Multiple Forms of Acidic Endopeptidase from Germinated Barley

An endopeptidase preparation from germinated barley Hordeum vulgare L., cv. Trophy, purified by affinity chromatography and density-gradient electrofocusing, consisted of three or four components. The preparation was only partly resolved by electrofocusing, with evidence of three possible components (pI 4.15, 4.28, and 4.37). Gel filtration on Sephadex G-75 yielded an asymmetrical peak, the major part of which corresponded to a molecular weight of 14,100, with evidence of one larger and two smaller components. The activity of the preparation was sulfhydryl-dependent; cysteine was the most effective of several sulfhydryl compounds tested. The preparation was sensitive to O(2) in the absence of metal chelating agents and was inhibited by sulfhydryl reagents. It showed very narrow concentration tolerances for both cysteine and a substrate, N,N-dimethylhemoglobin. The Km value on N,N-dimethylhemoglobin at pH 3.8 was 0.064 to 0.067% (w/v) substrate; V(max) was 0.80 to 0.83 A(340) per hour. Normal enzyme activity and molecular-size distribution were observed when the endopeptidases were extracted in the inhibited state and subsequently reactivated, thus ruling out the possibility that the enzymes might be autolytic artifacts that arose during extraction and purification.