8768
COMMUNICATIONS TO THE EDITOR
Vol. 81
In the double-irradiation experiments on CH?DOH the C13 perturbing field was such that ~ C I J H"/2n = 3-4 cps., and the high-field CI3 satellite of the methylene protons was observed by the usual field-sweep method with the magnetic field increasing from left to right. The extent of the field'I I sweep (about 4 cps.) in observing the spectrum cor- 32 -23 -10 0 * 13 t22 t37cps responds to a shift of the C13 resonance of only 1 cps., and thus does not lead to complications. Because the frequency of the perturbing field is constant to only f 1 cps. in the present experiments, the lines of the doublet are broadened and have -225 0 *225cps Fig. 1.-Top: spectra of the C13satellite of the methylene much less than their correct heights. It is, however, very easy to see which line of the original triprotons (60 Mc./sec.) of CHaDOH under conditions of rapid exchange of the hydroxyl protons and with double-irradiation plet is being perturbed (;.e., split into a doublet), a t about 15.1 Mc.jsec. The frequency of t h e second radio- as this line disappears from the spectrum. The frequency field relative t o the resonance frequency of the nine frequencies of the C13 resonance were located center C13 resonance is shown under each spectrum. The by such experiments and showed the correct spacings. Figure 1 shows the effect when the frequency arrows correspond t o t h e center of the satellite in the unperof the decoupling field is in the neighborhood of the turbed spectrum. Bottom: diagrammatic representation center triplet of the C13resonance. The three lines of the center triplrt of the C13 spectrum of CH2DOH at of the proton spectrum, and likewise of the C13 c m s t a n t field. spectrum, arise from the three possible ( + l , 0, to determine the relative signs of J c ~ and ~ H Jge,- HH -1) deuterium spin states. When the frequency (or J z . i c - 1 < ~ )This,4 . in effect, would allow an as- corresponds to the low-, middle- or high-frequency signment of absolute signs to the experimentally lines of the C13 triplet, i t is the low-, middle-, determined Juic, J,,, and other proton-proton or high-field lines, respectively, of the proton speccoupling constants. trum which are perturbed. Frequencies which are By using a modified spin-decoupling technique, different from these by more than 10 cps. are withand HJ g e m - - ~(and out appreciable effect. Since a low-field line in a we have now found that J C I ~ hence Jgem-") are of opposite signs in CH2DOH. sweep-field spectrum corresponds to a high-freIf the proton spectrum is to be observed, the double- quency line in a frequency-sweep spectrum, and irradiation method5 for the determination of rela- vice versa, the coupling constants JCLH and JEem- K D tive signs would involve, in the present case (or in are of opposite signs. Since the magnetic moments simpler systems, e.g., RRCI3 DH), "decoupling" of H and D have the same signs, JCLH and Jgern--~ the C13for a particular deuterium spin state. This are also of opposite signs. If the positive sign of is not possible, because of the relative magnitudes of J C ~ L H is accepted, then Jgern-" is negative in the coupling constants (e.g.>J c ~ ~140.6; - H >J c ~ ~ - methanol, D, and probably in general, provided the 22.5; J H D ) 1.7 cps. for CH2DOH). However, the carbon atom concerned is sp3hybridized. Thus, the desired result can be achieved if the CI3 is merely calculated sign of the geminal coupling constant is perturbed for a particular deuterium spin state, but in disagreement with experiment. not for others. This can be done by irradiating the The spectra were taken on a Varian V-4302 specC13 with a relatively weak magnetic field H" trometer equipped with a "spin-decoupler" made ( y c d 1 / / 2 ? r ao; are planned to test this possibility. More likely, -4 = 4Kao (bo - a0 K ) * ;and kl is the second- however, the variation in K1 reflects a heterogeneity order specific rate constant for the forward reaction. of Ab sites. Such evidence for Ab heterogeneity The results of one representative series of experi- is particularly significant in this case, in view of ments are shown in Fig. 1, plotted according to these considerations : (a) the unusually high degree equation (1). Each curve is drawn through the of uniformity of the antigenic determinant used points of three individual experiments with the to elicit anti-DNP Ab? (Ab heterogeneity in many same Ab and Hp solutions. The downward curva- other cases, especially where the antigen is prepared ture exhibited in Fig. 1 is reproducibile and real, by diazonium coupiing reactions, 9,10 may result and is independent of any possible uncertainty in primarily from the heterogeneity of the antigen) ; the values of K used in equation (1). The curva- (b) the use of a single rabbit antiserum, rather ture appears to reflect a decrease in the average than a pool; (c) the relatively mild treatment to value of K1 as the fraction of Ab sites reacted in- which the Ab was subjected; and (d) the similarity of the results with the zwitterionic Hp e-N-DNP(3) s. F. Velick, C. W. Parker and H. S. Eisen, Pvuc. N u l . A c a d . Sci. U . S., 4 6 , 1470 (1960). L-lysine and the anionic H p N-DNP-e-aminocaproic (4) J. M. Sturtevant, unpublished work. acid (which suggests that the gross heterogeneity klt = A
In
--L
+
+
( 5 ) F. S. Farah, M. Kern a n d H. S . Eiren, J . ExO. .%fed., 112, 1195 (19ciO). ( 6 ) We are indebted to Dr. Henry \[etzgpr for the pirification of the a n t i - D S P Ab (7) J. W. Baker anrl M I, ITcnitning, J f h ' m So?. (Tmndon), 144, 191 (1942).
(8) R. R. Porter, Biorhem. J . , 73, 119 (1939). (9) E. W. Gelewitz, W. L. Riedeman and I. M. Klotz, 4 ~ r h Rio. c h r i r i . Biophys., 63, 111 (1954). (10) M, Tahachnirk and H. Snbotka, J . B i d . Chem , 236, 1051 (1980).
3770
COMMUNICATIONS TO THE EDITOR TABLE I
Vol. 54
helical conformation have been shown to exhibit
APPARENTSECOND ORDERRATECONSTANTS AS A FUNCTIONa marked decrease in intensity of the absorption band (molar extinction coefficient approximately OF EXTENT OF REACTION 4,000) in the 190 mp region compared with the In units of M-l set.-' X lo-’, under conditions given in same peptide in random coil conformation (molar Text. cN-DNP-LLysine
Initial ratio [Hp]/[Ab sites] + Fraction of total Ab sites reacted 0.1 .2 .4 .6 .8 .9
0 . 5 2.0
9.0 7.9 5.8
.. ..
..
.. ..
8.4
..
..
6.8 . . 6.2 3.9 2.5 2.0 . . 1.4
N-DNP-cAminocaproate
0 . 5 2.0
8.1
13.2 . . .. 11.1 . . .. 9.9 9 . 0 . . 7 . 7 5.9 .. 5.1 2 . 1
..
..
..
1.5
in net electric charge of pure anti-DNP Abs is not primarily responsible for the apparent kinetic heterogeneity). The maximum value of kt for both haptens appears to be about 1 x IO8 M-l sec.-l, which makes this one of the most rapid bimolecular reactions in homogeneous solution known to biochemistry. It is interesting that the same k1 value was estimated’ from stopped-flow experiments with antiD N P Ab and an entirely different DNP-hapten, 2-(2,4-dinitrophenylazo)-1-naphthol- 3,6-disulfonic acid, by an independent spectrophotometric technique. One would predict1l*l2 for the present system that diffusion would limit the rate constant to a value of about lo9 M-‘ set.-'. Such a large value for l i l suggests that no substantial conformational rearrangement (requiring an appreciable free energy of activation) occurs within the Ab molecule, or active site, upon specific binding of the hapten. This research was aided by grants RG-4725 (J.M.S.) and E-4246 (S.J.S.) from the National Institutes of Health, U. S. Public Health Service. (11) J. H. Wang, J. P h y s . Chem., 69, 1115 (1955). (12) G. V. Schulz, 2. physik. Chem., 8 , 284 (1956).
