3482
COMMUNICATIONS TO THE EDITOR
weight compounds may be related to the effects observed when very thin liquid films are employed2; for example, cholesterol required only about 35 min. for elution at 0.68 atm. inlet pressure (30 ml./min. flow rate) with a 183 cm. column a t 222'. Stereochemical change in the A/B ring junction led to different retention times for otherwise similar compounds. Hydroxy compounds were eluted before the corresponding ketones, without trailing; ketones showed some trailing. A considerable difference in retention time was observed with increasing carbon content. Both colunins had about 2300 theoretical plates (cholestane) . The usefulness of this phase for other kinds of high molecular weight compounds has not been investigated. Silicone grease phases are known to have high thermal stability,3 - 4 and while the present phase may be used to 250-300 O its greatest value may lie in its unusual ability to resolve steroids rapidly a t relatively low t e m p e r a t ~ r e s . ~ (2) C. Hishta, J. P. Messerly and R. F. Reschke, Abstracts 137th A.C.S. Meeting, April, 1960, p. 29-B. (3) J. Cason and W. T. Millet, J . Org. Chcm., 24, 1814 (1959). (4) C. Asselineau, J. Asselineau, R . Ryhage, S. Stallberg-Steohayen and E. Stenhagen, A c f a Chcm. Scand., 13, 822 (1959). ( 5 ) Sterol analysis on a silicone column, operated a t 287'. has been described recently (R.IC. Beerthuis and J. H. Recourt, N a f w e , 186, 372 (1960)).
VOl. 82
in the neighborhood of PH 10. Here ImH+ represents the imidazolium ion and Im the neutral imidazole molecule. The relaxation time, T , in this case is r =
+
jk?: f K:~(COH-
cImHc))-'
(2)
Since the equilibrium constant is known,6both rate constants could be determined (at essentially zero ionic strength and 25 "). The system studied with the temperature jump consisted of imidazole, chlor phenol red (a PH indicator with p K (3.08, through which pH changes could be followed spectrophotometrically), and 0.1 Af KNOa. The measurements were carried out in the PH range of 5.5 to 6.5 a t 13'. The reaction mechanism can be written as I1 Im f H +
2Y
'u
ImH+
>x
+ In-Z
34
+ In-' 2I m + H I n 43
I I1
Iv
The state I1 in this system differs from that in equation 1 by the presence of dissociated indicator. LABORATORY OF CHEMISTRY O F NATURAL PRODUCTS W. J. A. VANDENHEUVELFurthermore, In-2 and HLn- represent the forms C. C. SWEELEYof the indicator important in the PH range under NATIONAL HEARTINSTITUTE BETHESDA 14, MARYLAND E. C. HORXING consideration. This mechanism is characterized RECEIVEDMAY17, 1960 by two relaxation times which are complicated (but known) functioiis of the six rate constants and the equilibrium concentrations. Only the longer FAST REACTIONS O F IMIDAZOLE STUDIED WITH relaxation time ( > l o psec.) could be measured, RELAXATION SPECTROMETRY although a shorter relaxation time ( < 5 psec.) Sir: The understanding of enzymatic reactions often could be detected. By varying concentrations is hindered because of the difficulty of isolating and using a trial and error procedure to fit the data, and studying individual steps in the mechanisms. all six rate constants were determined. The rate obtained are: In many enzymatic systems, it appears that constants W - 1 sec - I see - 1 % imidazole is involved intimately in the catalytic k1z = 2.3 X 10'0 K p l = 2.3 X l o 3 (4~15) process. For these reasons, studies of the kinetics kza 1.5 X 10'' kS2 = 1.5 X lo3 (&35) kza = 3 x 10'0 k12 = 2.4 X l o 4 (2~50) of the fast reactions of imidazole with hydrogen K 3 1 = 1.2 x 108 M-' (=tt35) kar = 9 X 108 ion, hydroxyl ion and a proton donor-acceptor system in the region of neutrality (chlor phenol The relatively large experimental error in the rate red) were undertaken with relaxation techniques. constants determined by the temperature jump Two experimental approaches were employed : method is due to the evaluation from the complithe dissociation field' and the temperature jump cated form of the relaxation time since individual methods2 Both utilize the same principle: per- relaxation times can be found to Z!Z 10%. turbation of a system a t chemical equilibrium and The rates of reaction of imidazolium ion and determination of the relaxation time (or times) imidazole with hydroxyl and hydrogen ions, renecessary for the reestablishment of equilibrium. spectively (&, &), are diffusion controlled. I t The measured spectrum of relaxation times can is interesting to note that a t PH 7 and a t salt be related to the rate constants of the chemical conceiitrations usually present in physiological reactions in the system.a Detailed descriptions of systems, the two rates would be about equal. the experimental apparatus and procedures can be The reported value of k12 is valid for zero ionic found e l ~ e a h e r e . ~ ~ ~ ~ ~ ~ ~ strength, whereas kz3 is determined at an ionic The dissociation field method was used to study strength of 0.1. The latter value, however, should the kinetics of the reaction be fairly independent of ionic strength since no net k12 change in charge is involved. The rate of combiImH+ OHIm HzO (1) nation of chlor phenol red and a proton (k24) is kzi also diffusion controlled and is identical with the I I1 value previously found for a similar reaction of (1) M. Eigen and J. Schoen, Z.Elckfrochcm.,69, 483 (1955). phenol red.7 On the other hand, the rate of pro-
+
+
(2) G. Czerlinski and M. Eigen, ibid., 63, 652 (1959). (3) M. Eigen, ibid.. 64, 115 (1960). (4) L. De Maeyer, ibid., 64, 65 (1960). ( 5 ) H. Diebler, Ph.U. Thesis, in prepurntion.
