PURIFICATION AND CHARACTERIZATION OF MULTIPLE FORMS OF THE PINEAPPLE-STEM-DERIVED CYSTEINE PROTEINASES ANANAIN AND COMOSAIN

A mixture of ananain (EC 3.4.22.31) and comosain purified from crude p ineapple stem extract was found to contain numerous closely related en zyme forms. Chromatographic separation of the major enzyme forms was a chieved after treatment of the mixture with thiol-modifying reagents: reversible modification with 2-hydroxyethyl disulphide provided enzyme for kinetic studies, and irreversible alkylation with bromotrifluoroa cetone or iodoacetamide gave enzyme for structural analyses by F-19-n. m.r. and electrospray mass spectrometry respectively. Structural and k inetic analyses revealed comosain to be closely related to stem bromel ain (EC 3.4.22.32), whereas ananain differed markedly from both comosa in and stem bromelain. Nevertheless, differences were seen between com osain and stem bromelain in amino acid composition and kinetic specifi city towards the epoxide inhibitor E-64. Differences between five isol atable alternative forms of ananain were characterized by amidolytic a ctivity, thiol stoichiometry and accurate mass determinations. Three o f the enzyme forms displayed ananain-like amidolytic activity, whereas the other two forms were inactive. Thiol-stoichiometry determinations revealed that the active enzyme forms contained one free thiol, where as the inactive forms lacked the reactive thiol required for enzyme ac tivity. M.s. provided direct evidence for oxidation of the active-site thiol to the corresponding sulphinic acid.