Analysis of GAPDH Enzyme Activity: A Quantitative and Qualitative Approach

Abstract

The enzymatic function of GAPDH (EC.1.2.1.12) is an integral component of glycolysis in glucose metabolism. Thus, GAPDH’s catalytic capacity profoundly influences the “bioenergetic signature” of any metabolically active cell. Determination of the enzymatic function of GAPDH has been instrumental in the assessment of the glycolytic or overall metabolic capacity of proliferating cells. In this chapter, we describe two different approaches to determine the activity of the GAPDH enzyme, based on quantitative and qualitative analytical methods. The quantitative approach is based on the spectrophotometric principle which relies on the net abundance of NADH, a coenzyme that is either generated or depleted depending upon the direction of the reversible reaction of GAPDH. The method outlined here is based on the reversible reaction, where a decrease in NADH is monitored at the absorption maximum (~340 nm). The advantage of this NADH-based assay is that it is specific to GAPDH. The qualitative approach uses an in-gel activity assay, in which the enzyme-dependent release of pyrophosphate is visualized as a calcium-phosphate precipitate. The qualitative assay is also relevant for kinetic analysis as the amount of calcium-phosphate precipitate relies on the level of GAPDH activity. Both these approaches are invaluable and rely on scientifically sound analytical principles.