Purification of Liver Flavokinase by Column Chromatography on Flavin-Cellulose Compounds

Flavokinase, which catalyzes the formation of flavin mononucleotide from riboflavin and adenosine triphosphate, has been partially purified from rat liver by such conventional means as fractionation with ammonium sulfate and chromatography on diethylaminoethyl cellulose (2). A greater separation of flavokinase from interfering acid phosphatases which catalyze hydrolysis of FMN’ (3, 4) was deemed desirable, but more extensive purification of the kinase necessitated the development of more selective treatments. Also it has been suggested recently that pure flavokinase has a specific activity of approximately 100 mpmoles of FMN synthesized per mg of protein per hour at 30”, regardless of the sources from which the enzyme is obtained (5). The apparent homogeneity of a preparation of a bean kinase upon electrophoresis and ultracentrifugation, together with comparable specific activities of this flavokinase and those partially purified by other investigators from several sources in&ding liver, were cited as evidence for this maximum specific activity. As specific activities considerably above that suggested for “pure” flavokinase had been obtained in this laboratory with the liver enzyme, even in the presence of some contaminating phosphatase (2), it seemed that greater purification of the kinase could be accomplished and would reveal the maximum specific activity suggested by others (5) to be unrealistically low. Chromatographic techniques which utilize the specific complexing of substrate-like compounds with enzymes have found occasional use in the purification of enzymes. An example of such a technique is afforded by the work of Lerman (6), who enriched mushroom tyrosinase by passage through columns of p-azophenol-cellulose derivatives. Studies on the substrate specificity of liver flavokinase (2, 7, 8) indicate that the enzyme exhibits moderate binding to several flavin substrates and competitive inhibitors. Among variations of the side chain in position 9 of the isoalloxazine system which permit reactivity are substrates with a I’-n-ribityl or competitive inhibitors with a methyl substituent present. Considerable tolerance in size and inductive effect of 6and 7-substituents is also observed. These findings suggested the chromatographic use of 9-(l’-n-ribityl)and 9-methylflavins bearing in either position 6 or 7 an acetamido