Purification of intact old yellow enzyme using an affinity matrix for the sole chromatographic step

Old Yellow Enzyme has been purified in high yield from crude extracts of brewers bottom yeast using an affinity matrix for the sole chromatographic step. The results of sodium dodecyl sulfate gel electrophoresis indicate that the enzyme is nearly homogeneous and consists of identically sized subunits of molecular weight of about 49,000. However, when the enzyme is isolated without the protective presence of a protease inhibitor, the electrophoretic pattern comprises three bands, which suggests limited, perhaps single bond cleavage of the polypeptide chain. This proteolytic cleavage results in weaker binding of ligands to the enzyme. The binding of a large number of compounds to the enzyme has been investigated, particularly with reference to the concomitant appearance of long wavelength absorption bands. The structural requirements for formation of long wavelength absorption are that the ligand must be aromatic in character, with an ionizable hydroxyl or thiol substituent.