Isolation of Old Yellow Enzyme in Free and Complexed Forms

A new method for the isolation of electrophoretically homogeneous old yellow enzyme (NADPH2 oxidoreductase, EC 1.6.99.1) from brewer's bottom yeast is described. Old yellow enzyme, as isolated by this method, is bound in a charge transfer complex to an unidentified compound of low molecular weight. This complex can be dissociated by dialysis of the reduced enzyme, and the complex is reformed upon addition of the dialysate to the oxidized enzyme. Preliminary studies on the chemical nature of the material bound to old yellow enzyme are described, and similar charge transfer complexes formed by the enzyme with quinoline derivatives are reported. Amino acid analysis data are reported for the purified, uncomplexed old yellow enzyme.