controlled 75.0C water into a 1000ml beaker, and record the actual temperature o

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controlled 75.0C water into a 1000ml beaker, and record the actual temperature of the water bath in Table 4 (keep To constant) bath Fgule u 17. Prepare a 75.0 C wat 18. When ready to proceed immediately add 5 drops of the enzyme solution from tube 4E to test tube 4 and repeat steps 5-13 under the enzyme concentration steps. 19. Answer questions of Part B in the Report Sheet pages 34 and 35 as a group. Part C. The Effecet of pH on an Enzymes' Reaction Rate Introduction The ph of the solution an enzyme is in can influence the three-dimensional shape of the enzyme. Every enzyme has an optimum pH at which it is most active. Most enzymes function best in solutions that are near neutral (pH 6-8), but several work in the more basic (pH 8-12) or the more acidic range (pH 2-6). In this experiment you will determine the optimum pH for the activity of catalase The Set Up 1. Follow steps 1, 2, and 3 under Set Up for all experiments on page 26 to begin. 2. All trials will be performed in a water bath at room temperature(20°C) Preparea water bath at room temperature (Figure 5) 3. Wear Safety Goggles when transferring solutions! Place five clean test tubes in a rack and label them pH 3, pH 5, pH 7, pH 9, and pH 11. Use a 10 ml graduated cylinder to add 3.0 ml of each pH buffer to each test tube, as in Table 3. Rinse cylinder with dH:0 between transfers. Test Tube Ph of buffer volume of bufferolu of3.0 % H O2 (mi) - - H 3 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 H 11 Table 3 Contents of test tubes 1-5 This table shows the volumes of the various solutions placed into the various test tubes for this experiment Add 5 drops of the enzyme suspension to 3.0 ml pH 3 buffer, place the test tube in the 4. wait a minimum o f 5 m inutes for the enzyme to be affected by the (Why equilibrate the enzyme and substrate separately to the buffer before the reaction then add 3.0 ml 3.0% H20, and repeat steps 5-13 under the enzyme concentration steps begins?), Remember to constantly and gently swirl each test tu be for each pH. Record data in Table 4. 5. in the tube labeled pH 5, add 5 drops of the enzyme solution, place the test tube in a er bath, and wait at least 5 minutes to allow the enzyme to be affected by the buffer 20°C wat Now add 3.0ml 3.0% H,02, and repeat steps 5-13 under the enzyme concentration steps data in Table 4 In the tube labeled pH 7, add 5 drops of the enzyme solution, place the test tube in the 20°C water bath, and wait at least 5 minutes. Now add 3.0ml 30% H,02, and repeat steps 5-13 under the enzyme concentration steps. Record data in Table 4 7. labeled pH 9, add 5 drops of the enzyme solution, place the test tube in the In the tube 20°C water bath, and wait at least 5 minutes. Now add 3.0ml 3.0% H20, and repeat st under the enzyme concentration steps. Record data in Table 4 eps 5-13 8. In the tube labeled pH 11, add 5 drops of the enzyme solution, place the test tube in the 30 Revised Spring 201 160 Laboratory Manual baugh

Explanation / Answer

Hypothesis: An enzyme will be most active at its optimum pH and any change in pH on either side of the optimum pH value will negatively affect the activity of the enzyme i.e. activity of the enzyme will be reduced if the pH is either increased or decreased from the pH of optimum activity of the enzyme.

Summary: There are two objectives in this experiment-

1) To find the optimum pH i.e. pH at which the activity of enzyme catalase is maximum.

2) The effect of change in pH on the activity of the enzyme.

Here we are testing activity of enzyme catalase in break down of hydrogen peroxide at different pH values ranging from pH 5 to 11 in an increment of 2. We have chosen these pH values so that we can make sure that we have pH values which fall on either side of the optimum pH value.

To test the activity of enzyme we equilibrate the enzyme and the substrate separately in the pH buffer before mixing them together. This is done so that any conformational changes that the pH of the solution (buffer) brings in the enzyme or substrate should bring it before enzyme and substrate get a chance to interact with each other. This way we will be able to make sure that whatever change in the activity that is observed in the experiment is due to the change in the pH and not because of any other factor.

Finally, we plot the enzyme activity of catalase Vs pH to know the "optimum pH" of the enzyme i.e the pH at which the activity of catalase is highest on the plot. Also, we will see that there is only one point or pH where activity of enzyme is highest and any pH value higher or lower than that pH should have the lower activity of the enzyme.

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