Peroxidase Experiment

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In this experiment, in the first part, the best concentration of enzyme was determined by recording the absorption over time. In the second part, the best concentration was selected from the previous experiment which was C and the optimum pH was determined.

Introduction

In an article, ‘Lab Presentation’ on Prezi, the peroxidase enzyme was tested on factors such as enzyme concentration and pH. It was measured using guaiacol as it turns orange when oxidized; spectrophotometer was used to determine the rate of absorbance. The article states that as the peroxidase enzyme concentration increases, the rate of reaction increases as well. This happens because the more the enzyme concentration, the more substrates will be catalyzed by peroxidase. …show more content…

An assay determines the enzyme activity. Guaiacol and hydrogen peroxide are key factors for this experiment. As the enzyme breaks down hydrogen peroxide, it gives out hydrogen and oxygen. Guaiacol turns the solution brown in the presence of oxygen, so as the oxygen was given out from the breakdown of hydrogen peroxide the solution turned brownish which proved that the enzyme was reacting. In the first part of this experiment, the concentration of the enzyme was found. In four test-tubes, labelled A-D, each test-tubes were diluted starting from the the test-tube A which had 5ml enzyme stock solution. 1 ml of the enzyme was added to the test-tube B containing 4ml buffer, which was 1:5 dilution after the mixture. 1 ml of the solution from test-tube B was then added to test-tube C, which then had 1:25 dilution. The same procedure was repeated with test-tube D, which had 1:125 dilution. Then, nine test-tubes were taken, out of them one was blank. The rest eight test-tubes had different volumes of buffer, enzyme, hydrogen peroxide and guaiacol. Guaiacol is a dye used to determine the presence of oxygen, which turns the solution brown if oxygen is present. Mixtures of two test-tubes were added and labelled A-D accordingly. The absorption rate of the four test-tubes were then determined over a total time of 120 seconds, having a gap of 20 seconds within the 120 seconds. The more dilute the solution was, the less was the absorption, which …show more content…

Test-tube C had the best concentration according to the results. Three test-tubes were labelled A-C. Test-tube A had 1ml enzyme solution which was added to test-tube B which had 4ml buffer (pH 5 was used). 1ml of the solution from test-tube B was then added to the test-tube C which also had 4ml buffer (pH 5). Test-tube C was used as the enzyme in all the reactions. Nine test-tubes were taken out of them one was used as the the blank, labelled as test-tube 9. The blank had 5ml buffer (pH 5), 2ml hydrogen peroxide, 1ml guaiacol and no enzyme. Then, 3ml of buffer (pH 3) and 2ml of enzyme were added to test-tube 1. Test-tube 2 had 2ml hydrogen peroxide and 1ml guaiacol. Test-tube 1 and 2 were mixed. The same procedure was used for test-tube 3 as test-tube 1, but this time the buffer was pH 5. Test-tube 4 was prepared the same way as test-tube 2. Then, Test-tube 3 and 4 were mixed. Test-tube 5 was prepared as test-tube 1 but with buffer of pH 7 and test-tube 6 was prepared as test-tube 2. Next, test-tube 5 and 6 were mixed. Last but not the least, test-tube 7 was prepared as test-tube 1 but with buffer of pH 9 and test-tube 8 was prepared as test-tube 2. Then, test-tube 7 and 8 were mixed. The spectrophotometer was set to 470nm and using the blank it was set to zero. The four test-tubes with different pH’s (pH 3, pH 5, pH 7, pH 9) were read

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