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The pK, values thus determined show remarkable strengths for the cyanocarbon acids. Pentacyanopropene and the first ionization of hexacyanoisobutylene are comparable with the stronger mineral acids. Hydrochloric acid, for example, is thought to have a pk’, in the neighborhood of - i . 7 Al high degree of resonance stabilization in the anions (which is not possible in protonated form) appears to be responsible for the high acidity.l Smoothed values of the acidity function Hcalculated according to
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that ethane formation could be inhibited effectively by radical scavengers in the gas phase photolysis, but not in the liquid, and that the ratio of CH4 2 CaH6/X2was nearly 2. We believe that since the cage effect is an iinportant concept in solution kinetics, i t is of value to confirm its existence directly and unequivocally. We wish in this paper to report such a confirmat ion. lye have photolyzed mixtures of azomethane and de-azomethane in the gas phase and in isooctane solution using a procedure essentially similar t o 11.. = pk‘a log CA-/CB.~ was that of Herk, Feld, and S ~ w a r c . Azomethane ~ are listed in Table I1 along with values of fic for prepared by oxidation of sym-dimethylhydrazine comparison.? The H- function presented here is with mercuric oxide, while d6-azomethane was based entirely on p - (tricyanovinyl)- phenyldicyano- purchased from lferck of Canada. Both materials methane, methyl dicyanoacetate and his-(tricyano- were proven to be chemically pure by gas chromatography. The isotopic purity of the de-azomethane vinyl) -amine. I-1- behaves in a maimer surprisingly similar to was checked by mass spectra. 3Yc CD3N&D2H H,. although there are quantitative differences. and no other impurities were found. The isooctane Xt lower concentrations ( < 5 M ) these appear to was spectroscopic grade, further purified by passing be of the kind expected on the basis of the differ- through silica gel. Its purity and the cleanliness ences in charge types. -4t higher concentrations of our handling procedure were checked by ultraH- roughly parallels Ho. The similarity of these violet spectra. two acidity functions is further evidence that these The reaction products were analyzed by mass indicator acidity functions depend primarily on spectroscopy. The cracking patterns of Nz, CD3the properties of the hydrogen ion in solution N S C D 3 , CH3NNCH3,and C2H6were determined relatively unencumbered by activity coefficient for our mass spectrometer, while the cracking patterns of C2D6: CaD5H, and CD3CH3 were effects of the indicators them~elves.~ kindly provided by l f r . J. Bell of Harvard Univer(7) R. P . Bell, “ T h e Proton in Chemistry,” Chaps. V I , V I I , ~ i t y . C~ z D ~ ,C P D ~ H ,CD3CH3, and C2H6 were Cornell University Press, Ithaca, N. Y . , 1869. determined from the mass 36, 35, 33, and 27 peaks, COSTRIBUTION S o . il4 respectively. From these peak heights, the reCENTRAL RESEARCH DEPARTMENT STATIOS RICHARD H. BOYD maining peaks in the 36 to 24 range could be EXPERIMENTAL E. I. DU PONTDE KEMOURS ASD COMPANY predicted within 4%. \ ~ T I L h f I S G T O N98, DELAWARE In the gas phase photolysis of azomethane one RECEIVED AKGUST29, 1961 would expect ethane to be formed by random recombination of methyl radical. For such a random process, the ethane is formed in proportions such DEMONSTRATION OF THE (‘CAGE’’ EFFECT that (CH3CD3)*”(CzH~)(C2D6) = 4. The obSir: In 1934, Franck and Kabinowitchl pointed out served ratio of parent peak heights is 4.1 f 0.8. However in the isooctane solution photolysis, that collision times of reactive molecules should be much longer in solution than in the gas phase. the inass 33 peak is less than 27, of the 36 peak. Further, in solution, after the colliding molecules This peak can be accounted for satisfactorily by the have diffused apart by a distance small compared contributions of isotopic carbon and CDsH. The to