Abstract
A Protease inhibitor from seeds of rice bean has been purified to apparent homogeneity as judged by native-PAGE with about 29% recovery using ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose, and gel filtration through Sephadex G-100. The purified preparation with molecular weight of 16.8 kD was found to be a monomer as revealed by SDS-PAGE under reducing and non-reducing conditions. The purified inhibitor in solution was stable upto 40°C. However, it lost its activity gradually and was completely inactive when heated at 100°C for 2 h and 125°C for 1 h. Heating at 125°C changed the conformation of the inhibitor as was evident from the altered UV spectrum compared to that of native. It had two pH optima at pH 6.0 and at 10.0 and was stable over a wide range of pH (pH 3.0 to 10.0). It lost its activity on exposure to 2-mercaptoethanol, indicating the role of S-S linkages in maintaining the three dimensional structure of the protein inhibitor. The inhibitor was completely inactive towards papain, while it inhibited pepsin only slightly. Trypsin and chymotrypsin were inhibited upto the extent of 60% and 30%, respectively. Trypsin inhibition was of non-competitive type with dissociation constant for the enzyme-inhibitor complex in the region of 2.07 mg ml-1. The rice bean inhibitor appears to be of Bowman-Birk type as it has molecular weight lower than that generally observed for Kunitz type inhibitors and seems to be double headed - a characteristic specific of Bowman - Birk type inhibitor.
References
Liener IE & Kakade ML, In The toxic constituents of plant foodstuffs, 2nd Edition, Academic Press, New York (1980) pp 7–71.
Bishnoi S & Khetarpaul N, J Food Sci Technol, 31 (1994) 73.
Ramamani S, Chandrasekhara HN & Murthy KN, J Food Sci Technol, 33 (1994) 197.
Tan-Wilson AL & Wilson KA, In Nutrirional and toxicological significance of enzyme inhibitors in plant foods (M Friedman, Editor), Plenum Press, New York (1986) pp 331–341.
Hilder VA, Gatehouse AMR, Sheermen SE, Barker RF & Boulter D, Nature, 330 (1987) 160.
Hilder VA, Gatehouse AMR & Boulter D, In Genetic engineering of crop plants (D Grierson, G Lycett, Editors), Butterworths (1980) pp 51–66.
Johnson R, Narvaez J, An G & Ryan CA, Proc Natl Acad Sci, USA, 86 (1989) 9871.
Jouanin L, Bottino MB, Girard C, Morrot G & Giband M, Plant Sci, 131 (1998) 1.
McMunnus MT, Laing WA & Christeller JT, Phytochemistry, 37 (1994) 921.
Wilson KA, In Advances in cellular and molecular biology of plants, Vol 4 (IK Vasil, Editor), Kluwer Academic Publishers, Netherlands (1997) pp 331–374.
Malhotra S, Malik D & Dhindsa KS, Plant Foods Humn Nutr, 38 (1988) 75.
Kakade ML, Simons N & Liener IE, Cereal Chem, 46 (1969) 518.
Davis BJ, Ann NY Acad Sci, 121 (1964) 404.
Laemmli UK, Nature, 227 (1970) 680.
Pichare MM & Kachole MS, Physiol Plant, 98 (1996) 845.
Ryan CA, Ann Rev Phytopathol, 28 (1990) 425.
Duarte PR, Bergeron D & Nielsen SS, J Agri Food Chem, 40 (1992) 32.
Chang CR & CC Tsen, Cereal Chem, 58 (1981) 207.
Annapurna SS, Ramadoss CS & Prasad DS, J Sci Food Agri, 54 (1991) 605.
Godebole SA, Krishna TG & Bhatia CR, J Sci Food Agri, 64 (1994) 87.
Wu C & Whitaker JR, J Agri Food Chem, 39 (1991) 1583.
Prakash B, Selvaraj S, Murthy MRN, Sreerama YN, Rama Sarma PR, Rajagopal Rao D & Gowda LR, J Mol Evol, 42 (1996) 560.
Norimura H, Hirashiki I, Ogata F, Yoshida N & Ito A, Agric Biol Chem, 54 (1990) 3029.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Maggo, S., Malhotra, S.P., Dhawan, K. et al. Purification and Characterization of Protease Inhibitor from Rice Bean (Vigna umbellata T.) Seeds. J. Plant Biochem. Biotechnol. 8, 61–64 (1999). https://doi.org/10.1007/BF03263060
Received:
Published:
Issue Date:
DOI: https://doi.org/10.1007/BF03263060