Enzymatic Activity Assay Development for Pyruvate Kinase

Pyruvate kinase (PK) is an enzyme that catalyzes the last step in the process of glycolysis. PK transfers the phosphate group from phosphoenolpyruvate to ADP, thereby generating one molecule of ATP and pyruvate. Pyruvate kinase deficiency (PKD) may result in the premature destruction of red blood cells (hemolytic anemia). The physiological significance of pyruvate kinase necessitates methods to determine the rate of pyruvate kinase activity in cells. However, few accessible and affordable PK screening methods are available on the market. We attempt to develop an affordable and sensitive pyruvate kinase activity assay in this proposed study. We will generate recombinant tissue-specific pyruvate kinase, purify them using affinity chromatography, and examine activities using a coupled absorbance assay. The method proposed may provide insights into the activity of pyruvate kinase isoforms, facilitate medical implications such as PK screening for new-born babies, and offer an inexpensive tool for research and educational purposes