BL483 Biochemistry Techniques

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

3. Applying Beer's Law to Continuous Enzyme Assays

To determine the rates of GOT (and GDH) assays, we will apply Beer's law to the continuous enzyme assay:

Rates obtained directly from spectrophotometer outputs in change in A-340 nm/min

  • To convert delta A-340nm/min to µmol/min, use Beer's law; where c = A/E
  • For this Expt we say, amount NADH oxidized/min = (delta Abs/min)/extinction coefficient
  • Extinction coefficient for NADH = 6.2/ml of assay

Conversion absorbance readings to amount of NADH is complicated by several factors of your assay conditions:

  • Total volume of assay = 3 ml
  • Volume of purification fraction used in the assay = 0.1 ml
  • Dilution factor for each fraction analyzed = variable (different for each assay)

These factors are then combined with the delta A-340 nm/min for each assay, to yield rates in terms of µmol OAA formed/min/ml purification fraction analyzed (remembering that for each NADH oxidized an OAA is reduced).

µmol/min/ml = (delta A-340 nm/min) X (3 ml assay volume/6.2) X (1/0.1 ml extract volume) X (dilution factor)

Defining 1 unit of GOT enzyme activity = 1 µmol OAA formed/min simplifies the reporting of your results, which can then be given as units GOT activity/ml purification fraction.

Example of how to calculate GOT Activity:

  • Assume for purification fraction F-1, you obtained delta A-340 nm/min = 0.1 and the F-1 had been diluted 1:100 before the assay was done
  • GOT units/ml F-1 = (0.1/min) X (3/6.2) X (10/ml) X (100)
  • GOT units/ml F-1 = 48 µmol OAA formed/min/ml F-1
  • This simplified to 48 units GOT activity/ml F-1, assuming 1 unit of GOT activity = 1 µmol/min

Calculation of Specific Activity (ie. units of enzyme activity / mg total protein):

Let's carry this example one step further.  You need to calculate specific activity for each step in the GOT purification to make a judgment about the improvement in purity of GOT achieved by each step in the purification.

Example of how to calculate GOT specific Activity:

  • Assume for purification fraction F-1, you got Folin assay A-500 nm  = 0.200 for the 0.1 ml of F-1 centrifuged extract diluted 1:10
  • Total protein for F-1 is found using the Folin standard curve and we'll assume it is (150 µg/0.1 ml) x 10 or 15 mg/ml
  • Specific Activity GOT units/mg F-1 = (48 µmol OAA formed/min/ml F-1) / (15 mg protein) or 3.2 µmol/min/mg protein
  • This simplified to 3.2 units GOT activity/mg for F-1, assuming 1 unit of GOT activity = 1 µmol/min

Summary: To determine Specific Activity for each step in the purification -

  1. Determine the GOT activity in µmol/min/ml of the fraction
  2. Determine the amount of total protein in that fraction in terms of mg/ml of the fraction
  3. Then divide the GOT activity/ml by the total protein/ml to yield the specific activity in GOT activity units/mg total protein.

Back To: Lecture 6 - Week #6

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

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