ISOLATION AND CHARACTERIZATION OF 2 FORMS OF AN ACIDIC BROMELAIN STEMPROTEINASE

Two forms of an acidic bromelain proteinase isolated from crude bromel ain, an extract from pineapple stem, were found by a two-step FPLC pur ification procedure. The basic main components were removed by cation exchange chromatography and the breakthrough fraction was further reso lved by anion exchange chromatography into 15 protein fractions, only two of which, called SBA/a and SBA/b, were proteolytically active. The se components were characterized by electrospray mass spectroscopy (ES MS), isoelectric focusing, N-terminal amino acid sequence analysis, mo nosaccharide analysis, and enzymatic parameters. The molecular masses of SBA/a and SBA/b, were determined by ESMS to be 23,550 and 23,560, r espectively. The isoelectric points (pI) of the two bands of SBA/a wer e 4.8 and 4.9; SBA/b, focused as a single band at pI = 4.8. Partial N- terminal amino acid sequences (11 residues) were identical to SBA/a an d SBA/b and identical with those of stem bromelain, the basic main pro teinase of the pineapple stem, and fruit bromelain, the acidic main pr oteinase of the pineapple fruit. Both components are highly glycosylat ed; hydrolysis of SBA/a yielded about twofold more monosaccharide per protein than SBA/b. The comparison of the catalytic properties of SBA/ a with those of SBA/b, revealed no relevant differences in the hydroly sis of three peptidyl-NH-Mec substrates and in the inhibition profiles using chicken cystatin and E-64, indicating that these components can be considered as two forms of a single enzyme. Both forms are scarcel y inhibited by chicken cystatin and slowly inactivated by E-64, hence are nontypical cysteine proteinases of the papain superfamily.