extinction coefficient approximately 7,500). Helical structures also exhibit a new absorption band or shoulder in the 205 mp region. Polylysine, polyglutamic acid and polyalanine are examples of synthetic polypeptides which have been studied by this technique. We have applied the study of far ultraviolet absorption spectra to oligomeric peptides derived from y-methyl-L-glutamate where the amino end is blocked by a benzyloxycarbonyl group. l s 7 All evidence which we have developed to date on the critical size necessary for helix formation has depended on optical activity measurements. The application of hypochromic studies from absorption spectra described in this paper provides the first data for the independent verification of the onset of helical conformations in the critical range of peptide chain length. The solvent for our investigations was 2,2,2trifluoroethanol which has low absorption in the 190 mp region. A Perkin Elmer model 350 spectrophotometer was used in this work. It was kept air free by continuous flushing with dry prepurified nitrogen. This enabled us to work in the region down to 189 mp with a slit width of less than 0.6 mm. and to 187 mp with a slit width of less than 1.5 mm. Matched quartz cells (0.1 mm. width) were used. The concentrations of the peptides studied were in the range of to mole/ liter. The molar extinction coefficients (E’) are shown in Fig. 1 as a function of wave length for various oligopeptides and for a high polymer of y-methylL-glutamate. These extinction coefficients (e’) have been corrected for the absorption of the benzyl group of the benzyloxycarbonyl blocking group in the oligopeptides.8 €’peptide
= cobserved
-
fCtH6CR)
x
ECEHKCHI
CONTRI~UTION No. 1709 DEPARTMENT OF CHEMISTRY LORENA. DAY wheree‘ is the molar extinction coefficient per residue YALEUNIVERSITY JULIAN M. STURTEVANT corrected for benzyl group absorption. €observed is NEW HAVEN, CONNECTICUT the observed molar extinction coefficient. fCsHaCH3 DEPARTMENT OF BIOLOGY is UNIVERSITY OF CALIFORNIA AT SANDIEGO S. J. SINGER the weight fraction of the benzyl group in the H ,the molar extinction coefficient molecule. E C ~ H ~ Cis LA JOLLA, CALIFORNIA of toluene in this region. RECEIVED AUGUST6, 1962 CONFORMATIONAL ASPECTS OF SYNTHETIC POLYPEPTIDES. VI. HYPOCHROMIC SPECTRAL STUDIES OF OLIGO-7-METHYL-LGLUTAMATE PEPTIDES
sir :
Absorption spectra of high molecular weight polypeptides and proteins in the far ultraviolet region have been employed for the study of their conformations in solution.2-6 Peptides in the (1) Previous paper in this series, M. Goodman, I. Listowsky and E. E. Schmitt, J. A m . Chem. Soc., 84, 1296 (1962). (2) I. Tinoco, A. Halpern and W. T. Simpson, in “Polyamino Acids, Polypeptides and Proteins,” ed. M. A. Stahmann, University of Wisconsin Press, Madison, Wis., 1962, p. 147. (3) P. Doty and W. B. Gratzer, ibid., p. 111. (4) K. Rosenheck and P. Doty, Proc. N d . Acad. Sci., 47, 1775 (1961).
With the pep tides studied the hypochromicity of the absorption band measured a t 189-190 mp commences at the nonamer, increasing in magnitude for the higher oligopeptides and the polymer. The appearance of the shoulder a t the 205 mp region also begins a t the nonamer, increasing in intensity with chain length. Assuming that the high polymer exists entirely in the helical state and the pentamer and heptamer possess no helical conformation in this solvent, we have calculated the helical content for any of the oligomers from the extinction coefficients a t
(5) W. B. Gratzer, G. M. Holzworth and P. Doty, ibid., 47, 1785
(1961).
(6) I. Tanaka and K.Imahori, J . Mol. Biol., 1, 359 (1959). (7) M. Goodman, E. E. Schmitt and 13. Yphantis, J. A m . Chem. Soc., 84, 1288 (1962). (8) J: R. Platt and H. B. Klevens, Chcm. Rer., 41, 301 (1947).
