Hemoglobin-digesting Acid Proteinases in Soybean Leaves

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Abstract
Three proteinases which digest hemoglobin rapidly at acid pH (3.5-4.5) were identified in crude extracts of soybean leaves and separated by chromatography on DEAE cellulose. All 3 enzymes were endopeptidases as judged by the ratio of .alpha.-amino-N plus peptide N over .alpha.-amino-N in the trichloroacetic acid-soluble portion of hemoglobin digests. Proteinase I did not bind to diethylaminoethyl cellulose and was not inhibited by any proteinase inhibitor tested. Proteinase II was partially inhibited by phenylmethylsulfonyl fluoride, N-ethylmaleimide and p-chloromercuribenzoate. The inhibition by phenylmethylsulfonyl fluoride may be due to the presence of contaminating carboxypeptidase. Proteinase III was the most anionic of the 3 and required the presence of sulfhydryl reagents to prevent the irreversible loss of activity. All proteinase preparations digested soybean ribulose bisphosphate carboxylase as shown by the disappearance of the large subunit of that protein, when partially digested preparations were subjected to sodium dodecyl sulfate gel electrophoresis. The 3 proteinases were endopeptidases. All 3 proteinases were present throughout leaf development; proteinase I predominated in expanding leaves; proteinase III became the predominant enzyme as the leaves matured. Senescence (yellowing) was associated with a decline in the activities of all 3 proteinases.