Purification of wheat proteases by affinity chromatography on hemoglobin-sepharose column

Abstract

A rapid chromatographic procedure for the isolation and purification of proteases from malted wheat flour is described. The separation was achieved by passing the crude extract through a hemoglobin-Sepharose column. Unadsorbed proteins were eluted with the starting buffer. The adsorbed proteases were then eluted with O.1N acetic acid. Recoveries of proteins and proteolytic activity were over 90%. A two-fold increase in specific activity was achieved by this purification technique. Disc electrophoretic analyses showed that all of the non-proteolytic components were separated from the proteolytically active proteins. The active peak comprised three major and one minor proteins of similar mobility.