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A low molecular weight active fragment of potato proteinase inhibitor IIPB was obtained by incubating the inhibitor with an equimolar amount of trypsin [EC 3.4.21.4] at pH 8 and 30 degrees for 16 hr, followed by gel filtration through Sephadex G-50, treatment with trichloroacetic acid, and CM-cellulose chromatography. The purified active fragment consisted of a single peptide chain with a molecular weight of 4,300, comprising 39 amino acid residues. It retained very strong inhibitory activity against chymotrypsin [EC 3.4.21.1] and subtilisin [EC 3.4.21.14]. However, the yield of this active fragment was rather low and was variable. On further incubation with trypsin, it was converted into smaller inactive peptides.

Four proteinase inhibitors, A-II, A-III, B-I, and B-II, were isolated from seeds of Albizzia julibrissin (silk tree) of the subfamily Mimosoideae, which is often regarded as the most primitive group of the Leguminosae plants. They were all of the high-molecular weight type (21,600 for A-II and A-III, and 19,000 for B-I and B-II), and composed of two polypeptide chains, linked together by a disulfide bond. A-II (A-III) inhibited bovine trypsin and alpha-chymotrypsin probably at an identical site. B-I (BII) inactivated bovine alpha-chymotrypsin and porcine elastase. Sequence analyses of A-II and B-II revealed a considerable homology with soybean trypsin inhibitor (Kunitz) but suggested the presence of an about 20-amino acid insertion in the A-II molecule.

Double-headed protease inhibitors I, IIa, and IIc (AB I, AB IIa, and AB IIc) have been purified from azuki beans "Takara" (Vigna angularis) by conventional chromatographic methods and their amino acid sequences have been determined. AB I, AB IIa, and AB IIc had molecular weights of 9,166, 8,661, and 8,756 daltons, consisting of 82, 78, 79 amino acid residues, respectively. The molecular weights of these inhibitors, determined by gel filtration at pH 8.0, were 18,000 for AB I and 17,000 for both AB IIa and AB IIc, indicating that the inhibitors are dimers. The inhibitors had isoelectric points of 4.7 (AB I), 6.8 (AB IIa), and 6.2 (AB IIc). AB I stoichiometrically inhibited both trypsin and chymotrypsin at a molar ratio of 1 : 1. On the other hand, AB IIa and AB IIc both inhibited trypsin at a molar ratio of about 1 : 2 and also inhibited chymotrypsin, though only weakly. Sequence comparison with other double-headed inhibitors indicated the reactive sites of AB IIa and AB IIc for trypsin to be Lys26-Ser27 and Arg53-Ser54, and those of AB I for trypsin and chymotrypsin to be Lys26-Ser27 and Tyr53-Ser54, respectively. The differences between AB IIa and AB IIc were that AB IIa lacked the C-terminal aspartic acid residue, and that Glu10 and Arg60 in AB IIa were replaced by Gln10 and His60 in AB IIc. A comparison between AB IIa and AB I revealed 25 variant amino acids among the 78 residues of AB IIa; further, Ab IIa lacked 4 amino acid residues in the C-terminal region of AB I.