The leaf protease of Trifolium repens

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Abstract
Nitrogen catabolism in sap expressed from leaves of T. repens has been studied, and the use of the increment in [alpha]-amino content as an index of protein hydrolysis in the leaves is discussed. A considerable variation in the rate of release of a-amino nitrogen in different samples of sap was found. A fraction showing about 5 times the protease activity of fresh sap has been prepared by removal of relatively inactive protein. Some characteristics of the hydrolysis of endogenous protein and gelatin by the crude enzyme, and of gelatin by the concentrated enzyme, were examined. With endogenous substrate, peptides accumulated briefly in the medium, then rapidly disappeared. With relatively high concentrations of gelatin substrate there was a marked accumulation of peptides in the medium. The protesase showed a broad pH optimum of 5-9-6-3; no activity could be detected at values lower than pH 4-0. The crude enzyme was rapidly inactivated at pH values below 5-0, but stability was not affected by pH values between 5''0 and 7''0. In the concentrated condition the enzyme was much more stable on storage. Reducing agents enhanced activity on endogenous substrate but not on gelatin. Chelating agents and heavy-metal ions were the most active of a number of possible inhibitors examined.