CHARACTERIZATION OF HEXOSE OXIDASE FROM THE RED SEAWEED CHONDRUS-CRISPUS

Hexose oxidase from the red seaweed, Chondrus crispus was purified to homogeneity. The enzyme appeared to be encapsulated in particles obtai ned after mechanical disintegration of the fronds. Liberation of the e nzyme in soluble form required either waiting for the spontaneous deve lopment of a suitable microbial flora in the suspension, or treatment with a mixture of proteases (pronase). As deduced from (SDS/)PAGE, the enzyme has a molecular mass of 87 kDa and probably consists of subuni ts of 36 kDa and 25 kDa. The low isoelectric point of 2.8 and the pres ence of 25% (by mass) sugars indicate that the enzyme is a strongly ac idic glycoprotein. The absorption spectrum of isolated enzyme minus th at of the substrate-reduced enzyme, and the EPR spectrum of the free r adical observed in the reduced enzyme revealed the presence of a flavi n. This cofactor is probably covalently bound since flavins were not r eleased upon denaturation of the enzyme by heat or acid treatment. Tak ing free FAD as a reference compound, the enzyme contains 1 mol flavin /mol enzyme. EPR spectroscopy of the purified preparation showed the p resence of Cu2+. However, since the amount was substoichiometric, subs trate addition did not affect the signal, and the addition of chelator or Cu2+ did not affect the activity, the presence of this metal ion s eems adventitious. It is concluded that the large discrepancies betwee n the presently and the previously reported [Sullivan, J. D. & Ikawa, M. (1973) Biochim. Biophys. Acta 309, 11-22] characteristics of the en zyme probably originate from the characterization of a contaminating p rotein in the latter case.