the mean distance between reactive molecules, authors estimate that if any CD3CH, is formed, it there remains a finite probability of re-encounter must be less than 0.3%, of the total ethane. before diffusion to the mean distance. Effectively (1) W e have recently becr,me aware of a n unpublished s t u d y by the solvent holds the reactive molecules toget her, .4usluos and co-workers a t the Sational Bureau of Standards, whose somewhat parallel our results. forming a “cage.” .Imore sophisticated treatment results ( 3 ) J. Bell, private communication. using the theory of random walks has been given RICHARD K. Lrox Esso RESEARCH & ENGINEERIXG Co. by Noyes.? DONALD H. LEVY LINDES,SEW JERSEY Numerous observatioiis have been explained by RECEIVED SEPTEMBER 11, 1901 invoking the cage effect with varying degrees of certainty. Probably the best is a recent study by Herk, Feld, and SzwarcJ3in which they reaffirm ROTATORY-DISPERSION CHANGES DURING THE THERMAL DENATURATION OF Szwarc’s previous conclusion that in the photolysis CHYMOTRYPSINOGEN AND CHYMOTRYPSIN of azomethane in liquid isooctane, ethane is formed chiefly by recombination of methyl radicals inside S i r : the solvent cage. This conclusion was inferred The effects oi denaturing reagents such as urea from three observations : that the CHI ,‘I\j2 ratio in producing drastic unfolding of proteins have was independent of azomethane concentration, become so well known as to direct our attention ( I ) Franck and Rabinowitch, 7 ‘ i . a ~ ~P .t r u n i i i i y .Cor., 30, 120, 9 away from the search for other interpretations of (1934). major denaturatiori reactions. In particular, the (2) IilrciJ . A m Chew S o , ’ , 83, 2 Y W ( I 9 t i l )
+
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Oct. 20. 1961 I
I
I
I
1
I
I
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-550'
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Transition Temperature
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1 20
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25
1 30
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35
40
45
50
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Temperature ("'3. Fig. 1.-Changes in rotatory dispersion constants of chymotrypsinogen during thermal denaturation a t pH 2.0. The system was completely reversible from 58" back to 20".
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I
20
25
30
35
40
-400'
45
Temperature PC). Fig. 2.-Changes in rotatory dispersion constants of chymotrypsin during thermal denaturation a t pH 2.0. Starred points show reversal back t o 20' from 49".
The data are well represented with ho chosen as 209 drastic transconformation processes involving the millimicrons. The results of our studies are polypeptide backbone and may reveal information shown in Figs. 1 and 2. The experiments were about quite different aspects of protein conforrna- carried out with no neutral salt added, pH being tion and interaction with solvent media. adjusted by nddition of HC1. Under these condiSchellman' has noted that the specific rotation of tions our agreement with Schellman on the [a]D chymotrypsinogen did not change significantly and 1, values for the native forms of the two through the temperature range of reversible de- proteins is good. In particular, for chymotrypsin, naturation at pH 2 even though the standard the transition temperature and the total changes entropy change for the process was found to be in [ a ]agree ~ within the total of errors. 