Reinvestigation of fractionation and some properties of the proteolytically active components of stem and fruit bromelains.
To check whether crude stem and fruit bromelains can be fractionated further or not, systematic separation procedures were applied to both enzymes. Six proteolytically active components, which were designated as SBB 1-5 and SBA, were fractionated from crude stem bromelain by successive use of gel filtration on Sephadex G-75, and chromatographies on CM-Sephadex and DEAE-Sephacel. One main and one minor active components, designated as FBA and FBB, respectively, were also separated from crude fruit bromelain by chromatographies on DEAE-Sephacel and then CM-Sephadex. Some of the physico-chemical and enzymatic properties of these eight components were compared. Each component migrated as a single band on SDS-polyacrylamide gel electrophoresis. Molecular weights determined by the same electrophoresis were about 27,000 for SBB 1-3 and FBB, and about 23,000 for the other four components. In terms of amino acid composition, FBB resembled SBB 1-3, which were remarkably similar to each other. FBA was also similar to SBA in amino acid composition, and contained much less basic amino acids than SBB 1 through 5. The principal amino-terminal residues determined by the cyanate method were valine in SBB 1-5 and SBA, and alanine in FBA and FBB. The principal carboxyl-terminal residues determined by the hydrazinolysis method were glycine in SBB 1-3, SBA and FBA, and serine in SBB 4-5 and FBB. However, fractional amounts of a few other amino- and carboxyl-terminal residues were also detected. As regards enzymatic activities, FBA and SBB 4 and 5 were much more active than the other five components against casein and some synthetic substrates [Bz-Arg-amide (at pH 6.1), Z-Gly-X, and Z-Ala-X (at pH 3.5)] with the notable exception that FBA was much less active than SBB 4 and 5 toward tripeptides (X-Gly-Gly).
D000596:Amino Acids / Q000032:analysis; D001963:Bromelains / Q000032:analysis*; D002241:Carbohydrates / Q000032:analysis; D005638:Fruit / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010944:Plants / Q000032:analysis*
D000596:Amino Acids; D002241:Carbohydrates; D001963:Bromelains