Methods for Effect of Temperature on Enzyme Activity - Lab Report Example

Enzymology Enzymes are protein-based molecules that facilitate biochemical reactions. This paper investigates the factors affecting the catalase enzyme activity to the conclusion that temperature, pH, substrate concentration, and presence of enzyme inhibitors influence activity of the catalase enzyme. Introduction Enzymology is a branch of science that studies the nature and functionality of enzymes. Catalase enzymes are susceptible to the environmental factors such as temperature, substrate concentration, pH level, enzyme concentration, and presence of enzyme inhibitors (Stocker, 2010; Saini, 2010). Substrate concentration is directly proportional to activity rate up to a point beyond which the reaction rate slows down and settles at a constant level because of the enzyme’s limiting factor (Yada, 2004; Whitehurst & Oort, 2009). Temperature and pH, however, increase the activity rate up to optimum levels beyond which the activity rate reduces. The optimum temperature and pH level for most enzymatic activity is room temperature and the pH level of seven (Rastogi, 2010). Enzyme inhibitors are another set of factors that reduce enzymes’ activity rate (Seager & Slabaugh, 2010). This report investigates the effects of substrate concentration, pH level, temperature and inhibitor hydrogen peroxide on bovine liver’s activity rate. It seeks to answer the question ‘how does the factors affect activity rate of Bovine liver?’ Materials The experimental apparatus included a computer set up, different substrate concentration levels, flasks, incubator, flask stopper, and the catalase. Methods Methods for Effect of Temperature on Enzyme Activity Care was taken to ensure that the oxygen gas sensor was plugged into the USB interface to launch the PAS portal window. This was followed by a click on the DataStudio icon to launch the program. The ‘create experiment’ button was then clicked to generate a ‘Digit’ box. A click on the setup button and a change of oxygen’s measurement unit to ppm then followed before calibration of the sensor. Amounts of liver extract were then incubated at given temperatures for five minutes before addition of substrate and push on the oxygen sensor stopper. With the stopper firmly held, the substrate was mixed and incubated while noting the amount of released oxygen. The experiment was repeated for different temperatures. Methods for the Effect of pH on Catalase Activity With the set ups at the room temperature, tests were conducted at the pH levels of two, four, six, seven, eight, ten, and twelve respectively. In each set up and for every pH level, 6 ml of buffer, 3 ml of liver extract and 4 percent of H2O2 were mixed in that consecutive order. This was followed by measurement of the level of generated oxygen for calculating reaction rates at each pH level. Methods for the Effect of Substrate Concentration on Catalase Activity With the experimental set up at the room temperature, a four percent concentrated substrate was mixed with 3 ml of liver extract and 1 ml of H2O2. This was repeated for different substrate concentrations at the following percentages: 8, 12, 16, 20, 24, and 28. Observation was then made for each set up. Methods for the effect of inhibitor hydroxylamine on catalase activity The experiment was set in different test tubes, the first test tube containing only water. Other test tubes had different volumes of water, different volumes of 1.5 percent hydrogen peroxide, and hydroxylamine inhibitors in all test tubes except test tubes one, two, three, and five. Repeating the experiments for each of the set ups, the amounts of released oxygen were recorded using the oxygen gas sensor and DataStudio. Results The following tables illustrate the results for the four sets of experiments. Results for the effect of temperature on catalase activity: Temperature tested (in °C) Reaction rate (in ppm/s) RT 71.8 ± 0.23 RT -7.02 ± 1.3 0 80 ± 0.51 RT 127 ± 0.27 40 88.6 ± 1.6 60 102 ± 2.1 80 30.2 ±0.46 Results for the effect of pH level on catalase activity: pH 2 4 6 7 8 10 12 Reaction Rate (in ppm/s) 17.2 ± 0.18 384 ± 3.1 140 ± 1.8 446 ± 7.0 249 ± 2.9 193 ± 1.4 129 ± 0.36 Results for the effect of substrate concentration on catalase activity: H2O2 concentration (%) H2O2 concentration (M) Reaction rate (ppm/s) 4 1.18 299 ±4.1 8 2.35 351 ±3.6 12 3.53 204 ±1.3 16 4.70 373 ±1.5 20 5.88 320 ±0.6 24 7.06 724 ±3.9 28 8.23 833 ±11 Results for the effect of hydroxylamine inhibitor on catalase activity: Flask Number Hydroxylamine amount (ml) Reaction Rate (ppm/s) 1 0.0 0.295 ±0.26 2 0.0 12.5 ±0.98 3 0.0 0.0±10-12 4 0.5 -0.197 ±0.19 5 0.0 229 ±1.4 6 0.5 5.79 ±0.17 7 0.5 25.8 ±0.29 8 0.5 18.0 ±0.24 9 0.5 -92.6 ±1.6 Analysis and Discussion Results or the effects of temperature on the activity rate identify a general increasing trend as temperature increases from zero to room temperature. Further increase in temperature beyond 400c, however, leads to a reduced activity rate. The following graph illustrates the effect