ROBERTCARLKRUEGER
LjS2
[CONTRIBUTION FROM THE
Vol. 72
DEPARTMENT O F CHEMISTRY, COLUMBIA UNIVERSITY]
The Effect of Ascorbic Acid on the Enzymatic Oxidation of Monohydric and o-Dihydric Phenols1 BY ROBERTCARLKRUEGER~
Several studies in these laboratories in recent years have dealt with the enzymatic action of tyrosinase on various substrates in the presence of 1ascorbic a ~ i d . ~ . ~ ' ~ * ~ In these studies, 1-ascorbic acid (AHZ) had been assigned the role of a reducing agent for any oquinone produced in the action of the enzyme on the various substrates. The AH2was assumed to act according to the scheme
.
Tyrosinase, Os
(1) Catechol
;---
1-Ascorbic Acid.-The ascorbic acid was obtained from Merck & Co., Inc., and was shown t o be more than 99% pure by titration with standard potassium iodatese The acid was weighed out on an analytical balance immediately before using. &(3,4-D&ydrorsphenyl) -alanine (Dopa).-The dopa for these experiments was isolated from the Georgia Velvet bean according t o the method of Miller.]@ AnaZ.ll Calcd. for C ~ H I ~ O , NC, : 54.6; H, 5.6; N, 7.1. Found: C, 54.4; H, 6.8; N, 7.0. 3,4-Xylenol.-The xylenol was a pureo grade from Fra$nkel and Landau, Berlin; m. p. 64-66 ; b. p. 225226
o-quinone
t2.
--+ Dehydroascorbic acid
Results The Effect of Ascorbic Acid on the Oxidation
of Monohydric Phenols.-Figure 1 shows the effect of ascorbic acid on the oxygen uptake in the In this investigation, the enzymatic oxidation enzymatic oxidation of tyrosine. The point C of tyrosine, $-(3,4dihydroxyphenyl)-alanine represents the point of coloration and thus the (dopa) and protocatechuic acid in the absence and point at which the ascorbic acid has become compresence of ascorbic acid has been measured by pletely oxidized. The oxygen uptake at point C manometric means. In other experiments pre- was found to be 140 * 5 cu. mm.a (4determinasented, the oxidation of tyrosine and 3,4-xylenol tions), 12 cu. mm.3 more than that needed to oxiin the absence and presence of AH2 has been fol- dize the ascorbic acid present according to equalowed by analyzing aliquots of the reaction mix- tion 2 (128 cu. mm.a). It is apparent that the ture. The results indicate that AH2, in addition dopa produced in the system does not bind the ento its function as a reducing agent for o-quinone zyme completely and stop the tyrosine from (reaction 2 ) , is acting in another capacity. AH1 oxidizing. This view is supported by the direct appears to function both as a pro-oxidant and an- analysis experiment (Fig. 3). These results are ti-oxidant in these systems and the indications are contrary to those of Robinson and Nelson6 who that it becoiiics oxidized in part by an induced re- showed that tyrosine was not oxidized and attrib:iction. uted this result to the complete binding of the tyrosinase by the small amount of dopa formed; Experimental The Enzyme Preparations. -These were prepared by the latter was oxidized by the enzyme and caused the oxidation of the ascorbic acid v i a a shuttling 11r. Stanley Lewis of these laboratories as described.' Preparation C259A2 contained 7000 catecholase units mechanism (equations 1and 2). i n d 194 cresolase unit.;/ml Preparation C259A3 conFurther evidence that dopa does not have a tained 3,500 catecholase units mid 97 cresolase units/ml. Later in the course of this work, this preparation was high affinity for the enzyme compared to the affound to have degraded and on the basis of tyrosine and finity of tyrosine for the enzyme has been obdopa oxygen uptake measurements had an activity of tained in this investigation by comparing the dis2740 eatecholase units/ml. sociation constants of the substrate-enzyme comI-Tyrosine.-The I-tyrosine used was Eastman R d a k Co. pure grade recrystallized from water. For the rcs- plexes. The Michaelis-Menten12 constants dejtirometer experiments, the tyrosine w a ~used a s it 2.0 termined by the method of Lineweaver and BurkI3 tng. /ml.suspension were found to be 0.0005 and 0.0006 mole/liter for the dopa-enzyme and tyrosine-enzyme complexes, ( 1 ) From a dissertation submitted in partial fulfillment of the rerespectively. quircments for the degree of Doctor of Philosophy in the Faculty of Science, Columbia University. Furthermore, the rate of oxygen uptake when Department a i Riological Chemistry, Co1li.w. of Medicine, tyrosine is oxidized in the presence of ascorbic acid T:mversity of Cincinntrti. Cincinnati. Ohio. is dependent on the tyrosine concentration. (31 IV. FI, Miller a n d i' K D a w s o r i 'I'IIIS .T
