Activity of a Protease
The Activity of a Protease (Trypsin)
Introduction
Enzymes catalyze reactions by creating alternate reaction mechanisms whose transition states are more thermodynamically stable than uncatalyzed reactions (Berg et al., 2002; UBC Department of Microbiology and Immunology, 2006). Increased thermodynamic stability in these transition states reduces the energy of activation, the minimum amount of energy input a chemical system requires for a reaction to occur (UBC Department of Microbiology and Immunology, 2006). Thus, substrate is converted to product using less energy at amplified rates when enzymes are involved.
Trypsin is a serine protease, a type of enzyme with a characteristic serine residue in its active site (Berg et al, 2002). Produced by the pancreas, trypsin acts in the small intestine to hydrolyze peptide bonds allowing for protein digestion (McDowell, 2007). As such, trypsin activity is very important in nutrition and health, with deficiencies in this and other digestive enzymes resulting in some of the symptoms of cystic fibrosis, among other diseases (Babar et al, 1988; Oliveira et al, 1999). Thus, the effect of various conditions on trypsin activity is of great interest and is further investigated here. The effects of enzyme and substrate concentrations, as well as the effects of pH, on the initial rates of BAPNA hydrolysis by trypsin are investigated.
Additionally, the inhibitory activity of the Jack Beans trypsin inhibitor is determined. This type of protease inhibitor in plants (particularly seeds) is widespread and well studied in the literature (Babar et al, 1988; Liener, 1962; Oliveira et al, 1999). Known as anti-nutritive factors these inhibitors cause the plant to be toxic when ingested (Babar et al, 1988; Oliveira et al, 1999) and are believed to have evolved as defense mechanisms against herbivores (Liener, 1962).
Methods
1) Calibration curve for p-nitroaniline solutions
According to the procedure presented on
Introduction
Enzymes catalyze reactions by creating alternate reaction mechanisms whose transition states are more thermodynamically stable than uncatalyzed reactions (Berg et al., 2002; UBC Department of Microbiology and Immunology, 2006). Increased thermodynamic stability in these transition states reduces the energy of activation, the minimum amount of energy input a chemical system requires for a reaction to occur (UBC Department of Microbiology and Immunology, 2006). Thus, substrate is converted to product using less energy at amplified rates when enzymes are involved.
Trypsin is a serine protease, a type of enzyme with a characteristic serine residue in its active site (Berg et al, 2002). Produced by the pancreas, trypsin acts in the small intestine to hydrolyze peptide bonds allowing for protein digestion (McDowell, 2007). As such, trypsin activity is very important in nutrition and health, with deficiencies in this and other digestive enzymes resulting in some of the symptoms of cystic fibrosis, among other diseases (Babar et al, 1988; Oliveira et al, 1999). Thus, the effect of various conditions on trypsin activity is of great interest and is further investigated here. The effects of enzyme and substrate concentrations, as well as the effects of pH, on the initial rates of BAPNA hydrolysis by trypsin are investigated.
Additionally, the inhibitory activity of the Jack Beans trypsin inhibitor is determined. This type of protease inhibitor in plants (particularly seeds) is widespread and well studied in the literature (Babar et al, 1988; Liener, 1962; Oliveira et al, 1999). Known as anti-nutritive factors these inhibitors cause the plant to be toxic when ingested (Babar et al, 1988; Oliveira et al, 1999) and are believed to have evolved as defense mechanisms against herbivores (Liener, 1962).
Methods
1) Calibration curve for p-nitroaniline solutions
According to the procedure presented on
References: Babar, V.S., Chavan, J.K. , Kadam, S.S. 1988. Effects of heat treatments and germination on trypsin inhibitor activity and polyphenols in jack bean (Canavalia ensiformis L. DC). Plant Foods for Human Nutrition, 38:319-324. Berg, J., Tymoczko, J., and Stryer, L., 2002. Biochemistry, 5th edition. W. H. Freeman and Company, NY, pp. 230-251. Calleri, E., Temporini, C., Perani, E., Stella, C., Rudaz, S., Lubda, D.,Mellerio, G., Veuthey, J. -L. , Caccialanza G., and Massolini G. 2004. Development of a bioreactor based on trypsin immobilized on monolithic support for the on-line digestion and identification of proteins. Journal of Chromatography A, 1045: 99-109. Liener, I. E. 1962. Toxic Factors in Edible Legumes and Their Elimination. American Journal of Clinical Nutrition, 11: 281-298. McDowell, J. http://www.ebi.ac.uk/interpro/potm/2003_5/Page1.htm [accessed 2007 October 20] Oliveira, A. E., Sales, M. P., Machado, O. L., Fernandes, K. V .S., and Xavier-Filho, J. 1999. The toxicity of Jack bean (Canavalia ensiformis) cotyledon and seed coat proteins to the cowpea weevil (Callosobruchus maculatus). Entomologia Experimentalis et Applicata, 92: 249-255.