BL4820 Lecture 4 - Glutamate Oxaloacetic Acid Transaminase (GOT) Purification -- Expt 3 Part A
3. GOT Enzyme Activity Assay
To assay the activity of GOT we will use a different type of enzyme assay from that used in Expt #2 to assay the phosphatase, which was an end-point type of assay. To assay GOT activity we will use a coupled assay and monitor it continuously:
Asp + 2-KG = OAA + Glu (GOT activity)
OAA + NADH + H+ = malate + NAD+ (MDH activity)
MDH (malate dehydrogenase) is added in excess so that it will not be limiting. This allows you to measure the activity of GOT since its activity level will limit the supply of OAA for the MDH activity. Thus, the OAA produced in the GOT catalyzed reaction will react with an equal amount of NADH. We will monitor the change in NADH using a spectrophotometer. The structure of NADH is shown below:
NADH is the reduced form of this cellular cofactor. When it donates its 2 electrons to OAA for its reduction to malate, NAD+ is formed. We can follow this process since NADH and NAD+ different in spectral properties. NADH with its reduced pyridine ring (shown in the upper right corner of the above graphic) absorbs light at 340 nm, while NAD+ has the oxidized ring normally found in pyridine and lacks absorbance at 340 nm. So as NADH is converted to NAD+ during the MDH catalyzed reaction where the rate limiting OAA is converted to malate, the A-340 nm decreases. You can follow the course of the reaction by monitoring the change in A-340 during the reaction.
You will assay GOT by following the A-340 nm change with time, which will shown as a digital graphic directly on the screen of your spectrophotometers in the lab. Thus, you will not need to do the procedure described in the text involving taking time points and estimating the initial and final concentration of NADH (as discussed on p. 112 and Fig. 10-2). I will discuss in the next lecture how we will convert the A-340 changes with time or absorbance rates for GOT to enzyme activity measured in terms of µmol OAA produced per min.
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