of temperature on the enzyme’s activity rate. From 00c to room temperature, the activity rate has an almost linearly increasing trend that influenced by enzyme kinetics up to room temperature, which is the optimum condition for the enzyme. Beyond this temperature, the enzymes are denatured to lower activity rates. The effects of pH on activity rate identify the same trend as temperature. The rate is directly proportional to pH level up to neutrality beyond which an inverse proportionality is registered. This is illustrated by the following graph. The catalase was most active between pH levels of six and eight with the highest activity at the level of seven. Both extremes denature the catalase through their unfavorable conditions. Effects of substrate concentration identify a non-linear effect on activity rate that increases with an increase in substrate concentration, an indication that the catalase was not limited. Though the trend is almost constant between 10 percent and 15 percent substrate concentration levels, this is attributable to experimental errors. The observed relationship between substrate concentration and activity rate is illustrated bellow. Derivation of the Michaelis-Menten equation From two primary equations, Ks=[E][S]/[ES]………………………………………….i and [Etotal]=[E]+[ES]…………………………………………ii Then equation I can be expresses as [E]= Ks[ES]/[S] And substitution in equation ii yields [Etotal]= (Ks[ES]/[S])+[ES] Dividing both sides by [ES] yields [Etotal]/[ES]= (Ks/[S])+1 A multiplication of the left hand side by k3/ k3 with the definitions, v= k3[ES] and Vmax= k3[Etotal]leads to the equation v=(Vmax[S])/(Ks +[S]) (Thomas, 2008). Vmax and Km are undefined for Michaelis-Menten equation in this experiment. This is because the reactivity rate promises an increasing trend beyond the experiment’s limits. Km is also undefined because it is a derivative of Vmax. The Lineweaver Burk plot is a reciprocal derivative of the Michaelis-Menten equation. Its equation is as follows. 1/v= 1/ Vmax +( Km/ Vmax)(1/[S]) It, however, has the limitation of unreliability in determination of the kinetics parameters. The extrapolated Lineweaver Burk plot from the experimental results is as follows. 1/v=0.0051+ 0.01765(1/[S]) From the equation, Vmax= 1/0.0051 = 196.07 And Km = -(1/-0.29) = 3.45 The Km and Vmax values vary for Michaelis-Menten equation and Lineweaver Burk plot equations, and this is expected because of two reasons. The Michaelis-Menten equation is limited to experimental observations while the Lineweaver Burk plot equation applies theoretical formula to estimate the values. Another theoretical explanation for the difference is the unreliability of the Lineweaver Burk plot equation (Shanmugam, 2009). The presence of hydroxylamine inhibitor, however, completely alters the activity rate of the enzyme. This is observed in the low activity rates and is because of the inhibitor’s effect of substituting the enzyme while it does not facilitate the reaction. The following graph explains the experimental effects of the inhibitor. These observations of characteristics of bovine liver catalase correspond to existing literary publications (Brenda, n.d.). Conclusion Temperature, pH level, substrate concentration, and presence of enzyme inhibitors affect enzyme catalase activity in different ways. Temperature and pH facilitate activity rate up to optimum levels beyond which activity rate reduces with an increase in the factors’ levels, because the extreme conditions denature enzymes. Increase in substrate concentration also increases the activity rate. However, the existence of the inhibitor, hydroxylamine, inactivates activity of the enzyme. ] References Brenda. (n.d.). The comprehensive enzyme information system. Retrieved from: http://www.brenda-enzymes.org/ Rastogi, S. (2010). Biochemistry 3E. New Delhi: Tata McGraw-Hill Education. Saini, B. (2010). Introduction to biotechnology. New Delhi: Laxmi Publications, Ltd. Seager, S., & Slabaugh, M. (2010). Organic and biochemistry for today. Belmont, CA: Cengage Learning. Shanmugam, S. (2009). Enzyme technology. New Delhi: I. K. International Pvt Ltd. Stocker, H. (2010). General, organic, and biological chemistry. Belmont, CA: Cengage Learning. Thomas, G. (2008). Medical chemistry: An introduction. New Jersey, NJ: John Wiley & Sons. Whitehurst, R., & Oort, M. (2009). Enzymes in food technology. Ames, IA: John Wiley & Sons. Yada, R. (2004). Proteins in food processing. Boca Ranton, FL: Woodhead Publishing. Appendix 1 Experimental data for hydroxylamine inhibitor on catalase activity Flask Number Hydroxylamine amount (ml) [Hydroxylamine] (M) [H2O2] (%) [H2O2] (M) [H2O2] (ml) Distilled H20 (ml) Reaction Rate (ppm/s) 1 0.0 0.0 0.0 0.0 0.0 12.0 0.295 ±0.26 2 0.0 0.0 1.5 0.44 3.0 9.0 12.5 ±0.98 3 0.0 0.0 0.0 0.0 0.0 9.0 0.0±10-12 4 0.5 3.66 0.0 0.0 0.0 11.5 -0.197 ±0.19 5 0.0 0.0 1.5 0.44 3.0 6.0 229 ±1.4 6 0.5 3.66 1.5 0.44 3.0 5.5 5.79 ±0.17 7 0.5 3.66 1.5 0.44 5.0 3.5 25.8 ±0.29 8 0.5 3.66 1.5 0.44 7.0 1.5 18.0 ±0.24 9 0.5 3.66 1.5 0.44 8.5 0.0 -92.6 ±1.6 Read More