BL4820 Biochemistry Techniques

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BL4820 Lecture 6 - Glutamate Oxaloacetic Acid Transaminase (GOT) Purification -- Expt 3 Part C

4. Summary Table of an Enzyme Purification

A key part of any enzyme purification is to make a summary table which brings together all the results from the various steps of the procedure into a single place for easy comparison. A summary table can be found in almost any paper on purifying an enzyme in a journal in the library. An example is given in the text in Expt #10, p. 116. You can use the text example as a guide for the one in your Lab Report, but I want one additional column in the Summary Table in your Lab Report, which is one that shows how much your specific activity has improved at each step - I will explain this below.

Your Purification Summary Table for Expt #3 - Purification of GOT should have the following columns:

1. Purification Step (called "Fraction" in text example)
2. Volume of the total sample obtained at each step of purification in ml
3. Protein for each sample in mg/ml - obtained in your Folin assays for most steps in purification, but use the A-280 nm for the last step (CMC fractions) where you arrive at mg/ml by assuming that an A-280nm = 1.0 is 1 mg/ml
4. GOT Activity in units/ml where units are defined as µmol OAA formed/min/ml
5. GOT Specific Activity in units/mg protein -- obtained by dividing column 4 by column 3
6. GOT Total Activity in units -- obtained by multiplying column 2 times column 4
7. Yield in Percent -- treat GOT Total Activity units for Fraction I (crude extract) as 100 % and find the Yield for other steps in purification by dividing their GOT Total Activity units by Fraction I GOT Total Activity Units to obtain a fraction which is then multiplied by 100 to get the percent Yield -- Overall Yield will decrease at each step in the purification
8. Fold Purified -- treat GOT Specific Activity for Fraction I as "1" and basis for calculating other steps increase in Purification -- find the Fold Purified for each step by dividing their GOT Specific Activity by Fraction I GOT Specific Activity, which should give a number greater than 1 if the GOT is more purified after that step -- Overall, Fold Purified should increase at the key steps of the purification.

Tricky Part of the Purification Summary Table: For Fraction I, you only took 5 ml of the total of 75 ml of pig heart extract you were given to obtain the data for Fraction I. However, you must treat this step in the purification as if you centrifuged all 75 ml of pig heart extract or your table will look messed up. In addition, you do not actually get 75 ml of Centrifuged Crude Extract, which is what we call Fraction I, since there is a large pellet after centrifugation. As shown below in the example Purification Summary, 45 ml is the volume of Fraction I, which is arrived at by taking the volume of Fraction I after you have centrifuged the 5 ml of pig heart extract to decide how much of the 75 ml would be Fraction I. In the data for the example Table below, 3 ml of Fraction I was obtained so the real volume = (3/5) X 75 ml = 45 ml.

Getting a Better Yield from CMC Step: You may find that in the last step (ie. Carboxy-Methyl Cellulose Column Chromatography), the GOT did not come out in one fraction. In this case, you still want to report your best fraction (ie. one with most GOT Activity) to get the Specific Activity. But you will get a better Yield if you sum up all the CMC fractions containing GOT Activity. To do this you multiply the volume (5 ml) times the GOT Activity (units/ml), then add up the total units of these fractions and calculate the Yield...Do Not calculate a Specific Activity for the Summed Total Units or a Fold Purification.

Example of Purification Summary for Pig Heart GOT (typical data - yours should be different):

• *Units are defined as µmol OAA produced/min

Back To: Lecture 6 - Week #6

Copyright ©1996, 1997, 1998, 1999, 2000, 2001 Wilbur H. Campbell, All Rights Reserved; wcampbel@mtu.edu

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