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

The increasing of PNA concentration will drive the initial velocity of the equation equal of lesser to Vmax and extent the linear portion of the graph. More trypsin would invariably provide more active sites to which BAPNA molecules can bind. The initial velocity of substrate hydrolysis is thus greater. Dropping the concentration would have the opposite effect, lowering the initial velocity of the reaction, limiting the linear region, as the former extends the linear region.

Part 2: Effect of Temperature on Velocity

Trypsin is also sensitive to temperature. Higher temperatures seemingly denature the enzyme, changing its structure and hence it is no longer able to fit in the substrates active site. Being a biological enzyme, it would assume to work well at temperatures associated with biological life, which it did, working optimally within the range of 36-54 degrees Celsius. Below this temperature, little activity was observed as the molecules were moving in a slower fashion, and the shape once again is changed.

The values of Km and Vmax (0.413 mM and 0.0627 respectively) obtained from Fig 1 imply that trypsin has a moderate affinity for its substrate.

Obtain constant amounts of 10X buffer, H2O, BAPNA, and enzyme and place into cuvettes, saving the addition of enzyme until last. Acquire prescribed temperature by lowering the bottom of the cuvette into a bath for two minuets. When removed, add the enzyme, place in the spectrometer with the same 410 nm setting and record absorbance's every 15 seconds for two and a half minuets. Repeat for the following temperatures (°C): 10, 38, 45, 47, 50, and 54. Use data to determine the ideal temperature for enzyme action.

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