PURIFICATION OF LACTATE DEHYDROGENASE FROM CHICKEN MUSCLE TISSUE Abstract The enzyme lactate dehydrogenase (LDH) catalyzes the last step of anaerobic glycolysis that is important for the normal function of the body. Purification of LDH is essential to understand its structure and function. The purpose of this experiment was to extract and purify LDH enzyme from chicken muscle tissue using a variety of various. Analytical methods such as activity and protein assay were employed to determine
Isolation and Characterization of Avian Lactate Dehydrogenase To whom correspondence should be addressed Sylvia DaoudKinze and James Proestes, Department of Biochemistry, Portland State University Professor, Portland Oregon, 97207-0751; E-mail: sylviakinzie@gmail.com and proestoj@gmail.com ------------------------------------------------- Keywords: Lactate, Dehydrogenase, Avian, Bradford Assay, Affinity Column Background: Lactate Dehydrogenase also known as LDH is an important NADH dependent
ligand affinity chromatography was given after the interactions that took place between Blue Dextran, a Cibaron Blue and dextran conjugate, which is used as a void marker in size-exclusion chromatography, and particular kinases. Until then, only purification of various proteins by size-exclusion chromatography with Blue Dextran, like for example, erythrocyte pyruvate kinase, phosphofructokinase, glutathione reductase, and several coagulation factors had been initiated. The final conclusion of these
are washed away before the bound proteins are eluted using the substrate, coenzyme or effector. Oxamate is a competitive inhibitor of Lactate dehydrogenase as this is not a substrate for the enzyme it is not metabolisable this means that the LDH will bind to the Oxamate and not release as quickly as it does from pyruvate. This is because pyruvate is converted to lactate in the LDH catalysed reaction and then is released from the enzyme. Oxamate cannot be released after reaction as it is not catalysed
was obtained, precisely minced to exclude extra fat, and then blended with pH 7.2 phosphate buffer. The purpose of blending the tissue with the buffer was to pulverize the cells, causing them to release their contents evenly—most importantly lactate dehydrogenase (LDH)—into the solution. Since other cell components such as proteases which reduce LDH were also released from the lysed cells, the slurry was kept on ice to minimize their kinetic activity. The solution was then centrifuged, creating a pellet