IMMUNOGLOBULIN AFFINITY CHROMATOGRAPHY OF MUSHROOM POLYPHENOL OXIDASE

Abstract

Immunoglobulin (IgG) against mushroom polyphenol oxidase (PPO) was produced in rabbits and its specificity for PPO was shown by double diffusion and Western blotting techniques. The use of IgG affinity chromatography in purification of PPO and in structural studies is described. Apparent pure enzyme, as judged by gel filtration, p-aminobenzoic acid-Sepharose affinity chromatography and by polyacrylamide gel electrophoresis (PAGE) on non-denaturing gel, was shown by PAGE—SDS and by IgG affinity chromatography to contain multiple protein bands. The multiple bands seen by SDS-PAGE are due to subunits, as well as to different isozymes present. The multibands obtained by IgG-Sepharose chromatography would appear to be due to differentiation among the different isozymes of PPO, as well as to possible subtle differences caused by activity of PPO on substrates in the mushroom during the initial phases of purification.