The multiple functions of thiooxidase.

Strains of P. oryxae were cultured, and crude filtrates of culture medium were prepared as described by Neufeld et al. (2). Protein was measured by the method of Lowry et al. (3). Assays--The increase in absorbancy at 320 rnp due to the formation of diphenyl disulfide was used to measure thiooxidase activity with thiophenol as substrate. The reaction rate was linear and proportional to enzyme concentration with a mixture of the following in a volume of 1.2 ml: sodium phosphate, pH 6.3, 42 mM; bovine albumin, 1.25 mg; diphenyl disulfide, 0.033 mM; enzyme; thiophenol, 0.83 mM. As discussed below, diphenyl disulfide eliminated an initial lag in reaction rate. One unit of enzyme caused a change in absorbancy of 1 .O per minute; i.e. 1.04 pmoles of diphenyl disulfide was formed per minute. The product of enzymatic reaction crystallized from solution, and after recrystallization from ethanol, it had an absorption spectrum identical with that of diphenyl disulfide prepared chemically with peroxide. Each product and a mixture of the two melted at 59.7”. As catechol was oxidized, the quinone that formed reacted with ethylenediamine, yielding a colored complex with absorption maximum at 572 mp. The increase in absorbancy at this wave length was used as a measure of catechol oxidase with the following mixture in a volume of 1.2 ml: sodium succinate, pH 5.5, 42 mM; ethylenediamine, 17 mrvr; enzyme; catechol, 2.1 mM. After a short lag, the rate of color formation was linear and proportional to enzyme concentration; 1 unit of catecholase caused a change in absorbancy of 1 .O per minute. Pur$cation--Four liters of filtered culture medium were stirred with 24 g of Celite, 400 ml of 1 To protamine sulfate were added, and the black precipitate was removed by filtration. Twelve grams of Celite, 100 ml of saturated sodium chloride, and 4 liters of acetone were added to the filtrate, and the mixture was stirred for 30 minutes. The adsorbent, collected on sintered glass, was eluted by stirring for 20 minutes with three successive 50-ml portions of 0.1 M sodium chloride. Acetone (65 ml) was added to the combined eluates (151 ml), the precipitate was removed by centrifugation, and the enzyme was precipitated from the supernatant solution by the addition of 120 ml of acetone. The precipitate was dissolved in 16 ml of 0.04 M potassium phosphate, pH 7.5; 60 mg of sodium pyrophosphate were added, and, after centrifugation, the solution was dialyzed against 2 liters of 0.002 M Tris, pH 8.0. The dialyzed solution was charged onto a 2X 30-cm column of DEAE-cellulose1 equilibrated with 0.002 M Tris, pH 8.4. After 100 ml of the latter fluid had passed through the column, the enzyme was eluted by changing the buffer to 0.1 M potassium phosphate-O.2 M sodium chloride at pH 7.6. The yields of these procedures, carried out at 4”, appear in Table I. Part of the chromatographically purified enzyme was dialyzed against distilled water and dried while frozen. Properties of Enzyme-Approximately 4 mg of purified enzyme (in 1 ml of 0.05 M Tris, pH 7.5) were examined with the Spinco model E ultracentrifuge at 60,000 r.p.m. From the results obtained (Fig. l), a sedimentation constant, szL = 2.42 S, was estimated, and the preparation seemed homogeneous. In another experiment, dried purified enzyme (2.7 mg) was dissolved in 0.12 ml of 0.01 M Tris at pH 7.5, layered on a gradient of 5 to 20% solution of sucrose in 0.01 M Tris at pH 7.5, and centrifuged at 38,000 r.p.m. for 13 hours at 4” with an S.W. 39 rotor with a model L Spinco centrifuge (4). Forty-6ve serial 0.1.ml fractions were obtained and analyzed for thiooxidase and catecholase activity (Fig. 2). In a further effort to separate the two enzymatic activities, enzyme was eluted from DEAE-cellulose with a gradient solution made by adding 0.2 M Tris-0.1 M sodium chloride at pH 7.2 to 0.002 M Tris at pI-I 8.4 in a 250-ml mixing chamber. The relative amounts of thiooxidase and catecholase found in the several effluent fractions are shown in Table II. The curves in Fig. 3 give pH optima for the two activities; both activities were stable to heating at 60” for 5 minutes but were destroyed by boiling. A plant tyrosinase, active with phenol and catechol, did not catalyze the oxidation of thiophenol. The K, for thiophenol was 4.1 x 10m4 M, and for catechol it was 9.2 X 10e4 M, as calculated from the Lineweaver-Burk plots shown in Fig. 4. When tested manometrically, the purified enzyme also catalyzed the oxidation of diethyldithiocarbamate, methylmercaptoimidazol, and resorcinol; thiouracil was not oxidized.