Purification of flavin mononucleotide-dependent enzymes by column chromatography on flavin phosphate cellulose compounds.

The chemical syntheses of monoand dimethyl esters of riboflavin phosphate were accomplished by allowing riboflavin to react with the appropriate monoand dimethylchlorophosphoric acids. Riboflavin monomethyl phosphate was found to be coenzymatically active with nicotinamide adenine dinucleotide phosphate-cytochrome c aporeductase, pyridoxine (or pyridoxamine) phosphate apooxidase, and glycolate apooxidase. Riboflavin phosphate esters of cellulose phosphate and diethylaminoethyl cellulose were prepared by reactions with riboflavin dichlorophosphate in pyridine; the corresponding derivative of cellulose was prepared by reaction with riboflavin phosphate and dicyclohexylcarbodiimide in pyridine. The riboflavin phosphate cellulose compounds were used to retain the riboflavin phosphate-dependent apoenzymes selectively during their chromatographic purification from extraneous protein. Extensive enrichment of glycolate apooxidase was achieved by chromatography on riboflavin phosphate cellulose.