Isolation and Characterization of a New Trypsin Inhibitor from Crotalaria paulina Seeds

Abstract

SummaryA new trypsin inhibitor (CPTI) has been isolated from Crotalaria paulina seeds. Puri cation of the inhibitor was carried out by gel ltration, ion-exchange chromatography, and subsequent reversed-phase HPLC. The presence of a single polypeptide chain, with a molecular mass of 20 kDa and isoelectric point 4.0, was detected. The trypsin inhibitor had a K i value of 4.5 £ 10 ¡ 8 M and was capable of acting on human, bovine, and porcine trypsin and weakly on bovine chymotrypsin. Amino acid analysis showed that CPTI has a high content of aspartate, glutamate, leucine, serine, and glycine, having 177 amino acid residues in its composition. These data suggest that the protein belongs to the Kunitz-type trypsin inhibitors. IUBMB Life, 48: 519-523, 1999

Purification and characterization of a trypsin inhibitor from the seeds of <italic>Artocarpus heterophyllus</italic> Lam.

“…Some inhibitors of this family coming from Psophocarpus tetragonolobus (L) Dc and Bauhinia rufa can weakly inhibit trypsin, but are effective for chymotrypsin or elastase [26,27]. A few Kunitz-type inhibitors from Peltophorum dubium, Crotalaria paulina, and Poecilanthe parviflora only inhibit trypsin and have no inhibition activity toward chymotrypsin and elastase [21,28,29]. However, some Kunitz inhibitors not only exhibit potent inhibitory activity toward trypsin but also inhibit other proteases, such as chymotrypsin, elastase, and papain [5,12,20].…”

Peltophorum dubium and soybean Kunitz-type trypsin inhibitors induce human Jurkat cell apoptosis

“…Such protease inhibitors (PI) are pseudosubstrates with affinity toward the catalytic site of enzymes [1]. They are widely distributed in living organisms, and many studies have been performed on plant PI, especially on those isolated from the Leguminosae family [2][3][4][5]. Legume seeds are known to contain high levels of PI, such as those belonging to the Kunitz and Bowman-Birk-type families.…”

Affinity Chromatography as a Key Tool to Purify Protein Protease Inhibitors from Plants

“…The addition of calcium into of enzyme solution beyond stabilizing enzymes prevents the process of autolysis (Sipos & Merkel, 1970). The formation of p-nitroaniline (bright yellow) from amide substrate (BApNA, BTpNA) by trypsin and chymotrypsin is monitored at 405-410 nm Macedo et al, 2007;Mello et al, 2001;Pando et al, 1999;Oliveira et al, 2007Oliveira et al, , 2009Oliva et al, 1996).…”

“…Trypsin and chymotrypsin from bovine pancreas are serine protease more used in vitro assays for determination of the presence of inhibitory activity by the crude extracts of several origins, by accompaniment of inhibitory activity during all the isolation process, as well as to characterize inhibitor purified, including determination of dissociation constant (Ki), formation of inhibitor-serine protease complex and studies about stability of the inhibitory activity (Mello et al, 2001;Macedo et al, 2002Macedo et al, , 2003Macedo et al, , 2007Pando et al, 1999;Araujo et al, 2005;Oliveira et al, 2002Oliveira et al, , 2007aOliveira et al, ,b, 2009). Mature trypsin is composed for 223 amino acid residues with His57, Asp102 and Ser195 residues forming its catalytic triad (figure 2).…”

“…Hydrolyze of azocasein (Xavier-Filho et al, 1989) and release of p-nitroanilide from amide synthetic substrate (BTpNA, N-Benzoyl-L-tyrosine p-nitroanilide; N-Succinyl-Lphenylalanine-p-nitroanilide) are commonly used to assay chymotrypsin inhibitory activity Macedo et al, 2007;Mello et al, 2001;Pando et al, 1999) as well as the hydrolyze of ester linkages of BTEE, N-Benzoyl-L-tyrosine ethyl ester (Bhattacharyy et al, 2006(Bhattacharyy et al, , 2007(Bhattacharyy et al, , 2009). …”