Enzyme Lab Protocol
Hypothesis: The rate of decomposition of .15M NaCl at a pH of 8 is dependent on temperature. Prediction: The highest amount of decomposition will occur at 37 degrees Celsius. Rationale: The use of the enzyme at 37 degrees Celsius will be the most favorable because that is its natural habitat. Independent Variable: The different temperatures- 25,37, and 49 degrees Celsius. Dependent Variable: Rate of decomposition of the side chains. Negative Control: The buffer’s with Azocasein but lacking Trypsin. Experimental Controls: set pH of 8, .15M of NaCl, and the same concentrations of the buffers, Azocasein, Trypsin, and TCA in each replicate (not the same amount of each in each replicate!). Procedures:
1. Align 18 centrifuge tubes. To all 18 these tubes, add 450 microliters of the buffer- 0.15M NaCl, 20mM Tris-HCl, pH8 to the tube as well as 500 microliters of Azocasein. To only 9 of these 18 tubes, add 50 microliters of Trypsin. The tubes without the Trypsin will act as the controls. 2. Incubate six tubes (three with Trypsin, three without) at 25 degrees Celsius, another six at 37 degrees Celsius, and the last six at 49 degrees Celsius each for ten minutes. 3. Add 500 microliters of a 10% TCA.
4. Separate what is left of the solution from the unused solution by place the test tube in a clinical centrifuge for two minutes. 5. Blank the supernatant using a spectrophotometer.
6. Decipher the rate of decomposition of the R-group by reading the absorbance found by the spectrophotometer. *The negative controls will define which side chains were lost from natural causes and which were lost because of the addition of Trypsin.