Oct. 20, 1964
COMMUNICATIONS TO THE EDITOR
on neopentyl chloride as the abundance of the parent peak was found to be 5 10-47c relative to the base peak Liven by the t-butyl ion. The A.P. of the lower chloroalkanes are given in Table I. Evidently they depend primarily on carbon
4499
mechanism (eq. 1) is that reported for imidazole itself.' protolysis
Im
+ H + + I = + HzO
*/ 'H,
kw
h\
direct transfer
TABLE I APPEARANCE POTENTIALS OF THE CHLOROALKANES, RC1
R
r---A.P.(RCI+), E I.a
e.v.--P.I.h
A.P. (CHaCCI) - A.P. (RCI)
0 -21 243 1 1 . 4 4 r!r 0 . 0 2 d 11.28 10.97 0.34 -26 230 1 1 . 1 0 i 0.06" 10 82 0.66 -32 217 1 0 . 7 8 =k 0 04' 10 77 rt 0 . 0 3 10.78 0.67 -35 214 10.67 0.95 10.50 i: 0 . 0 7 -37 205 10.66 0.94 -38 204 10 48 i 0 . 1 10 65 0 91 10.52 =t0 . 1 -40 203 IO 61 1.14 -43 195 L-C~HE 10 3 i 0 . 1 a Electron impact, this work. b Photoionization, ref. 2. c JH,(KCl) from ref. 5. d Mean value in ref. 2 is 11.37 e.v. J Mean value in ref. 2 is 11.83 e Mean value in ref. 2 is 11.19 e . v e.v
CHI CzHs n-CXH7 i-CsH? n-C4Hu t-CdHg ser-C4Hg
number, although t-butyl chloride shows an additional effect owing to chain branching. Heats of formation of the parent ions, AHf(RCI+),can be found by combining literature values5 of AHr(RC1) and our A.P. ( R C l I ) . Now it is well known that in homologous series, lengthening or branching the chain confers stability; that is, AHf decreases. This is the trend found for the chloroalkane ions. Alternatively, one can regard the A.P.(RCl+j relative to A.P.(CH3Clf) as a measure of stabilization. These A.P. increments suggest that even if a localized electron on chlorine were removed in process 1, the charge on the resulting ion, C,tH2n+lC1+, is then essentially delocalized as might be expected for the analogous ion, CnHzn+z+. Finally, it is usual that A.P. obtained by photoionization are lower than those found by electron impact.2 As the reverse was found here for the chlorobutanes, this is a discrepancy which needs to be resolved.
+
+
IniH+ HIOHhydrolysis ImH = imidazoliuni ion, H I - = protonated indicator
A H f , kcal. mole-' RClc RC1+
+
The experimental data are quantitatively fit by eq. 2 and 3, derived using an appropriate steady-state treatment for [ H + ]and [OH-]. 7-1
rm-1 =
= rm-1
+
rp-1
+
(2)
TH-1
+ [T) + k ~ ( [ I m l+ JHI-1)
k~([ImH+l
kll[HI-] where TDT
7
=
+ k43[ImH+]
1
over-all relaxation time (10 to 70 psec.),
= direct transfer relaxation time, r p = protolysis
relaxation time, and T H = hydrolysis relaxation time. The bars over the concentrations indicate equilibrium concentrations a t the upper temperature (10'). Our experimental system consisted of imidazole (or protein), phenol red (indicator), and 0.1 M KN03. The pH dependence of r for imidazole and several proteins (Fig. 1) is well fitted by eq. 2 and shows a dominance of TDT. There was no complication from carboxyl or ammonium protonic processes in the pH range studied. ( 5 ) S. \V. Benson and A . N. Bose, J . Chem Phys., 39, 3463 (1963). The dependence of 7-l on total protein concentration (6) National Science Foundation Senior Postdoctoral Fellow, 1963-1964. is linear'with an intercept value of (1.00 i 0.05) X io4 Correspondence should be addressed t o the Department of Chemistry, 111inois Institute of Technology, Chicago 16, Ill. set.-' for all the proteins a t pH 7.5 and 4.6 x 10-5 M M. BALDWIK total phenol red. -4ccording to eq. 3, the intercept is a DEPARTMESTOF CHEMISTRY ALLAXMACCOLL function of all the rate constants and the indicator conVXIVERSITY COLLEGE SIDSEYI . MILLER^ LOSDONW . C . l . centration. Although small variations in the protolytic RECEIVED A U G U S T 24, 1964 and hydrolytic rate constants would not be detected, the constancy of the intercepts demands approximate constancy of all the rate constants or a set of accidental A Kinetic Study of the Imidazole Groups of compensations yielding a constant intercept. AcciChymotrypsinogen, Chymotrypsin, and Some dental compensation appears improbable and if it is Derivatives, Using the Temperature- Jump Method rejected, k13, k31, k,,, k z l , k34, and k43 are essentially inSir : variant for all the "available" imidazole groups of Ionizable groups of proteins are best characterized chymotrypsin and its derivatives. The slope of a T - I by the rate constants of their protonic processes. I t is v s . protein concentration plot thus measures the "availof interest t o learn through the values of these conable" concentration of imidazole groups, defined as stants how the factors of protein structure modify the those groups which are capable of reacting with indiproperties of these groups relative to those of small cator. The direct transfer constants k13 and kal are then model compounds. Relaxation methods can provide a measure of the extent to which the environment prorate constants when the protein has only a few groups vided by the protein hinders the direct transfer reaction of a given type. The two imidazole groups of chymoin eq. 1. Although the rate constants have not yet trypsin (CT) form such a case and in the presence of a been refined with statistical methods, estimates of pH indicator are the source of a large chemical relaxak13 and k31 are 2.9 X 108 and 5.8 X lo7 M-I set.-', tion transient measurable with a precision of 3YG or (1) ,M. Eigen, G. G. H a m m e s , and K . Kustin, J. A m . Chem. Soc., 82, better using the temperature-jump technique. The 3482 (1960).
