Anionic Proteinase from Actinidia chinensis

1 A method is described for the preparation from Actinidia chinensis (Chinese gooseberry) fruit of a crystalline proteinase which catalyzes the hydrolysis of N 2 ‐benzoyl‐ l ‐arginine ethylester, N 2 ‐ p ‐toluene‐sulphonyl‐ l ‐glutamine p ‐nitrophenyl ester (used in the routine assay) and at least 15% of the peptide bonds of gelatin. The synthesis of the tosyl‐glutamine p ‐nitrophenyl ester is described. The enzyme resembles papain in its action against benzoyl‐ l ‐arginine ethyl ester, having a broad pH‐optimum from pH 5–7, with K m (app) of 89 mM and k cat of about 2.6 sec −1 at pH 5.6, 25° in 0.3 M KCl. 2 The enzymic activity elutes from Sephadex G‐50 as a protein of molecular weight 12800 ± 700, and is partially resolved from inactive protein of apparent molecular wight 15400 ± 800. The preparation has minimum solubility near pH 3.1 and migrates at both pH 8.3 and 5.0 on polyacrylamide gel electrophoresis as two closely‐spaced anionic bands. These two electrophoretic components are partially resolved by chromatography on DEAE‐cellulose and both are enzymically active against tosyl‐ l ‐glutamine p ‐nitrophenyl ester. 3 The inhibitions of enzymic activity by 5,5′‐dithiobis‐(2‐nitrobenzoic acid) (DTNB) and 4,4′‐dithiodipyridine, both reversible by 1,4‐dithiothreitol, show that enzymic activity is dependent on an intact sulphhydryl group assaying about 0.23 moles per 12800 g protein. The enzyme is rapidly inactivated by iodoacetate and thereafter gives a low sulphhydryl assay with dithiodipyridine. Conversely, pretreatment with DTNB gives substantial protection against iodoacetate.