Isolation of Subunits of α-Amylase Inhibitors from Phaseolus Beans

Subunit structures were examined for proteinaceous α-amylase inhibitors, TAI, UAI, DAI, OAI-2, MAI-2, and SAI-2, from respective Phaseolus beans of Toramame, Uzuramame, Daifuku, Otebou, Murasakihanamame, and Shirohanamame. Firstly, TAI was denatured by heating at 50℃ for 30 min in 50 mM imidazol-HCl buffer(pH 7.2)containing 6M guanidine HCl. The denatured protein was chromatographed on DEAE-Sephacel in the presence of 8M urea, resulting in a separation of two protein components, α and β. Size-exclusion HPLC also separated the denatured inhibitor into two protein peaks, I and II. N-Terminal 20 amino acid sequences showed that the protein components, α and β, corresponded to II and I, respectively. The other inhibitors denatured by the above procedure were also analyzed for their subunit structures by HPLC, and found to be composed of two subunits, α and β like that TAI consisted of α and β. Comparison of N-terminal 20 amino acid sequences of subunits indicated the presence of a few amino acid substitutions among the inhibitors