Horseradish Peroxidase - Lab Report Example

Effect of pH on the activity of horseradish peroxidase 2nd March Effect of pH on the activity of horseradish peroxidase Introduction Enzymes aresignificant biological catalysts. They speed up chemical reactions in the body by providing an alternate pathway for a reaction to follow with a lower activation energy. The enzymes do not take part in the actual chemical reactions and are therefore unchanged when the reaction ends. They only change the rate of reaction (Kovarik & Allbritton, 2011). The structures of enzymes comprise of a globular protein and a cofactor. The bonds holding the proteins in their structures are affected by temperature and pH changes. Such changes affect the shape of an enzyme, thus the catalytic action of an enzyme is dependent on the pH and temperature. For each enzyme there is a small of range of pH within which it works optimally. Enzymes have active sites in their structures. The active site is the part of the enzyme that has the correct shape and the functional groups required to bind to the substrate (Dunford, 1999). Enzyme activity can be measured in any one of these two ways: observing the rate at which the substrate disappears during a reaction or measuring the rate at which the product is formed. Enzyme assays are used in such measurements. There are two methods that have been developed for use in measuring the amount of substrates or products in a chemical reaction: continuous and fixed-timed assays. Continuous assay make use of a spectrophotometer to measure the rates at which the substrate disappears and products form in real-time (Leskovac, 2003). This experiment will measure the enzyme activity of peroxidase. Peroxidase is an enzyme that takes part in breaking down peroxides, for instance hydrogen peroxide In biological processes, peroxide is a poisonous by product of aerobic respiration. The reaction proceeds as follows: Peroxidase + Hydrogen peroxide -------------------> Peroxidase + Water + Oxygen 2H­2O2 (aq) ------------------------> 2H­2O (l) + O2 (g) To measure the peroxidase activity a change in the amount of product formed will be evaluated over time. For the breakdown of peroxide by peroxidase, the simplest molecule that can be measured is O2 gas, the product of the decomposition of peroxide. To accomplish this the real volume of O2 gas produced is measured by use of an indicator. For this experiment an indicator (pyrogallol) that shows the presence of O2 gas will be used (Dunford, 2010). Method 2.50 cm3, 0.35 cm3, 0.10 cm3, and 0.35 cm3 of deionized water, buffer solution (at a pH of 6.0), hydrogen peroxide, and pyrogallol respectively were pipetted into two separate cuvettes labelled Cuvette 1 and Cuvette 2. The contents of the cuvettes were then mixed well using a small glass rod. The spectrophotometer was set to 420 nm after which Cuvette 1 was placed into it. 0.1 ml of the buffer solution was added to the cuvette and then stirred using a small glass rod. The readings of the spectrophotometer were recorded every 10 seconds for 5 minutes. Cuvette 2 (blank) was placed into the spectrophotometer. 0.1 ml of the enzyme was added to it and stirred using a small glass rod. The readings of the spectrophotometer were recorded every 10 seconds for 5 minutes. The assay was repeated using buffers at different pH. A graph of absorbance against time was plotted. The maximum change in absorbance for 20 seconds was obtained using maximum linear rate for both the test and the blank. Results pH Enzyme activity (Abs/second) 6.0 20 8.0 40.12 10.0 60.28 12.0 120.05 Table 1: Table showing the results of the absorbance of peroxidase at a wavelength of 420nm. Determining the Rate of the Enzyme-catalyzed Reaction (the Enzyme Activity) For the complete analysis to be done, enzyme activity was determined by graphical means. First, a graph of absorbance versus time was prepared, and then the best straight line that can be drawn from the data subsequently plotted. After the best straight line was drawn enzyme activity can be easily determined from the slope of the line.  