CHOHHAOLI
1716
appeared in their: effect on the physiological function of nitrogen fixation in Azotobacter. It is coneluded, therefore, that an explanation based on the relative physical properties of hydrogen and nitrogen is unlikely.
[CONTRIBUTION FROM
THE INSTITUTE OF
Vol. 70
Nitrous oxide was found to be a specific inhibitor for nitrogen fixation by Azotobacter. Its inhibition may have important implications for the mechanism of the reaction. MADISON 6, WISCONSIN
RECEIVEDDECEMBER 9, 1947
EXPERIMENTAL BIOLOGY, UNIVERSITY OF CALIFORNIA]
Kinetics of the Reactions between Iodine and Certain Substituted Phenols BY CHOHHAOLI In previous studies' it was shown that the reaction of tyrosine with iodine follows a bimolecular rate law and that the most reactive iodinating agent is hypoiodous acid. It was also demon&rated2 that the formation of diiodotyrosine is catalyzed by phosphate and other basic ions. The present investigation extends such studies with other para substituted phenols. Reactions were carried out a t 25' in acetate buffers of pH 5.23 and 5.65 containing different iodide ion concentrations. p-Chlorophenol and p-hydroxyphenylethylamine (tyramine), C. P. crystalline preparations, were employed without further purification ; glycyl-tyrosine was kindly supplied by Dr. J. S. Fruton and the late Dr. Max Bergmann. The reaction rates were followed in the manner previously' described. The rate law was found to be identical with that for the formation of diiodotyrosine and may be expressed by the equation
- d(Phenol)/dt
= kz(phenol)(Ia-)
(1)
where kz is the specific rate constant for the reaction Phenol 212 + diiodophenol 2H+ 21 Table I summarizes the specific rate constants for the formation of diiodophenolsa; the values
+
+
+
for the reaction between iodine and tyrosine were estimated from previous studies. The concentrations are in moles per liter, and time in minutes. It may be noted that the product of R2(I-)2in each buffer appears to be rather constant at the range of iodide concentration studied. In concentrations of iodide beyond the range studied, was not found to be constant. In the study of diiodotyrosine formation,' it was found that the reaction involves four paths: namely, iodine and phenol, iodine and phenolate, hypoiodous acid and phenol, and hypoiodous acid and phenolate. It was further noted that the most reactive pair is hypoiodous acid and phenolate, whereas the reaction between iodine and phenol is the least reactive. For first approximations, the reaction between iodine and phenol may be represented by equation (la). -d(phenol) /dt = &'(phenol)(HOI)
+ k"(phenolate)(HOI) (la)
From the equilibria 1-8-
-1
2
+I-
+ Hs0 I_ HOI + H + + IR-O-OH _T ~ 0 - 0 +- H + 12
(2) (3) (4)
TABLE I SPECIFICREACTION RATE,kz, OF IODINATING p-CHLOROPHENOL, GLYCYLTYROSINE, TYRAMINE AND TYROSINE'" I N ACETATE DIFFERENTIODIDE CONCENTRATION AT 25' BUFFERSOF pH 5.23 AND pH 5.65 CONTAINING k2 in gram moles per liter per minute (I-)
m X 102
--p-ChlorophenolpH 6 2 3
pH 5.65
--
p H 5.23
TyraminepH 5.65
--Glycyl-tyrosinepH 5.23 pH 5.65
--TyrosinepH 5.23
pH 5.65
3.34 0.135(0.128)b 0.40(0.34) 0.70(0.72) 2.03(2 29) 0.91(0.94) 3.15(3.21) 0.52(0.53) 1 82(1.94'1 4.08 .081( ,085) .26( .26) .45( .45) 1.63(1.49) .63( .63) 2.1q2.14) .38( .36) 1.20(1.301 4.84 .060( .061) .18( .18) .35( .38) 1.09(1.07) .45( .49) 1.55(1.55) .28( .261 0 YO(0.93) .20( .22) 0.63(0.62) .16( .15) 0.53(0.541 6.34 .033( .035) .11( .11) Reaction rates for tyrosine are from a previous paper (see ref. 1). * The figures in parentheses are computed values from Equations 7,8, 9 and 10. 0
(1) Li, THISJOURNAL, 64, 1147 (1942). (2) Li, ibid., 66, 228 (1944).
