Purification of GOT from Pig Heart and Pig Liver
by Joe Petersen
Faculty advisor: Dr. Brian Lenzmeier
Glutamate-Oxaloacetate Transaminase (GOT) is an enzyme that catalyzes the reversible transfer of an amino group from aspartate to alpha-ketoglutarate to produce oxaloacetate and glutamate. In my experiment I have been testing the specific activity of GOT using activity assays. In the presence of malic dehydrogenase, oxaloacetate and NADH will be converted to malate and NAD+. Since NADH absorbs light at 340 nm I was able to measure the rate of oxidation of NADH to NAD+ by following the decrease in absorbance at 340 nm over time. The change in absorbance over time is directly proportional to the change in concentration of oxaloacetate over time providing an accurate measure of GOT activity. For my research I have purified and tested the activity of the GOT enzyme obtained from pig heart and liver. The first step in the purification process was to convert the GOT enzyme to its heat stable pyridoxal form. Once the GOT enzyme had been converted, I was able to use heat and centrifugation to further the purification process. This step was followed by a large scale ammonium sulfate fractionation. After each purification step I performed GOT activity assays to determine the specific and total enzymatic activity of the sample. The goal of this project is to achieve 80-100 fold purification of the GOT enzyme with a greater than 5% yield. I am currently examining chromatography methods to reach this level of purification.