Isolation and characterization of the chemically cross-linked vegetable proteinase inhibitor B ( Sagittoria sagittifolia linn) by using bifunctional reagents and by high pressure liquid chromatography

Abstract

1. 1. Proteinase inhibitor B from the root of the arrowhead vegetable legume has been cross-linked with DMA, DMS and EGS. The cross-linked AI-B with DMA resulted only in the monomer inhibitor, but with DMS and EGS, the dimer, trimer and a small amount of tetramer were obtained besides the monomer. 2. 2. Difficulty in the isolation and separation of monomer, dimer, trimer and tetramers of AI-B by classical column (TSK-2000-SW) on HPLC was encountered. The successful separation and isolation of monomer and dimer was obtained using an advanced column (Shim-Pack-Diol-300) for exclusion HPLC. 3. 3. The assay of inhibitory activity showed that the monomer and dimer had a much lower activity than the native AI-B. Furthermore, the monomeric activity was decreased more drastically than those of the dimeric. The circular dichroism (CD) spectra showed that the monomeric conformation had been changed, but the dimeric conformation had not.