Iwasaki T, Kiyohara T, Yoshikawa M
J Biochem. 1976 Feb;79(2):381-91. doi: 10.1093/oxfordjournals.jbchem.a131081.
The complete amino acid sequence of an active fragment of potato proteinase inhibitor IIa has been established by the Edman degradation procedure and the carboxypeptidase technique. Sequence analyses were carried out on the reduced and carboxymethylated active fragment and its tryptic peptides. To aid in the alignment of some tryptic peptides, the partial sequences of two fragments obtained by selective tryptic cleavage of the reactive site peptide bond of inhibitor IIa at acidic pH, with subsequent reduction and carboxymethylation, were also analyzed. The active fragment consisted of 45 amino acid residues including 6 half-cystine residues. Degradation of the intact active fragment by subtilisin [EC 3.4.21.14.] at pH 6.5. yielded 3 cystine-containing peptides. Sequence analyses of these peptides revealed that the 3 disulfide linkages were located between Cys(10) and Cys(24), Cys(14) and Cys(35), and Cys(20) and Cys(43). The reactive site peptide bond of inhibitor IIa, a Lys-Ser bond, was located between positions 32 and 33 of the active fragment. The overall sequence of the active fragment was quite different from those of potato chymotrypsin inhibitor I (subunit A) and potato carboxypeptidase inhibitor.
通过埃德曼降解法和羧肽酶技术确定了马铃薯蛋白酶抑制剂IIa活性片段的完整氨基酸序列。对还原和羧甲基化的活性片段及其胰蛋白酶肽段进行了序列分析。为了有助于一些胰蛋白酶肽段的比对，还分析了通过在酸性pH下选择性胰蛋白酶切割抑制剂IIa的活性位点肽键、随后进行还原和羧甲基化得到的两个片段的部分序列。活性片段由45个氨基酸残基组成，包括6个半胱氨酸残基。在pH 6.5下，用枯草杆菌蛋白酶[EC 3.4.21.14.]降解完整的活性片段，产生了3个含胱氨酸的肽段。对这些肽段的序列分析表明，3个二硫键位于Cys(10)与Cys(24)、Cys(14)与Cys(35)以及Cys(20)与Cys(43)之间。抑制剂IIa的活性位点肽键，即赖氨酸-丝氨酸键，位于活性片段的32位和33位之间。活性片段的整体序列与马铃薯胰凝乳蛋白酶抑制剂I（亚基A）和马铃薯羧肽酶抑制剂的序列有很大不同。
