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

Abstract

A new trypsin inhibitor (CPTI) has been isolated from Crotalaria paulina seeds. Purification of the inhibitor was carried out by gel filtration, 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 Ki value of 4.5 x 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.

Two Kunitz-Type Inhibitors with Activity Against Trypsin and Papain from Pithecellobium dumosum Seeds: Purification, Characterization, and Activity Towards Pest Insect Digestive Enzyme

“…In addition, other members of this family inhibit preferentially trypsin but also inhibit chymotrypsin to varying degrees [12,13]. The dissociation constant establishes a higher affinity between the trypsin and PdKI-3.2 than with PdKI-3.1 and is in agreement with Ki values reported for other trypsin inhibitors [18,42,43]. Table 2 shows the in vitro inhibition of the inhibitors purified here against serine proteinases.…”

Identification of a Kunitz-Type Proteinase Inhibitor from Pithecellobium dumosum Seeds with Insecticidal Properties and Double Activity

“…In this study, a related Kunitz inhibitor from P. domusum seeds was purified and characterized and its effects on digestive proteinsases from insect pests were examined in vitro. The PdKI purified is a protein with a unique polypeptide chain of molecular mass of 19.7 kDa, in agreement with the molecular mass of other trypsin inhibitors ( − ). Thermal inactivation of PdKI at different temperatures resulted in a progressive loss of trypsin-inhibiting activity at temperatures >40 °C and an ∼40% decrease at 100 °C.…”

Trypsin Isoinhibitors with Antiproliferative Activity toward Leukemia Cells fromPhaseolus vulgariscv “White Cloud Bean”

“…The unadsorbed fraction (B1) was dialyzed against 10 mM NH 4 OAc buffer (pH 5) and then applied to a 2.5 × 20 cm column of SP-Sepharose (GE Healthcare). After elution of unadsorbed proteins (fraction S1), the column was eluted with a 0-1 M NaCl concentration gradient in the NH 4 OAc buffer. The first and second adsorbed fractions (SP2 and SP3) were then further purified by gel filtration on a Superdex 75 HR 10/30 column (GE Healthcare) in 0.2 M NH 4 HCO 3 buffer (pH 8.5) using an AKTA Purifier (GE Healthcare).…”

“…Protease inhibitors have been purified from an array of leguminous and nonleguminous species encompassing Torresea cearensis [1], Erythrina caffra [2], Dolichos lablab [3], Crotalaria paulina [4], Medicago scutellata [5,6], Canavalia gladiata [7], Pisum sativum [8], Dimorphandra mollis [9], Swartzia pickellii [10], Psophocarpus tetragonolobus [11], Delonix regina [12], Poecilanthe parviflora [13], Adenanthera pavonina [14], Cajanus cajan [15], Dolichos biflorus [16], Phaseolus acutifolius [17], Arachis hypogaea [18], Leucaena leucocephala [19], Bauhinia bauhinioides [20], Bauhinia variegata [21], Bauhinia ungulata [22], Vigna unguiculata [23], Lens culinaris [24], Glycine max [25], Peltophorum dubium [26], Pithecellobium dulce [27], Glycine soja [28], and barley [29].…”

“…Starting buffer for SP-Sepharose chromatography is 10 mM NH 4 OAc buffer (pH 5). Broken line across the right half of the chromatography represents the linear 0-1 M NaCl gradient in 10 mM NH4 OAc buffer (pH 5) employed to elute adsorbed proteins. Trypsin inhibitory activity was found in both fractions SP2 and SP3.…”