Purification of Glutamate Oxaloacetate Transaminase (GOT) from Pig Heart

BIOCHEMISTRY 3723 Experiment 6

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60 Procedural Changes: Indicate any changes you made from the procedure in the lab manual. Explain why these changes were made and how they affect the results.

Results: 1. Fractionation: a. Pig heart homogenate: Record the measured volume of Fraction I: ______Record the total volume of "homogenate" removed for assays: ______Record the exact volume of “Fr. I aliquot”: ______Calculate and record the actual supernatant volume of Fraction I: ______

b. Heat denaturation: Record the time when the temperature reaches 60° C: ______. Record the time when the temperature reaches 73-75 °C: ______. Record the time when the sample was placed on ice: ______. Calculate and record the total time above 60 °C: ______. Record the volume of Fraction II: ______. Explain why maleate is in the buffer used for the heat step.

Why is -ketoglutarate added?

c. Ammonium sulfate fractionation:Tabulate the volumes of Fraction II and all ammonium sulfate supernatant and precipitate fractions. In the same table, record the amount of ammonium sulfate added to the supernatant fractions to produce the next ammonium sulfate precipitate. Show all calculations (not just a sample). Table I: Ammonium Sulfate Fractionation

Fraction Volume g (NH4)2SO4 (mL) added Fr. II 55%P ------55%S 75%P ------75%S ------2. Enzyme assays: Pipetting for assays was done by: ______

a. Submit one ∆A340/time graph. On this graph show how the reaction rate is calculated. b. For all fractions, tabulate dilutions, aliquots assayed and observed initial reaction rates (∆ A340/time). Include in table GDH activity in Fractions I and II.

61 Table II: GOT activity measurement Dln Aliquot of Initial Avg. Undil. Initial Initial Rate of diluted rate initial rate Rate GOT only Fractionfraction fraction ∆ A340 ∆ A340 (µmol/min.) (µmol/min.) ** µL min min mL enzyme mL enzyme Fr. I Fr. I Fr. I-GDH ------Fr. II Fr. II Fr. II-GDH ------55%P 75%P 75%S ** If you had to dilute the fraction to assay it, record the dilution here. d. Calculate and include in the table the enzyme activity in each undiluted fraction in units of (µmole NADH reacted/min.)/mL enzyme. Show a sample calculation. The extinction coefficient for NADH is 6.22 L/(mmol•cm)

e. Calculate and tabulate initial rates (µmol/min./ml) in Fractions I and II due to GOT only.

Explain why the GOT assay also measures GDH activity and why the GDH assay measures GDH but not GOT activity.

3. Bradford protein concentrations: Pipetting was done by: ______a. Tabulate volumes used to make dilutions for all fractions. Table III: Protein Dilution

Fraction Sample (µL) H2O (µL) Dilution Fr. I Fr. II 55%P 75%P 75%S

62 b. Tabulate A595 and plot a standard curve for Bradford protein determination. Show sample calculation for g protein in standards. Remember that each sample contains 5.00 mL of Bradford reagent. Table IV: Protein Determination

Volume final[Protein] Average Original Sample sample2O 595 proteinAssayed [Protein] Dilution Protein (µL)(µL) (µg/mL) µg/mL µg/mL (mg/mL) (C2) (C1) (Ave. C1) 0.000100. 0.000 1400. 1400. ------1400. 1400. 1400.

c. Use the standard curve to determine the protein concentration (mg/mL) in each undiluted fraction. This will involve determining the average protein concentration of the samples diluted for the Bradford assay and then correcting for the dilution made before running the Bradford assay. Do not use A595 values that are higher than the standard curve. If the A595 values from both the 0.05 mL and the 0.10 mL aliquots are on the standard curve, calculate protein concentration from each value and average the two. Show calculations and add values to Table IV.

63 4. Record data in Purification Table. (Save a copy for use in the next experiment). Show sample calculations as needed. 5. Choose the ammonium sulfate fraction to use for the next experiment. Justify your choice.

6. Discuss: a. How worthwhile was the heat step; i.e., was purification achieved with reasonable yield? Use numbers to justify your conclusion.

64 b. Sum the of mg protein in the three ammonium sulfate fractions. Calculate the % recovery of protein in the 3 fractions compared to Fraction II. Explain your results (you should have near 100% recovery of protein).

c. Sum the total activities in the three ammonium sulfate fractions. Calculate the % recovery of enzyme activity in the 3 fractions compared to Fraction II? Does the loss in GOT activity parallel the loss in total protein? Justify your answer.

d. Was the ammonium sulfate fractionation worthwhile? Justify your answer. How could the purification by ammonium sulfate fractionation (of Fraction 75%P) be improved? How could the yield of Fraction 75%P be improved?

7. Before submitting this report, enter necessary data in Exp. 7, Results 1. ENTER THESE VALUES NOW!