(6) A. H . M. Kirby and A. Neuberger, Biochem. J.,32, 1146 (1938); (7) H. Diebler, M. Eigen and G G. Harnmes. 2. Nalurforsch., (b) in press (ISGO).
COMMUNICATIONS TO THE EDITOR
July 5, 1960
3483
ton transfer between imidazole and chlor phenol meric compounds derived from y-methyl-L-glutamic red (&), appears to be slightly smaller than the acid. The optical activities were determined in value expected for a diffusion controlled p r o ~ e s s . ~both random coil and helix-forming solvents. Weller* also found similar rates for reactions with However, association complicated interpretation 5 x 10' &I--1 sec.-l) which suggests of the results.','" Molecular weight determinations acridine (k that this rate either is characteristic of proton using short column equilibrium ultracentrifugation transfers involving a nitrogen atom or depends confirmed association.'l In this communication somewhat on the pK-values of the proton donor- we wish to report optical activity enhancements acceptor system. For such transfer processes, and abnormal rotatory dispersions for the oligodiffusion controlled rates ( k - 3 X l o 9 'If-' sec.-l) meric peptides in solvents where no association have been founds only when the proton was trans- occurs. When the oligomeric peptides are studied ferred between two oxygen atoms which were more in dimethylformamide and m-cresol, concentration acidic (pK--5) than the systems considered above. independent optical activity enhancements are The value of k,z should be compared to previous re- noted (Table 11). The lack of association was sults obtained for similar reactions between a cation confirmed by molecular weight determination in Table I contains a list and hydroxyl ion; these are listed in Table I. All dimethylf~rmamide.~~ these rate constants are characteristic of diffusion of the optical rotations and rotatory dispersion bo controlled processEs; the differences in rates are due values from the Moffitt equationl2 for polymers of to steric factors. Thus, ammonia is the fastest be- poly- y-methyl-L-glutamates in ditnethylformainide cause it has four sites for proton transfer. Tri- and dichloroacetic acid. methylamine ion, on the other hand, is sterically TABLE I hindered by the methyl groups and has a corDATAO N POLY-7-METHYL-L-GLUTAMATES respondingly slower rate of reaction. The fact OPTICALROTATION
-
Solvent
TABLE I Cation "a
+
Acridinium (CHa)a?iIIf
Reference
kir( M - 1 ser.-i)
3 . 0 X 1Olo 1 . 8 5 x 10'0 1 . 0 x 10'0
1 8
9
that the imidazole rate constant is twice as large as that for trimethylamine indicates that the reaction is able to take place a t both nitrogen atoms in the imidazoliurn ion ring. This occurrence is due either to resonance stabilization which makes the two nitrogens equivalent, or to a fast tautomerization of the ring after reaction a t the neutral nitrogen. Acridine would be expected to be less sterically hindered than trimethylamine, but should react more slowly than imidazole, which has two reactive nitrogen atoms. This fact is in accord with the intermediate value of the rate constant that was found. This investigation was carried out during the tenure of Postdoctoral Fellowships from the National Cancer Institute, United States Public Health Service (K.K.) ; and National Science Foundation (G.G.H.). ( 8 ) A . Weller, Z . Rieklrorhrin., 6 4 , 5: (19iiOi. (9) h l . Rigeri, 3 . S. Jhnson,E. K u \ t i n nnd A . E i t t i g , in prepn.
