Abstract
A trypsin inhibitor was isolated from Cassia obtusifolia by ammonium sulfate precipitation, Sepharose 4B-trypsin affinity and Sephadex G-75 chromatography. The inhibitor consisted of a single polypeptide chain with a molecular mass of 19, 812.55 Da. It was stable from pH 2 to 12 for 24 h, whereas it was unstable either above 70°C for 10 min or under reduced conditions. The inhibitor, which inhibited trypsin activity with an apparent Ki of 0.3 μM, had one reactive site involving a lysine residue. The native inhibitor was resistant to pepsin digestion, whereas the heated inhibitor produced 40% degree of susceptibility. The disulfide linkage and lysine residue were important in maintaining its conformation. Partial amino acid sequence of the purified protein showed a high degree of homology with various members of the Kunitz inhibitor family. Moreover, the inhibitor showed significant inhibitory activity against trypsin-like proteases present in the larval midgut on Pieris rapae and could suppress the growth of larvae.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Beekwilder J, Schipper B, Bakker P et al (2000) Characterization of potato proteinase inhibitor II reactive site mutants. Eur J Biochem 267:1975–1984
Broadway RM (1995) Are insects resistant to plant proteinase inhibitors? J Insect Physiol 41:107–116
Erlanger BE, Kokowsky N, Cohen W (1961) The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys 95:271–278
Felicioli R, Garzelli B, Vaccari L et al (1997) Activity staining of protein inhibitors of proteases on gelatin-containing polyacrylamide gel electrophoresis. Anal Biochem 244:176–179
Gatehouse AMR, Norton E, Davison GM et al (1999) Digestive proteolytic activity in larvae of tomato moth, Lacanobia oleracea; effects of plant proteinase inhibitor in vitro and in vivo. J Insect Physiol 45:545–558
Giri AP, Harsulkara AM, Ku MSB et al (2003) Identification of potent inhibitors of Helicoverpa armigera gut proteinases from winged bean seeds. Phytochemistry 63:523–532
Guo HZ, Chang ZZ, Yang JR et al (1998) Anthraquinones from hairy root cultures of Cassia Obtusifolia. Phytochemistry 49:1623–1625
Harsulkar AM, Giri AP, Patankar AG et al (1999) Successive use of non-host plant proteinase inhibitors required for effective inhibition of gut proteinases and larval growth of Helicoverpa armigera. Plant Physiol 121:497–506
Knights RJ, Light A (1976) Disulfide bond-modified trypsinogen. J Biol Chem 251:222–228
Laskowski Jr M, Qasim MA (2000) What can the structures of enzyme-inhibitor complexes tell us about the structures of enzyme substrate complexes? Biochim Biophys Acta 1477:324–337
Roychaudhuri R, Sarath G, Zeece M et al (2003) Reversible denaturation of the soybean Kunitz trypsin inhibitor. Arch Biochem Biophys 412:20–26
Roychaudhuri R, Sarath G, Zeece M et al (2004) Stability of the allergenic soybean Kunitz trypsin inhibitor. Biochim Biophys Acta 1699:207–212
Sreerama N, Venyaminov SY, Woody RW (2000) Estimation of protein secondary structure from circular dichroism spectra: inclusion of denatured proteins with native proteins in the analysis. Anal Biochem 287:243–251
Vadivel V, Janardhanan K (2002) Nutritional and antinutritional characteristics of seven south Indian wild legumes. Plant Food Hum Nutr 57:151–164
Acknowledgement
This work was supported by Grant (NCET040861 and 20006502) from Ministry of Education, People’s Republic of China.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Liao, H., Ren, W., Kang, Z. et al. A trypsin inhibitor from Cassia obtusifolia seeds: isolation, characterization and activity against Pieris rapae . Biotechnol Lett 29, 653–658 (2007). https://doi.org/10.1007/s10529-006-9281-6
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10529-006-9281-6