Journal of Protein Chemistry, Vol. 17, No. 8, 1998 The Complete Ammo Acid Sequence of a Trypsin Inhibitor from Bauhinia variegata var. Candida Seeds Trypsin inhibitors of two varieties of Bauhinia variegata seeds have been isolated and character- ized. Bauhinia variegata Candida trypsin inhibitor (Bvc TI) and B. variegata lilac trypsin inhibitor (BvlTI) are proteins with M r of about 20,000 without free sulfhydryl groups. Amino acid analysis shows a high content of aspartic acid, glutamic acid, serine, and glycine, and a low content of histidine, tyrosine, methionine, and lysine in both inhibitors. Isoelectric focusing for both varieties detected three isoforms (pI 4.85, 5.00, and 5.15), which were resolved by HPLC procedure. The trypsin inhibitors show K i values of 6.9 and 1.2 nM for BvcTI and BvlTI, respectively. The N- terminal sequences of the three trypsin inhibitor isoforms from both varieties of Bauhinia variegata and the complete amino acid sequence of B. variegata var. Candida L. trypsin inhibitor isoform 3 (BvcTI-3) are presented. The sequences have been determined by automated Edman degradation of the reduced and carboxymethylated proteins of the peptides resulting from Staphylococcus au- reus protease and trypsin digestion. BvcTI-3 is composed of 167 residues and has a calculated molecular mass of 18,529. Homology studies with other trypsin inhibitors show that BvcTI-3 belongs to the Kunitz family. The putative active site encompasses Arg (63)–Ile (64). KEY WORDS: Trypsin inhibitor; Bauhinia variegata seeds; primary structure; sequence homology. 0277-8033/98/1100-0827$15.00/0 © 1998 Plenum Publishing Corporation Luciana Di Ciero, 1 Maria L. V. Oliva, 4 Ricardo Torquato, 4 Peter Kohler, 2 Jurgen K. P. Weder, 3 Jose Camillo Novello, 1 Claudio A. M. Sampaio, 4,5 Benedito Oliveira, 1 and Sergio Marangoni 1 Received January 9, 1998; revised April 21, 1998 1. INTRODUCTION Serine protease inhibitors have been isolated from vari- ous Leguminosae seeds (Richardson, 1991). Considering nutritional aspects, they are important in reducing pro- tein digestion in animals and humans (Richardson, 1977). As the role of inhibitors in plant seeds is not fully understood, further knowledge concerning trypsin inhib- 1 Department of Biochemistry, Biology Institute, Universidade Estad- ual de Campinas, 13083-970, Campinas, SP, Brazil. 2 Deutsche Forchungsanstalt fur Lebensmittelchemie, D-85748 Garch- ing, Germany. 3 Institut fur Lebensmittelchemie, Technische Universitat Munchen, D-85748 Garching, Germany. 4 Department of Biochemistry, Escola Paulista de Medicina, UNIFESP, 04044-900, Sao Paulo, Brazil. 5 To whom correspondence should be addressed; e-mail:sampaio. bioq@epm.br. itors in plants seeds such as Bauhinia variegata may contribute to the understanding of the interactions be- tween plant protease inhibitors and endogenous animal proteases (Xavier-Filho and Campos, 1989). Serine proteinase inhibitors from plants inactivate several enzymes, and are classified in families: the Kun- itz trypsin, Bowman-Birk proteinase, potato I, potato II, barley trypsin, and squash inhibitor families (Weder, 1991). Kunitz-type inhibitors are proteins (M, 18,000- 22,000) with one or two polypeptide chains and low cys- teine content, usually with four Cys residues arranged in two disulfide bridges, and containing one reactive site, located in the loop formed by the disulfide bridge that is close to the N-terminus of the molecule with 170–180 amino acids (Richardson, 1991). The trees of the genus Bauhinia (Leguminosae- Caesalpinioideae) are distributed throughout Brazil, and are used as ornamental trees, as medicinal plants, and as 827