Agric. Biol Chem., 47 (7), 1627- 1628, 1983 1627

Note tested for whether they oxidized bilirubin. In the same way, the activities for A^N-dimethyl-/?-phenylenediamine, L-tyrosine, ascorbic acid and bilirubin of bilirubin oxidase Difference between Various Copper- were measured. The reaction mixtures consisted of 8 fimol of substrate containing Enzymes (Polyporus and 80^g of enzyme in 0.2m triethylamine-acetic acid Laccase, MushroomTyrosinase and buffer (pH 5.6) for Polyporus laccase, 0.2m potassium CucumberAscorbate Oxidase) phosphate buffer (pH 6.5) for tyrosinase, 0.2 mpotassium- and Bilirubin Oxidase Noriaki Tanaka and Sawao Murao Department of Agricultural Chemistry, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka 591, Japan Received July 5, 1982

Bilirubin oxidase is a copper-containing enzyme which oxidizes bilirubin more rapidly than any other sub- stances.^ In the course of investigations, it has been found that phenolic substances and ascorbic acid are oxidized by this enzyme. On the other hand, laccase (EC 1.10.3.2), tyrosinase (EC 1.10.3.1) and coeruloplasmin (EC 1.16.3.1) are copper-containing enzymes which oxidize phenolic substances.2) Ascorbate oxidase (EC 1.10.3.3) is also a copper-containing enzyme which oxidizes ascorbic acid. Many phenolic substances are quite good substrates for ascorbate oxidase.3) Consequently, we examined wheth- er laccase, tyrosinase and ascorbate oxidase can oxidize bilirubin. Brodersen and Bartels have reported that co- eruloplasmin cannot oxidize bilirubin.4) Laccase was purified from a culture filtrate of Polyporus Fig. 1. Polyacrylamide Gel Disc Electrophoresis of versicolor IFO 9791 by the procedure of Fahraeus and Purified Tyrosinase. Reinhammar.5) The homogeneity of the purified enzyme Purified tyrosinase (10/xg protein) was applied to elec- was examined by means of polyacrylamide gel disc elec- trophoresis with 7% gel at pH 8.0. The protein was stained trophoresis. A single protein band was obtained, so that it with Coomassie Brilliant Blue. could be regarded as homogeneous.1* Tyrosinase (grade III) from mushrooms was purchased Table I. Bilirubin Reactive Activities from Sigma Chemical Co. and purified by a procedure of Copper-containing Enzymesand involving DEAE-Sephadex A-50, hydroxyapatite and Substrate Specificity of DEAE-Sephadex A-50 column chromatographies.6)7) The Bilirubin Oxidase purified enzyme gave a single band on polyacrylamide gel disc electrophoresis (Fig. 1), so that it could be regarded as Enzymes homogeneous. Substrate Bilirubin T^å . Ascorbate Ascorbate oxidase (grade III), which was a pale blue .. Laccase fyrosinase . , powder from cucumber, was purchased from Toyobo Co., oxidase oxidase Ltd. (/xmol oxygen/80 /ig protein/min) Bilirubin oxidase was purified as described in a previous paper.1] The purified enzyme gave a single band on Bilirubin 1.3 ± ± ± 7V, A^-Dimethyl - polyacrylamide gel disc electrophoresis.8) ^-phenylene- 0.22 0. 8 By manometric methods, the activities of iV,Af-dimethyl- diamine /?-phenylenediamine of Polyporus laccase, L-tyrosine of Tyrosine 0 0. 7 mushroom tyrosinase and ascorbic acid of cucumber Ascorbic acid 0. 1 0.7 ascorbate oxidase were measured and these enzymes were 1628 N. Tanaka and S. Murao sodium phosphate buffer (pH 5.6) for ascorbate oxidase, 2) B. G. Malmstrom, L.-E. Andreasson and B. and 0.2m potassium phosphate buffer (pH 8.0) for bili- Reinhammar, "The Enzymes," Vol. XII, ed. by P. D. rubin oxidase. Bilirubin is dissolved only on the alkaline Boyer, Academic Press Inc., New York, N. Y., 1975, side, so the activity for bilirubin of each enzymewas p.507. measured at pH 8.0. 3) C. R. Dawson, "The Biochemistry of Copper," ed. As shown in Table I, bilirubin was little oxidized by by J. Peisach, P. Aisen and W. E. Blumberg, Polyporus laccase, mushroomtyrosinase and cucumber Academic Press Inc., New York, N. Y., 1966, p. 305. ascorbate oxidase, but bilirubin oxidase catalyzed the 4) R. Brodersen and P. Bartels, Eur. J. Biochem., 10, oxidation of bilirubin muchmore strongly than the other 468 (1969). enzymes. Bilirubin oxidase, Polyporus laccase, mushroom 5) G. Fahraeus and B. Reinhammar, Ada Chem. tyrosinase, cucumber ascorbate oxidase and coeruloplas- Scand., 21, 2367 (1967). min are copper-containing enzymes which oxidize pheno- 6) R. M. Nelson and H. S. Mason, "Methods in lic substances, but bilirubin oxidase differs from them in Enzymology," Vol. XVIIA, ed. by H. Tabor and C. the context of reactivity to bilirubin. W. Tabor, Academic Press Inc., New York, N. Y., 1970, p. 626. REFERENCES 7) W. D. Harry and E. C. Joseph, J. BioL Chem., 245, 1613 (1970). 1) N. Tanaka and S. Murao, Agric. Biol. Chem., 46, 8) S. Murao and N. Tanaka, Agric. BioL Chem., 45, 2499 (1982). 2383 (1981).