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 H2O. 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 H2O 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 H2O was added and mixed and placed in the spectrophotometer. This was the control to measure the hydrolysis of BAPNA in the absence of enzyme. In the second cuvette 0.1 ml of enzyme was added and mixed, then placed into the spectrophotometer. Readings of the absorbencies were taken every 15 seconds for ten minuets. The extinction coefficient was then used to convert each absorbance reading to PNA concentration.
Seven tubes were prepared with the following a constant of 10X buffer, water, and enzyme. Added to the mixture were the following amounts (in ml) of BABNA before placing into the spectrophotometer: 0.05, 0.10, 0.20, 0.30, 0.45, 0.60, and 0.80. Corresponding amounts of H2O were then added in the following amounts (ml): 0.75, .70, .60, .50, .35, .20, and .00. The absorbencies were read every 15 seconds for 2.5 minuets.
The PNA concentration was then plotted as a function of time. The slope of the linear portion of the graph represented the initial velocity of substrate hydrolysis as a function of time. The linear properties of the graph begin to wane as the BAPNA supply decreases over time.
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
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.
Results:
The reaction rate against the BAPNA concentration of the hydrolysis of BAPNA displays a preliminary linear increase in the rate of reaction with a gradual decrease in the change of rate with substrate concentration to Vmax. The Lineweaver-Burke plot graph (Fig 1) estimated the value of Vmax to be 0.0627 mM/min, while the Km estimated was 0.413 mM. The equation for the double reciprocal was 1/velocity=(6.586) 1/substrate conc.+15.947.
The curve representing the rate of reaction versus time demonstrated a low rate of reaction for the low temperature extremes, including 10?C. The most efficient temperature demonstrated by our experiment was that of 54?C. However when the temperature was increased to 56?C, the reaction declined. Each graphical representation of the individual temperatures carried with it similar characteristics. Each possessed an initial linear relationship, and then each began to level off as the extinction coefficient was reached.
Discussion:
The results of our first experiment displayed that as the concentration of substrate in a solution of enzyme increases, the rate of reaction increases. Enzymes work on the principal that substrate is formed by random collisions between enzyme and substrate. Hence more of either will increase the production of product. Our data showed this too is true, as product was formed at a faster rate with more enzymes, than of those solutions containing less.
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.
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.
References:
Karp, G. (1996) Bioenergetics. Pages 91-103 in Karp, G., Cell and Molecular Biology: Concepts and Experiments Second Edition. John Wiley & Sons Inc., New York
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