316 e.u.2which, if attributable to helical unfolding, In the case of chymotrypsinogen, we found very would correspond to a t least ten per cent. of the small changes in specific rotations and dispersion residues changing from helix to coil. The lack of constants though the temperature dependencies change in rotation means that there is either no of the dispersion constants, usually thought to change in backbone conformation, or optical rota- measure rigidity of conformation, change drastition is not to be interpreted as current opinion cally. The situation for chymotrypsin is somewhat suggests. Vile have measured the rotatory dis- different and may be analyzed a t least tentatively persion of chymotrypsinogen in order to confirm using the popular method based on the studies on and extend Schellman's findings. The rotatory poly-amino acid helix-coil transitions. On this dispersion of chymotrypsin also was studied since basis, and using our XO, we expect a change in bo its denaturation appears more typical of other from 0 to -650' as coil goes to helix. bo is thus proteins in that the changes in rotation are fairly to be considered a measure of the helix content large.' The data suggest, however, that the of a protein and leads to estimates of the helix chymotrypsin denaturation is to a first approxi- content of chymotrypsin as 237, in the native mation the sum of the effects present in chymo- protein and 19y0 in the denatured form or a loss of trypsinogen plus effects from the uncoiling of a helix folding in nine residues. In the case of small segment of a-helix. chymotrypsinogen, the very small changes in bo Rotatory-dispersion data were obtained using a indicate no unfolding during denaturation. Rudolph spectropolarimeter sensitive to 0.00&' Neurath and Dixon4 have postulated that chymoof rotation and accurate in wave length to 1 A. trypsin differs from chymotrypsinogen in having Rotations were measured a t these lines of the a small additional segment of helix. Imahorij mercury emission spectrum: 365, 405, 436, 546, has since established this state of affairs for the and 578 millimicrons. The protein samples were two proteins a t pH 7.7. Our results support this obtained from the Worthington Biochemicals interpretation. Corporation. Only chymotrypsinogen was examThe measured changes in the entropy and enined carefully for impurities and was found to con- thalpy of denaturation for the two proteins are tain less than 1% foreign protein contamination. given in Table I. Using Schellman's estimates6 We have used the Moffitt equation (Eq. 1) for the entropy and enthalpy changes per residue in for the analysis of our data. helix formation and our estimate of the number of residues unfolded in the chymotrypsin denatura(1) J. A. Schellman, Compt. r e n d . L a b . Carlsberg, Sev. Chim., 30, 450-461 (1958). (2) M. A . Eisenberg and G. W. Schwert, J . Cen. Physiology, 34, 583-606 (1951).
(3) K. Imahori, Biochim. Biophys. Acta, 3 1 , 336-341 (1958). (4) H. Neurath and G. H. Dixon, Federafioit Proc., 16, 791 11957). (5) K. Imabori. A. Yoshida and H . Hashizume, Biochim. Bioahys. Acta, 46, 380-381 (1960). ( G ) W. F. Harrinpton and J. A. Schellman, Compt. rend. L a b . Carlsberg, Ser. Chim., 30, 21 (19%).
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tion we would expect the difference in A S to be 45 e.u. and in AH to be 18 kcal. These are in good agreement with the observed differences (Table I) when one considers that the estimates of both the number of residues involved and the changes per residue must necessarily be crude since the end effects will be large when dealing with such short segments of helix. We also have oversimplified the problem by neglecting side chain interactions. However, the denaturation region lies a t lower temperatures for chymotrypsin than for chymotrypsinogen, indicating that more profound changes in the stability of the structure occur during activation of t h e zymogen than are revealed by the production of a new helix segment. Nevertheless, a t temperatures above the denaturation region, the two proteins are very nearly identical in a0 and bo values, and are thus probably in very similar conformational states. bG is about - 130' for both denatured proteins a t 50' and in both cases there is only a small temperature dependence. a. for denatured chymotrypsinogen is -538' a t 50°, while a0 for denatured chymotrypsin is -5548' a t the same temperature. a. also shows nearly the same temperature dependence for the two denatured proteins.
