Isolation and Characterization of Lactate Dehydrogenase

Abstract

Lactate Dehydrogenase is an enzyme found mostly in all living cells, that catalyzes the reversible conversion of pyruvate and lactate; the isolation and purification of the enzyme is commonly studied in biochemistry labs. Based on the current procedure at Purchase College, the enzyme lactate dehydrogenase has been extracted from an Idaho potato and isolated using numerous methods, including salting out, dialysis and affinity chromatography. The total protein concentration is measured by Bradford Assay. Also, the purified enzyme's activity was then analyzed by protein activity assay using a UV-vis spectrophotometer. However, in the past, the activity assays presented unclear results, as the spectra produced a lot of noise and blips that impacted the slope values; this ultimately posed a problem since the activity of LDH is determined by slope, which translates to the rate loss of NADH over a minute. Due to the poor results of the current activity assay, it was worth investigating the activity assay parameters, such as the pH and the concentrations of reagents. The change of parameters was tested and compared to the original procedure. Results indicated that the new parameters presented clearer spectra and more accurately quantified units of lactate dehydrogenase amongst the samples than the original parameters that are currently being used in the Biochemistry Lab at Purchase College.