On the Mechanism of Peroxidation of Uric Acids by Hemoproteins

This study of the mechanism of peroxidation of purines by hemoproteins was stimulated by an observation that uric acid was unstable when formed from inosine in the presence of erythrocyte nucleoside phosphorylase, xanthine oxidase, and phosphate buffer.Since the conversion of hypoxanthine to uric acid by xanthine oxidase generates 2 molecules of hydrogen peroxide, a peroxidation was suspected.When catalase was included in the system, the yield of uric acid from inosine was quantitative, and uric acid was stable.The peroxidative catalyst remained inseparable from hemoglobin, and it was eventually concluded that the active principle was methemoglobin.A number of other hemoproteins are also known to catalyze peroxidation of uric acid.These include myeloperoxidase (4,5), horseradish peroxidase (6), lactoperoxidase, verdoperoxidase (7), and cytochrome c (8).The component common to these proteins and of methemoglobm is a trivalent iron porphyrin prosthetic group.Hematin itself was found to be an excellent catalyst for uric acid peroxidation, but the pH optima of methemoglobin and of horseradish peroxidase were quite different from that of hematin.The studies described herein show that any purine with an unsubstitued N-7 or N-9 position and an 8-0~0 substituent is susceptible to peroxidation in the presence of hemoprotein (or hematin) and peroxide.A mechanism of peroxidation of uric acids catalyzed by hemoproteins is proposed. EXPERIMENTAL PROCEDUREMaterials-Beef liver catalase, uricase, milk xanthine oxidase, and horseradish peroxidase were obtained from Worthington Biochemicals, Inc. Crystalline cytochrome c (horse heart) and beef hemoglobin (twice recrystallized) were obtained from Sigma Chemical Corporation.The common purines were commercial products.Uric acid was recrystallized twice from Li&Os solution (9).8-Oxyadenine, 2, gdioxyadenine, 8-oxyguanine, and 8-oxy-2,6diaminopurine were synthesized according to Cavalieri and Bendich (10).7-Methyl-g-oxyguanine was isolated from human urine as previously described (11).Methyluric acids were a gift of Dr. Sidney Udenfriend, except that tetramethyluric acid waz a gift of Dr. F. Bergmann of Jerusalem.Met~--Spectrophotometric assays were performed at room temperature in a Beckman model DU spectrophotometer with use of 3-ml quartz cuvettes with a l-cm light path.Spectra were obtained with a Cary recording spectrophotometer, model