A Reversible Association of Bovine Carbonic Anhydrase with Milk Xanthine Oxidase

Bovine carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) was observed to act as a powerful competitive inhibitor of the reduction of cytochrome c by xanthine oxidase. Carbonic anhydrase did not compete with the substrates of xanthine oxidase or with electron acceptors other than cytochrome c. Ki for carbonic anhydrase was influenced by changes in dielectric constant, ionic strength, and pH in a manner which suggested that its interaction with xanthine oxidase was primarily based on electrostatic forces. Neither sulfonamide inhibitors of carbonic anhydrase nor the removal of zinc from carbonic anhydrase interfered with its ability to compete with cytochrome c, but heat denaturation completely destroyed its inhibitory capacity. A variety of proteins chosen at random were found unable to inhibit the reduction of cytochrome c by xanthine oxidase, and carbonic anhydrase did not inhibit the reduction of cytochrome c by microsomal reduced triphosphopyridine nucleotide-cytochrome c reductase. Human erythrocyte carbonic anhydrase B did not substitute for the mixed bovine carbonic anhydrase. Ki for carbonic anhydrase was invariant with temperature in the range from 24.5° to 51°. The value of ΔF° for the association of carbonic anhydrase with xanthine oxidase was -11.7 kcal per mole, and the corresponding value of ΔS° was +59 e.u. per mole. The binding of 1 molecule of carbonic anhydrase appeared capable of preventing the binding of somewhat more than 1 molecule of cytochrome c to xanthine oxidase.