COMMUNICATIONS TO THE EDITOR
4500
1 -
lo
6.0
65
7.0 PH
7.5
8.0
85
9.0
Fig. 1.-pH dependence of 7 - l . A11 concentrations are total concentrations; temperature 10 f lo, ionic strength 0.1 [ K S O n ) Af pheno Curve 1, 0.8 X model imidazole, 2 . 3 X red: curve 2, A, 8 X C T , 4.6 X J I phenol red; curve 3 , 0,8 X 1 0 ~ CGX, 6 4.6 X Mphenol red; curve 4, v,8 X 10-5 M T P C K -CT (94.5% inactive), 4.6 X 10-5 JM :If DIP-CT, 4.6 X l O - 6 M phenol red; and curve 5 , @ 8 X phenol red
respectively, for the proteins, to be compared with 7 X lo8and kS1 = 1.3 X IORA P 1 sec.-l for model imidazole. These results suggest that the “available” imidazole groups of these proteins experience relatively slight environmental restrictions. The “available” concentration of imidazole groups in C T is twice the total protein concentration as expected from titration data.2 The ~-1-tosylamido-2phenylethyl chloromethyl ketone derivative of CT (TPCK- CT) has only one “available” imidazole group by the present method of analysis, in agreement with other evidence indicating that the catalytic-site imidazole in TPCK- C T is eliminated.3 The relaxation time profiles in the several variables were identical for diisopropylphosphorylchymotrypsin ( D I P - - C T ) and T P C K - C T when corrected for a C T impurity of 5.5% in our TPCK-CT samples. This suggests that the D I P group blocks indicator attack on imidazole, consistent with a close approach of the imidazole and serine groups a t the catalytic site. Since TPCK--CT has lost the protonic processes of the catalytic-site imidazole, it is possible that this group is also not free for protonic reactions in DIP-CT. By these tests T P C K - C T and C T differ only in the concentration of “available” imidazole groups.. Rejecting accidental compensation, i t is possible to conclude that both imidazoles of C T are essentially identical in behavior with the single “available” imidazole group of TPCK- C T and. consequently, that the catalytic-site imidazole group shows no special ionization properties. The behavior of chymotrypsinogen ( C G S )is peculiar and will require additional study. The intercept on a - I “,. ‘s. C G S concentration plot is identical with t h a t ob-
tained for CT, TPCK-CT, and DIP-CT. This suggests t h a t the rate constants for “available” imidazoles are the same for CGX as for the other proteins. If so, the “available” imidazole concentration in CGN is about 1.5 (rather than 2 or 1) times the total protein concentration. The effects of substrates and competitive inhibitors on the rate constants and “available” imidazole concentrations are currently being investigated. We have found t h a t 2 X lop3-I.I indole increases r by 3XyGwhen total [ C T ] = l o p 4 M . Indole is a competitive inhibitor having a dissociation equilibrium constant of 7.0 x -11.The increase in r expected if indole binding prevents the direct transfer reaction a t one of the two CT imidazoles can be calculated to be 37%. Consequently, it appears that the binding of indole causes some type of protective infolding of one imidazole group, reducing its “available” concentration. This finding strongly suggests t h a t the binding of substrate side chains, through changes in folding, exerts direct control on a functional group participating in acylation or deacylation. The method is thus able to provide detailed information about the catalytic process even a t this early stage of development. DIP-CT and TPCK-CT were prepared from Worthington a-CT, and Worthington CGN was used. Acknowledgment.-This work was supported by the National Science Foundation. LABORATORY FOR BIOPHYSICAL CHEMISTRY DEPARTMEXT O F CHEMISTRY UNIVERSITY 3~ MINNESOTA h$INNEAPOLI:S 14, MINNESOTA
k13 =
& L f . ..\ M a r i n i a n d C Wunsch. Biorhrintsli
‘,P(‘, Schiiellmann a n d E . Shaw. tb2d
,
2 , 252
Vol. S(i
YAPEL RUFUS LUMRY
!LNTHONY
RECEIVED AUGUST14, 1964
Phenylbis (dimethylamino)fluorophosphonium
Phenylpentafluorophosphate Sir: -4 recent report2 on some aryldialkylaminotrifluorophosphoranes, -4rPF3NR2, led us to include this type of compound in a general study of five-coordinate stereochemistry, conducted by means of Fl9 n.m.r. spectroscopy.3 BoththeF19and P314n.m.r.spectrafor anyR2PFs species, except the perfluoroalkyl derivatives, (Rf)2PF3, could be interpreted in terms of a trigonal bipyramidal arrangement of the ligands around the phosphorus atom, the fluorine atoms occupying the two axial and one equatorial positions. Among a number of alkyl- (aryl-) dialkylaminotrifluorophosphoranes, which we have investigated, one clearly stands out with respect to an unusual transformation occurring upon storage of this compound. Phenyldimethylaminotrifluorophosphorane,C6HaPF3N(CH3),, not previously reported, was prepared using the method of the Russian workers,2 via C6H5PF3H and CaH5PF3C1, the latter being allowed to react with dimethylamine. %:e have also obtained the compound less expediently by the reaction of phenyldimethylaniinochlorophosphine with arsenic or antimony tri( l j Phosphorus-Fluorine Chemistry. X I . F o r X in this series s e e . I< Schmutzler, Z . .Valur.forsch.. In press. ( 2 ) Z h . X5 Ivanova atid A . V. K i r s a n o v , Z h u r . Obshch. K h i m . , 32, 2R42 (1962) (a: E. J.. Muetterties, XV h l a h l e r , a n d I
