mercury dimer very tion at higher pH values, in the absence of a derapidly," and has no effect on the disulfide dimer; naturating agent, may be correlated with the and (c) behavior toward cysteine, which has no numerous indications that BSA undergoes a reversieffect on the low pH dimer, but reverts both the ble conformational change, which may be described mercury and disulfide dimers to monomer. as a swelling, a t low PH. According to Tanford, et The erratic behavior of the low pH aggregation uZ.,~~ BSA exists in a compact form a t PH values reaction makes it appear likely that there is in- between 4.3 and 10.5. Just below pH 4.3 it changes volved a catalytic mechanism which we have been to an expandable form which then undergoes a unable to identify or control. Nevertheless, there continuous expansion which increases with charge is definite evidence that the sulfhydryl group of and decreases with ionic strength. Large changes mercaptalbumin is involved, since: (a) a stoichio- in viscosity, sedimentation constant and other metric excess of Hg++ inhibits and slowly reverses properties, resulting from this expansion, set in as the dimerization reaction; (b) the maximum ex- the PH is dropped below about 3.5. Presumably tent of aggregation is greater with pure BMA than the disulfide bond which is involved in the aggrewith a mixture of 57y0BMA and 43y0 BSA; and gation reaction is masked in some way until the (c) BSA with its sulfhydryl blocked does not aggre(17) A. P. Rylc and F. Sanger, Hiochem. J . . 60, 535 (1955). gate. Furthermore, the monomerization of the (IS) A. Fava, A. Iliceto and E. Camera, THIS JOURNAL, 79, 833 dimer on a thioglycolate column suggests that the (1957). aggregation is the result of the formation of inter(19) C. Huggins, D. F. Tapley and E. V. Jensen, Nnturr, 167, 592 molecular disulfide bonds. Since the reaction (1951). (20) W. Kauzmann and K. G. Douglas, Jr., Arch. Biochem. Biotakes place in the absence of any oxidizing agent y s , 65, 108 (1956). other than the protein itself, it is proposed that a b k (21) C. Tanford, J. G. Buzzell, D. G. Rands and S. A. Swanson, thiol-disulfide exchange is involved. THISJOURNAL, 77, 6421 (1955). IO
I
I
!
Jan. 20, 1958
POLY-HYDROXY-L-PROLINE
molecule has undergone a significant degree of swelling. It may be pointed out that there are other reactions which take place in weakly acid solutions which may involve thiol-disulfide interchange. The sulfhydryl enzymes ficinz2 and papain2a require activation by a reducing agent such as cysteine, and this activation can be produced a t PH values as low as 4. Gorin, Dougherty and Tobolsky2‘ have postulated that in general thiol-disulfide interchange is an intermediate step in the reduction of a disulfide by an excess of mercaptan. If this is true also in the activation of ficin and papain, these reactions serve as examples of exchange reactions proceeding rapidly in weakly acidic solution. The slow reversal of the aggregation by raising the pH or by addition of HgClz suggests that a monomer-dimer equilibrium exists, although it is evident that unknown factors exert an apparently erratic influence on either the rate of attainment of equilibrium or the extent of reaction a t equilibrium, or in both. However on the assumptions that only the protein components are involved in the equilibrium state, we can try to distinguish between the following two possibilities: (1) BMA can react with itself or with BSA to give aggregates; or (2) BMA can react only with itself. The experimental data indicate that with pure BMA a maximum of 87% of dimer is formed a t “equilibrium,” while with ordinary BSA (5770 BMA and 43% BSA), a maximum of 65% dimer is formed. For case 1, the figure for pure BMA leads to an equilibrium constant for the reaction (eq. 5 ) of 2K = 1.72 X lo5liters per mole, where K is the intrinsic constant for the reaction, assumed in what follows to be the same for
the disulfide group of both BMA and BSA. From
393