h l ~ PLASCK x INSTITL~T
;\rASFRED B I G E N (;ORUOS [.; IIAMMES
FL'ER P*IVSIKAI,ISCHE C H E R l I l i
G ~ E T T I N G EGERMASY S, RticErvsn
KENNETI-I KUSTIN J ~ - N E2 ,
1060
CONFORMATIONAL ASPECTS O F SYNTHETIC POLYPEPTIDES. 11. CRITICAL RANGE FOR INTRAMOLECULAR HYDROGEN BONDING
Sir: In our previous report,' we employed optical activity to measure the conf~rmation*-~ of oligo(1) M. Goodmanand E. E. Schmitt, THIS J O U R N A L , 81.6507 (1959). (2) J. T. Y a n g a n d P. Doty, i b i d . , 79, 761 (1957). (3) P. D o t y , A. M . Holtzer, J. H. Bradbury. E. K.Blout, ibid., 76, 4493 (1954), (4) P. Doty and J. T. Yang, ibid., 78, 498 (1933). ( 5 ) P . D o t y and R. D. Lundberg, Proc. N u l l . Acad. Sci., 43, 213
(1957).
A/I
[ a 1"D
Dirnetliylformamide High +lo Dimethyliormamide 93 +lo Dimeth ylformamide 22 8 Dichloroacetic acid 104 -33 Corrected for refractive index of solvent; a 212 m p was used as XO.
+
boa
-544 -532 -4486 56 value of
+
Rotatory dispersion as a criterion for helix content has been established and examined by Xloffitt,'2 Doty,2 Cohen,13 Tinoco,14 Blout15 and Elliott.IG Moffitt expanded the Drude equation to include a higher order term.'? He suggested that a plot of [ a ](X2-Xr,2) us. ( X 2 - X 2 ~ ) - ' with other than a zero slope exhibits abnormal rotatory dispersion, indicating helical structures. Doty2 suggested the use of A," as a criterion of helicity for some synthetic and naturally occurring polypeptides. We determined the optical rotatory dispersion of the oligomeric compounds in helix and random coil solvents. In dichloroacetic acid Moffitt plots indicated material essentially of a random coil nature. In dimethylformamide, however, increasingly negative abnormal dispersions were observed in going from the trimer to the undecamer (Fig. 1) (Table 11). ( 6 ) A . R . I h m n i e , 4 . Elliott, W. I?. Hnnby a n d R . R . n l : c l c . ~ ~ l m , P r o i R o y . SOL.( r n v d o i i ) , A242, :12h (1957). ( 7 ) I. Tinoco, THISJOTJRNAL, 81, 1 5 4 1 (1959). ( 8 ) C . Cohen, N n t i w r , 1'75, 129 (1955). (9) E. K. Blout, P. Duty and J. T. Yang, T H E J C J U R N A I . . 7 9 , 719 (195i). (10) P. Doty, J , H. Bradbury nnd A . h l . IIoltzer, ibici., 78, !417 (195Ij). ( 1 1) 17. Yphantic, A n n . .\'. 1'. A r o d . Sei., to be piibliahed (12) (a) W hloffitt, J. C h e f s . Phys., 2 5 . 4R7 (195fj); (b) W. \loOift and J. T . Yang, Proc. T o l l . A c a d . S c i . , 4 2 , 5 O l i (1956); (c) W .hloffitt.
;bid., 42, 736 (1956); (d) W. Moffitt. D. D. F i t t s and J. G. Kirkwood, ibid.. 43, 723 (1957). (13) C. Cohen and A . G. Szent-Gyorgi, THIS J O U R N A L , 79, 218 (1957). (14) I. Tinoco and R . E'. Woody, J. Chenz. P h y s . , 3 2 , 461 (19fiOi. (15) E. R . Blout in C. Djerassi. "Optical Rotatory Dispersion." Chap. 17, hIcGraw-Hill Book Company, Inc., New Ymk, N. Xr.,19GO. (16) E. hl. Biadbury, L. Brown, A. R. Downie, A . Elliott, W. E. H a n h y and T. R. R. McDonald, Nature, 183, 1736 (19.59). (17) Derived from the square root of the slope obtained from a plot Of
[Q]
VS. xy[P].
