Lactate Dehydrogenase Lab Report

Lactate Dehydrogenase (LDH) is an cytosolic enzyme that participates in anaerobic glycolysis. LDH couples the reduction of pyruvate into lactic acid to the oxidation of NADH to NAD+, which allows glycolysis to continue to produce ATP in the absence of oxygen. LDH is a tetramer, a protein complex of 4 polypeptide subunits, composed either a subunit expressed strongly in the heart, H type,or a subunit that is highly expressed in the muscle, M type. There are 5 isozymes of LDH composed the 2 subunit types. The molecular weight of each subunit is approximately 36.6 kDa and a total of 146 kDa for the enzyme. The isoelectric point of the H type is 5 and the M type subunit is 8. Using the different pI of these isozymes, LDH determined the identify
The supernatant was decanted into a clean pre-chilled beaker and the pellet was discarded. A 0.5 mL sample was frozen down for later analysis of purification method. The clarified homogenate was rapidly stirred with a pre-chilled stir bar while 19.77 grams of ammonium sulfate was added slowly for a 40% cut precipitation. The clarified homogenate was stirred for 15 minutes after the ammonium sulfate was dissolved. The clarified homogenate was centrifuged for 15 minutes at 15,000 gx. The volume of the 40% cut supernatant was measured and the pellet was discarded. The 40% cut was stirred rapidly while 14.61 grams of ammonium sulfate was slowly added for a 65% cut precipitate and was stirred slowly for an additional 15 minutes. The 65% cut was centrifuged for 15 minutes at 15,000 xg. The supernatant was discarded. The pellet was resuspended in cold 4 mL of pH 7.5 phosphate buffer and transferred to a pre-chilled test tube for further purification. 200 uL was frozen down for later
First, the amount of total protein removed by the Affinity Chromatography is difficult to quantify because there appears to be an error in the analysis data for 65% cut precipitate. The protein from the previous purification step limits the amount of protein that can be recovered by the subsequent purification step. In this case, the total protein recovered by 65% cut was 159 mg, but Affinity Chromatography recovered 350 mg of protein. Purification steps cannot gain protein unless protein is added during the purification process, which none was. The effectiveness of 65% cut precipitation cannot be determined solely by the difference between the difference between the total protein for Clarified Homogenate and 65% cut. However, the total Activity units approximates the selectivity of each purification step when compared to total protein recovered. The difference between the Activity units shows the amount of desired protein lost, which describes the discrimination between other protein. The higher the difference the less effective the purification step was. The difference between 65% cut and Clarified Homogenate activity is the largest and therefore the least effective. For lack of correct data for the amount of protein recovered by 65% cut and Affinity Chromatography this can be qualitatively seen by analyzing the SDS-PAGE. The more bands removed between the steps the more effective. The 65% cut removed only