(3) The preparation of N-glycyl-3,5-diiodotyrosine has been reported by Abderhalden and Guggenheim [ B e . , 41, 1241 (1908)l. Diiodotpramine has also been prepared by Abderhalden and his COworkers [Arch. ECS. Phrsiol., 195, 167 (1922)l. As far as we are aware there is no report concerning the preparation of P-chloro-3,5diiodophenol. In the present experiments we have observed that Pchloro-3,5-diiodophenol is very insoluble in acetate buffers and it is gradually crystallized out in fine needles as the reaction proceeds. The colorless crystals have a melting point at 108.5O. Anal. Calcd for HQCsHdzCl. J , 66.74. Found: I . 66.82
Equation (la) becomes
-= (1- 1K *(H g +) [K'& + dt d(phenol)
s] (phenol) (11-1
(5)
where &, & and Kq are the equilibrium Constants
of equations 2, 3, and 4, respectively, Thus, by 'Omparing the equations (l) and ( 5 ) , the Observed specific rate constant, kz, i s a function of iodide and
May, 1948
KINETICS OF REACTIONS BETWEEN IODINEAND SUBSTITUTED PHENOLS
1717
hydrogen ion concentration as shown by the expression
The computed specific rates are listed in Table I; it may be noted that the agreements between the calculated and observed values are satisfactory. From Table 11,it is evident that, in each phenol studied, the value of k’ is always much smaller It is obvious from equation (6) that when the hydrogen-ion concentration is maintained un- than that of k”. This indicates that the phenolate changed, the product of k ~ ( 1 - ) ~becomes a con- ion is far more reactive than the undissociated stant. Since the equilibrium constants4 of equa- phenol. It is also clear that the smaller is the tions (2) and (3) and the dissociation constants of dissociation constant of a phenol, the faster is tyramineJ6 gly~yltyrosine~ and the reaction rate. For instance, the dissociation tyrosine8 are known, the specific rate constants k’ constant of p-chlorophenol is fifty times higher and k” can be computed from the data in Table I. than that of tyramine and yet the latter reacts The results are summarized in Table 11. There- with hypoiodous acid much faster than p-chlorophenol. TABLE I1 It is of interest to note that the specific rate for THE COMPUTED VALUESOF k’ AND k” FOR p-CHLORO- glycyltyrosine is greater than the value for tyrosine. It appears to indicate that the presence of a PHENOL, TYRAMI!iE, TYROSINE AND GLYCYLTYROSINE IN peptide linkage enhances the rate of iodination. ILCETATE BUFFERAT 25’ This inference may also be arrived a t by an experiDissociation constant ment using carbobenzoxyglutamyltyrosine. In Phenols K (1011) k’ X (10-0) k” X (IO-lo) acetate buffer of pH 5.65 containing an iodide con+Chlorophenol 66.0 (5)“ 1.7 0.2 m a t 25’, the biomolecucentration of 3.34 X Tyrosine 8 . 5 (8) 8.1 1.1 lar specific rate, k2, of iodinating carbobenzoxygluGlycyltyrosine 4.0 (7) 12.0 40.0 tamyltyrosine was found to be 4.24 gram-mols per Tyramine 1.26 (6) 10.0 58.0 liter per minute which is more than twice the speNumbers in parentheses refer to the reference in the cific rate for tyrosine. Whether the conclusion text. can be generalized for other peptides requires furfore, in a solution of known hydrogen-ion and ther investigation. iodide-ion concentrations, the observed specific The absence of the carboxyl group in tyrosine rates for iodinating p-chlorophenol, tyrosine, tyra- causes an increase in iodination rate. Thus, k” mine and glycyltyrosine may be computed from for tyramine is about 50 times larger than that for the equations tyrosine. On the other hand, a replacement of -CH2CH2NH2 group in tyramine by -C1 radical #-Chlorophenol : greatly diminishes the specific rates of iodination. No satisfactory explanation can be offered a t present to correlate the structure of substituents on the Tyrosine : iodination rate of phenols.
Summary
Tyramine :
ka = -(I-)*(€€+) [4.2
x
10-9
+ 3.06(;+;
0-
151
(9)
Glycyltyrosine :
The rate of the reactions between iodine and pchlorophenol, tyramine and glycyltyrosine have been determined a t 25’ in acetate buffers. In the range of iodide concentrations studied, the rate can be represented by: rate = k’ (phenol)(HOI) k” (phenolate)(HOI). The specific rate of the reaction between phenolate and hypoiodous acid decreases in the order : tyramine > glycyltyrosine > tyrosine > p-chlorophenol.
+
(4) (a) Bray and MacKay, THISJOURNAL, 82, 914 (1910); (b) Bray, ibid., 84, 932 (1910). ( 5 ) Murray and Cordon, ibid., 57, 110 (1335). (6) Ogston, J . Chsm. Soc., 1713 (1336). (7) Greenstein, J . B i d . Chem., 95, 485 (1332). (5) Hitchcock, J . Gen. Phyriol., 6 , 747 (1925).
BERKELEY, CALIFORNIA
RECEIVED M A Y 29, 1947
