Fractionation and Some Properties of the Proteolytically Active Components of Bromelains in the Stem and the Fruit of the Pineapple Plant*

Abstract

One main and five minor proteolytically active components were separated from crude stem bromelain [EC 3. 4. 4. 24] by gel filtration on Sephadex G-75 and ion-exchange chromatographies on DEAE-cellulose and sulfoethyl-Sephadex. One main and two minor active components were also separated from crude fruit bromelain by chromatographies on DEAE-cellulose and sulfoethyl-Sephadex. The seven components, but not components III and IV of stem bromelain, were analyzed for enzymatic activities, molecular weights, amino acid compositions and amino- and carboxyl-terminal residues. Components I-1 and II-1 of stem bromelain were very similar to fruit bromelains B and A, respectively, with respects to chromatographic behavior and molecular weight, but slight differences in other properties were noted. In enzymatic activities, the k3 (app) values of components I-1 and I-2 for BAA** (0.0046 and 0.0039 sec−1) were much lower than those of other components in stem and fruit bromelains. The molecular weights determined by Sephadex G-75 gel filtration were about 28,000 for stem component I-1 and fruit bromelain B, about 19,000 for fruit bromelain C and about 18,000 for stem components I-2, II-1 and II-2 and fruit bromelain A, respectively. In amino acid composition, stem component I-1 was similar to fruit bromelain B, while other components were somewhat different. Stem component I-1 gave valine and other six components gave alanine as the major amino-terminal residue. On the other hand, stem components I-1 and II-1, and fruit bromelains A and B gave glycine, and residual three components gave serine as the major carboxyl-terminal residue. Although the procedure for purification of the main enzyme (component I-1) from the stem was much improved than that reported previously, endgroup analyses indicated that the preparation contained still extra amino- and carboxyl-terminal residues. These additional terminal residues were not decreased even though the main component isolated from the completely inactivated crude stem bromelain with iodoacetamide was used. Additional small amounts of end groups were also found in other six components.