Studies on Milk Xanthine Oxidase

Milk xanthine oxidase has been isolated in good yield and purity from pasteurized buttermilk and found to contain FAD, molybdenum, iron, and labile sulfide in the ratio of 1:1:4:4. Analytical data on the spectral characteristics of the enzyme in the oxidized and various reduced states are presented. The anaerobic reduction of the enzyme by substrates proceeds in two phases: a catalytically significant fast phase and a very much slower secondary phase. Anaerobic titration studies indicate that per eq of enzyme-bound FAD a 4-electron reduction occurs in the fast phase: 3 additional electrons may be accepted from substrates in the slow phase, and an 8th electron from dithionite. Rapid reaction studies indicate that, with all substrates tested, with the possible exception of purine, the rate-limiting step in catalysis is the 4-electron reduction of the enzyme, the reaction of the reduced enzyme with O2 being considerably more rapid.