Two peptide chains A_1 and A_2 of the Lys active fragment, linked via a couple ofinter-disulfide bonds, could be separated from each other after reduction with dithiothreitoland gel filtration on Sephadex G-25. Reoxidation of the reduced peptide chain A_1 resultedin recovering the inhibitory activity with 25% yield, based on the original activity of theLys fragment. The A_1 active fragment was further purified by affinity chromatographywith immobilized trypsin. Sephadex G-25 gel filtration produced two forms of the A_1 activefragment, the major fraction being a monomer and the minor one being a dimeer with loweractivity. The results obtained offered evidence of the evolution of mung bean inhibitorfrom an ancestral single-headed inhibitor by fused gene duplication with A_2 as a connectingpeptide. The CD spectra of the Lys fragment and the reoxidized peptide chain A_1 werealso compared. Two peptide chains A_1 and A_2 of the Lys active fragment, linked via a couple ofinter-disulfide bonds, could be separated from each other after reduction with dithiothreitoland gel filtration on Sephadex G-25. Reoxidation of the reduced peptide chain A_1 resulted in recovering the inhibitory activity with 25% yield, based on the original activity of the Lys fragment. The A_1 active fragment was further purified by affinity chromatography with immobilized trypsin. Sephadex G-25 gel filtration produced two forms of the A_1 active fragment, the major fraction being a monomer and the The results obtained was evidenced by the evolution of mung bean inhibitor from an ancestral single-headed inhibitor by fused gene duplication with A_2 as a connecting peptide. The CD spectra of the Lys fragment and the reoxidized peptide chain A_1 were also compared