Previous work has indicated that coenzyme A reacts in vitro with oxidation products of epinephrine and norepinephrine (1). The present study suggests that it reacts, in addition, with an oxidation product intermediate between 0-3,4-dihydroxyphenyl-L-alanine and dopachrome. Aqueous solutions containing 0.72 X 10~8 M coenzyme A, 0.76 X 10~3 M dihydroxyphenyl-alanine, and 100 units of tyrosinase per ml of distilled water were prepared immediately before each experiment. The reactions proceeded at 25° C in a total volume of 4 ml of solution buffered at pH 7.4 by 1 ml of 0.1 M sodium phosphate. 220 230 240 250 260 270 280 290 300 WAVE-LENGTH IN MILLIMICRONS-* 310 Fig. 1. Absorbancies of dopachrome, coenzyme A, and the product formed from coenzyme A and an oxidation product of dihydroxyphenylalanine at various wave lengths. Curve 1 : Twelve minutes after addition of tyrosinase, a solution containing 0.19 X 108 M dihydroxyphenylalanine and 25 units of tyrosinase per ml, in 0.025 M sodium phosphate at pH 7.4, was diluted 5-fold with water and the absorbancy determined. Curve 2: The general procedure was similar to that of Curve 1, but the initial solution contained only 0.18 X 10~3 M coenzyme A in the phosphate buffer. Curve 3: Identical to Curve 1, but with the additional presence of 0.18 X 10~3 M coenzyme A initially. Such a reaction implies that pigmentation may be intimately related to the basic metabolic processes of skin. MATERIALS AND METHODS The tyrosinase and coenzyme A were the same preparations used in previous studies (1, 2). Previously described technics of measurement were used (3). Measurements were made between eight and seventeen minutes after the addition of tyrosinase. The RESULTS ultraviolet absorbancies of separate solutions containing dihydroxyphenylalanine and tyrosinase, coenzyme A alone, and dihydroxyphenylalanine and coenzyme A in essentially equal molarity together with tyrosinase are presented in Fig. 1. Tyrosinase had no effect upon the absorbancy of coenzyme A alone. In the 277 278 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY O.6OO-1 0.50o4 COENZYME A IN MOLES PER LITER 10*-* Fig. 2 Curve 1 : Absorbancy at 305 im* of dopachrome formed after twelve minutes in solutions containing initially 38 X 106 M dihy-droxyphenylalanine, coenzyme A, and 25 units of tyrosinase per ml in 0.025 M sodium phosphate at pH 7.4. The absorbancy was plotted as a function of coenzyme A. Curve 2: Absorbancy at 412 m of the sulfhy-dryl-containing compound of Ellman formed from 4 ml of separate solutions identical to those of Curve 1. Curve 3: Absorbancy at 412 m/* of the sulfhy-dryl-containing compound of Ellman formed from 4 ml of coenzyme A in the indicated concentrations. absence of tyrosinase, the absorbancy of solutions containing dihydroxyphenylalanine and coenzyme A was equal to the sum of the absorbancy of each substance alone. Addition of coenzyme A to pre-formed dopachrome in the presence of tyrosinase also yielded solutions in which the absorbancies of the individual components were additive. At 305 m/x, the absorbancy of dopachrome was at a maximum, while the absorbancies of coenzyme A and the coenzyme A-derived compound were comparatively negligible. The presence of coenzyme A and tyrosinase led to a decrease in the formation of dopachrome, as determined by the absorbancy at 305 m/x, (Fig. 2). No dopachrome was formed in solutions which initially contained essentially equivalent amounts of coenzyme A and dihydroxyphenylalanine. The free sulfhydryl group disappeared when the molarity of coenzyme A was less than that of dihydroxyphenylalanine, (Fig. 2). Excess of coenzyme A over dihydroxyphenylalanine led to the expected increase in free sulfhydryl group. DISCUSSION The results in the present study are strongly suggestive of a chemical reaction between the sulfhydryl group of coenzyme A and an oxidation product intermediate between dihydroxyphenylalanine and dopachrome. With formation of the coenzyme A-derived compound, the sulfhydryl group disappears from the free state and simultaneously the formation of dopachrome decreases. The new sulfhydryl compound has an absorbancy pattern which is distinctly different from that of coenzyme A (4), dihydroxyphenylalanine (3), and dopachrome. Its pattern partially resembles the one previously found for acetyl-coenzyme A, especially in the region of 230 m/x (4). The sulfhydryl groups of cysteine and glutathione, as well as coenzyme A, react with oxidation products of dihydroxyphenylalanine (3, 5). The glutathione reaction product may be an orthodiphenolic thioether (5). In vivo, cysteine and gluthathione may control the rate of inacti-vation of coenzyme A and formation of dopachrome by means of their combination with dihydroxyphenylalanine.