this equilibrium constant we can calculate that the expected maximum yield of dimer from ordinary BSA is 70%. For case 2 a similar calculation leads to a maximum yield of only 47%. Thus this interpretation supports the view that the disulfide involved in the exchange can be supplied by either BSA or BMA. Markus and K a r ~ s have h ~ ~shown that a certain one of the 17 disulfide bonds in human serum albumin is particularly susceptible to reduction. Only this bond is reduced by mercaptoethylamine at p H 7 in the absence of detergent, whereas all the others undergo reduction when detergent is added. If bovine and human albumins behave similarly in this respect, it is an attractive hypothesis to assume that the thiol-disulfide exchange leading to aggregation a t low PH involves the one sensitive disulfide bond in BSA. The question arises as to the sources of the favorable free energy change accompanying the low pH aggregation reaction. In the mechanism proposed in equation 2, only an exchange of bonds occurs, and no new bonds are formed. Furthermore, there are factors which are adverse to the dimerization reaction. At low pH, BSA carries a large net positive charge, so that there is an electrostatic repulsion between BSA molecules which must be overcome in the formation of a dimer. In addition, one expects a considerable decrease in entropy in a protein dimerization reaction, primarily as the result of loss of translational and rotational entropy. One may speculate that any of a variety of additional factors may be involved, short-range dipole interactions, “hydrophobic” forces, etc., which have been proposed in other protein-protein interactions, but it is further possible in this case that when the intramolecular S-S bond is broken and replaced by an intermolecular bond, conformational changes may occur in the polypeptide chains near the original bond, which contribute to a favorable free energy change for the dimerization reaction.
(22) S. A. Bernhard and H. Gutfreund, Biochcm. J., 63, 61 (1956). (23) J. R. Kimmel and E. L. Smith, J . B i d . Chcm., 207, 515 (1954). (25) G. Markus and F. Karush, ibid., 1 9 , 134 (1957). Tobolsky, THISJOURNAL, (24) G. Gorin, G. Doughcrty and A. 71, 3551 (1049). N E W HAVEN,CONN.
V.
[CONTRIBUTION FROhl
THE
DEPARTMENT O F BIOPHYSICS, WEIZMANN INSTITUTE O F SCIENCE]
Poly-hy droxy-L-proline BY JOSEPH KURTZ,GERALD D. FASMAN,’ ARIEHBERGERAND EPHRAIMKATCHALSKI RECEIVED JULY 31, 1957 0-Acetyl-hydroxy-L-proline (11) yielded on treatment with phosgene the intermediate N-carbonyl chloride I11 which was cyclized by means of silver oxide to 0.acetyl-N-carboxyhydroxy-L-prolineanhydride (IV). Poly-O-acetylhydroxy-Lproline (V) was derived from IV on polymerization in pyridine. Deacetylation of V with aqueous ammonia gave rise to polyhydroxy-L-proline (VI). Osmotic and sedimentation measurements in aqueous solution gave average molecular weights of 10,600 and 10,700, respectively, for the sample of VI synthesized. 0-p-Tolylsulfonylhydroxy-L-proline( X ) was synthesized and converted into the corresponding N-carboxyanhydride XI, by means of phosgene and silver oxide. Poly-0-p-tolylsulfonylhydroxy-L-proline (XII) was obtained by polymerization of X I in pyridine: V showed mutarotation in formic acid. The specific optical rotation changed within several hours from +25” to -172 . The form with [ a I e 6 D -175’ could be reversed to the form with [ L Y ] ~ +25” ~D by means of dimethylformamide. Mutarotation in acetic acid was observed also with XII. In this case reversal could be effected by means of pyridine.
In a previous publicationZ the synthesis of poly(1) Children’s Cancer Research Foundation, 35 Binney Street, Boston, Mass. Weizmann Fellow, 1954-1955. (2) A. Berger, J. Kurtz and E. Katchalski, THISJOURNAL, 76, 5552 (1855).
L-proline was reported. This compound proved to be a useful high molecular weight model in the study of collagen and gelatin.3 As collagen also (3) P. M . Cowan and S. McGavin, Nalrcrc, 176, 501 (1955); P. M. Cowan, S. McGavin and A. C. T . North, ibid., 176, 1062 (1955).