From the equation in the graph plotted and the best straight line, the slope of the curve is given by the equation y = 32.031x – 19.965. The slope of the best straight line is 32.031. This is the enzyme activity. Discussion Pyrogallol is the trivial name for the compound 1,2,3-trihydroxybenzene. Normally benzene-derived systems are activated by hydroxyl- groups. The 3 hydroxyl groups located on pyrogallol’s benzene nucleus mean that the compound is very reactive, even to the point where it can easily react with oxygen in the air (Bacha, et al., 2013). The reaction is very complicated, and free radicals are thought to take part in the reaction. Such free radical reactions that involve activated benzene rings regularly lead to coloured products that have unusually high molecular weights that cannot be easily characterized (Roy & Abraham, 2006).  Pyrogallol reacts with the oxygen that is liberated to form orthoquinone that is coloured, and this reacts further to form more coloured products that comprise mainly of purpurogallin. The compound pyrogallol has a very high affinity for oxygen, and while in solution, it instantly attaches to free oxygen molecules to form orthoquinone and subsequently purpurogallin, which has an orange-brown colour (Feldman, 1997). In these experiment pyrogallol was chosen because it is the perfect substrate for horseradish peroxidase. The sharp and intense orange-brown colour of purpurogallin that is formed when pyrogallol is oxidized by hydrogen peroxide and horseradish peroxidase was then measured using the spectrophotometer to measure how much light passed through the purpurogallin solution (Roy & Abraham, 2006). From the results obtained, as the pH rose steadily, the enzyme activity also rose. This showed that the optimum pH at which horseradish peroxidase works is an alkaline pH. In the reaction pyrogallol becomes oxidized by horseradish peroxidase and it then undergoes dehydrogenation to form orthoquinone, that also subsequently undergoes further dehydrogenation that is coupled with the loss of C, is then paired with one pyrogallol molecule to yield the end-product; purpurogallin (Tauber, 1953). The reaction describing the first oxidation of pyrogallol to the orthoquinone can be written down as follows: 2 pyrogallol + O2 (g) ------------------------> 2 orthoquinone+2H2O (l) Conclusion The experiment successfully determined that rate at which horseradish peroxidase works is affected by changes in the pH. As the pH rose the amount of oxygen produced as a result of the hydrogen peroxide being broken down by the enzyme rose. This showed that an increase in pH fosters better working conditions for peroxidase-derived enzymes. During the experiment it is possible that errors were introduced into the experiment that affected the readings ad results that were obtained. For instance during the time the various quantities of reagents were being pipetted into the test tubes, it is possible that incorrect amounts of the reagents were pipetted, or they became contaminated and affected their roles in subsequent reactions. It is also possible that the time measurements were not properly taken and this led to inconsistencies in the measurements taken. Such possible errors can be easily avoided in future experiments by ensuring that proper pipetting technique is followed that ensures that the correct amount of reagent is drawn and then mixed with other reagents. References Bacha, C. et al., 2013. Measuring phenol oxidase and peroxidase activities with pyrogallol, l-DOPA, and ABTS: Effect of assay conditions and soil type. Soil Biology and Biochemistry, 12, Volume 63, pp. 183-191. Dunford, B., 1999. Heme peroxidases. s.l.:John Wiley. Dunford, B., 2010. Peroxidases and Catalases: Biochemistry, Biophysics, Biotechnology and Physiology. s.l.:John Wiley & Sons. Feldman, K., 1997. Cyclization Pathways of a (Z)-Stilbene-Derived Bis(orthoquinone monoketal). Journal of Organic Chemistry, 7, 65(15), pp. 4983-4990. Kovarik, M. & Allbritton, N., 2011. Measuring enzyme activity in single cells. Trends in Biotechnology, 5, 29(5), pp. 222-230. Leskovac, V., 2003. Comprehensive Enzyme Kinetics. s.l.:Springer Science & Business Media. Roy, J. & Abraham, E., 2006. Continuous biotransformation of pyrogallol to purpurogallin using cross-linked enzyme crystals of laccase as catalyst in a packed-bed reactor. Journal of Chemical Technology and Biotechnology, 11, 81(11), pp. 1836-1839. Tauber, H., 1953. Oxidation of pyrogallol to purpurogallin by crystalline catalase. The Journal of Biological Chemistry, pp. 395-400. Read More