Trypsin Lab Essay Research Paper Title The

Trypsin Lab Essay, Research Paper Title: The Effects of Substrate Concentration and Temperature on the Rate of Hydrolysis of the Enzyme Trypsin. Abstract: Quantitative measurements can relate both temperature and substrate concentration to the enzymatic activity of trypsin. By analyzing the data, it is suggested that at BAPNA concentrations below those corresponding to Vmax are rate limiting, as less active sights are available for adhesion. The values of Vmax and Km relate a temperate catalytic efficiency of trypsin. The temperature range of most efficiency for the enzyme was those between 36 and 54 degrees Celsius. Introduction: Enzymes are specialized proteins that aid in formation or breakdown of larger protein or multi-protein complexes. Trypsin is a pancreatic protease

that digests proteins by hydrolyzing the peptide bonds in proteins. It has a high degree of specificity and will only hydrolize the peptide bonds that occur on the carboxyl side of the amino acids lysine or arginine. Generally hydrolytic reactions occur with the addition of water to breakdown a large protein into two protein fragments. Substrate concentration and temperature both would interfere and affect with the hydrolysis of Na-benzol-L-arginly-p-nitroanalide (BAPNA) into arginina and p-nitroaniline (PNA). An increase in the substrate concentration would most likely enhance the conversion into PNA, as collisions between the enzyme and substrate would increase. Temperature and pH can both influence the kinetics of an enzyme (Karp 100). Trypsin, being an organic enzyme, would

probably work most effectively at temperatures consistent with biological life, falling in the ranges of 34?C and 40?C. The change in PNA concentration can be plotted against BAPNA concentration or temperature. To measure the kinetics of an enzyme, two variables can be found, Vmax and Km. Km is the estimated substrate concentration required for the reaction to advance at one half Vmax. Vmax is the maximal velocity of the reaction. These two values can be determined from the double reciprocal of the Michalelis-Menton equation or the Lineweaver-Burke Plot, with the y intercept being 1/ Vmax, and the x intercept being -1/ Km. the equations are as follows: Michalelis-Menton velocity of reaction= Vmax (substrate concentration)/( Km’s) Lineweaver-Burke plot 1/velocity= Km/

Vmax*1/sibstrate concentration+1/ Vmax Methods: Part 1: Effect of Substrate Concentration on Velocity Cuvette one was placed into the spectrophotometer containing the following: 0.1 ml of 10X buffer (400 mM Tris-HCl and 160 mM CaCl2), and 0.9 ml H3O. The absorbance was then read using a wavelength of 410 nm, and the absorbance number was used as a blank for the rest of the lab. The cuvette contained no PNA (the colored substrate) and hence is the reading when no reaction is taking place. The wavelength was chosen because the substrate is colored yellow, and a color other than yellow was needed to penetrate the cuvette, (410 nm is blue light). The absorbencies were then found using the following concentrations (in mM): 0.020, 0.040, 0.060, 0.080, 0.100, 0.120, 0.160, and 0.200.

The results were then plotted with the absorbance being the dependent variable and the concentration the independent. The extinction coefficient, also called the molar absorption coefficient, could then calculated using the equation provided by the Biology 152 Lab Manual, E=A/cl were “E” is the extinction coefficient, “A” the absorbance, “c” the product of concentration, and “l” the length of the light path. With the extinction coefficient found, the rate of reaction could be found. 0.1 ml of 10X buffer and 0.4 ml of H3O were added to two cuvettes and gently mixed. 0.4 ml of 1 mM BAPNA was then added to each. To cuvette one, an additional 0.1 ml of H3O was added and mixed and placed in the spectrophotometer. This was the control to measure the hydrolysis of BAPNA