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 obtained after mechanical disintegration of the fronds. Liberation of the enzyme in soluble form required either waiting for the spontaneous development 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 subunits of 36 kDa and 25 kDa. The low isoelectric point of 2.8 and the presence of 25% (by mass) sugars indicate that the enzyme is a strongly acidic glycoprotein. The absorption spectrum of isolated enzyme minus that of the substrate-reduced enzyme, and the EPR spectrum of the free radical observed in the reduced enzyme revealed the presence of a flavin. This cofactor is probably covalently bound since flavins were not released upon denaturation of the enzyme by heat or acid treatment. Taking free FAD as a reference compound, the enzyme contains 1 mol flavin/mol enzyme. EPR spectroscopy of the purified preparation showed the presence of Cu2+. However, since the amount was substoichiometric, substrate addition did not affect the signal, and the addition of chelator or Cu2+ did not affect the activity, the presence of this metal ion seems adventitious. It is concluded that the large discrepancies between the presently and the previously reported [Sullivan, J. D. & Ikawa, M. (1973) Biochim. Biophys. Acta 309, 11-22] characteristics of the enzyme probably originate from the characterization of a contaminating protein in the latter case.
Groen
B W
BW
Department of Microbiology and Enzymology, Delft University of Technology, The Netherlands.
De Vries
S
S
Duine
J A
JA
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D003300:Copper / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D005415:Flavins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D015394:Molecular Structure; D008970:Molecular Weight; D011487:Protein Conformation; D012624:Seaweed / Q000201:enzymology*; D013053:Spectrophotometry; D013379:Substrate Specificity
D005415:Flavins; D003300:Copper; D000429:Alcohol Oxidoreductases; C021354:hexose oxidase