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'THE EFFECT OF LITHIUM BROMIDE ON THE STRUCTURAL TRANSITION OF RIBONUCLEASE IN SOLUTION
Sir:
Interest has evidenced itself recently on the effect of aqueous lithium bromide solutions on the thermodynamic stability of the ordered structures in the fibrous and globular proteins. Contrary conclusions have been reached by different investigators. l 4 Harrington and Schellman' reported a decrease in the levorotation of varioue proteins dissolved in such solutions, with particula . attention being given to the behavior of ribonuclease. Based upon the correlations obtained be tween chain conformation and optical rotation for the simple homopolypeptides by Doty and cow o r k e r ~ , *it~was ~ deduced that the helical structures were stabilized by the action of lithium bromide. Since it is well known that the activity of water is abnormally low in such solutions,s the stabilization inferred could be attributed conveniently to this cause. These deductions have, however, been seriously questioned recently as a result of more extensive solution measurements, which unfortunately were restricted to a single temperature. On the other hand, investigations of the dimensional changes exhibited by a variety of fibrous TABLE I proteins, when immersed in a large excess of CHANGESIN THERMODYNAMIC FUNCTIONS DTJRING DEaqueous lithium bromide solutions, have led to the NATURATION conclusion that a t appropriate temperatures a Chymotryptransformation from the ordered to disordered Chymotrypsin' sinogena Difference state o c c u r ~ . ~ ,The ~ J transformation in these AS, e.u. 360 316 44 cases is accompanied by an axial contraction. A 110 99.6 10.4 A H , kcal. significant disparity exists, therefore, between these latter results, typical of higher polymer It is quite clear for both proteins that the major concentrations, and the widely accepted interpretacontributions to the entropy and enthalpy change in tion placed on the optical rotation data obtained reversible denaturation are not associated with large in dilute solution. The suggestion has been made changes in optical rotation. The best description that the nature of the lithium bronide interaction of the denaturation reactions reconciling the may be strongly dependent on the protein concenthermodynamic and rotatory dispersion data which tration, and a mechanistic model supporting this we have been able t o find is this: The entropy hypothesis has been offered.Y Since the structural transformation of ribochange is the result of a cooperative, first-orderlike melting of the tangle of side chain interactions, nuclease that occurs in pure aqueous solutions has the apparent probably predominantly hydrophobic in nature, a t been studied extensively,1,10.11,12 the protein-solvent intefrace. The melted sections discrepancy cited above and the associated speculaare similar to liquid condensed phases in surface tion should be resolvable by studying any variafilm studies. Water is able to penetrate the forest tions that take place in the transformation temof side chains after denaturation, but infrequently perature caused by the addition of lithium bromide before. Although the side chains in the melted (1) W. F. Harrington and J. A. Schellman, Compl. rend. f r a o . Lab. sections gain considerable freedom, there is little Carlsbcvg, Ser. chim., SO, 167 (1957). unfolding of the protein in the usual sense and the (2) C. C. Bigelow and I. I. Geschwind, ibid., 32, 89 (1961). (3) L. Mandelkern, J. C . Halpin, A. P. Diorio, and A. S. Posner, conformation of the backbone is essentially unJ . A m . Chem. SOC., in press. altered in the process so that a. and bo do not change. (4) I,. Mandelkern. J. C . Halpin, and A. F. Diorio, J. Polymer sci., Other studies to be reported shortly support this in press. interpretation. ( 5 ) L. Mandelkern, W. T. Meyer, and A. F. Diorio, J . Phys. Chem., in press. This research was supported by grants from the ( 6 ) J. T. Y a n g and P. Doty, J. A m . Che?it. Soc., 79, 761 (1957). Louis and Maud Hill Family Foundation and the (7) P. Doty, Rev. o f M o d . Phys., 31, 107 (1959). (8) R. A. Robinson and R . H. Stokes, "Electrolyte Solutions," National Institutes of Health, U. S. Public Health London, 1955. Service and by fellowship support to J.B. from the Butterworth, ( 9 ) A. R. Haly and J. W. Smith, Biochim. e1 Biophys. Acta, 44, National Science Foundation. 180 (1'2GO). SCHOOL OF CHEMISTRY
UNIVERSITY OF MINNESOTA MINNEAPOLIS 14, MINNESOTA RECEIVED JULY 5, 1961
BRANDTS RUFUSLUMRY
JOHN
(10) J. G. Foss and J. A. Schellman, J . P k y s . Ckem., 6 3 , 2007 (196'2). (11) J. Hermans, Jr., and H. A. Scheraga, Biochim. el Biophys.
Acla, 3 6 , 534 (1959). (12) J. Hermans and H. Scheraga, J . A m . Chem. Soc.. 83,3283(1'261).
