Immunoaffinity purification and characterization of glyceraldehyde-3-phosphate dehydrogenase from human erythrocytes.

A new procedure utilizing immunoaffinity column chromatography has been used for the purification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) from human erythrocytes. The comparison between this rapid method (one step) and the traditional procedure including ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography shows that the new method gives a highest specific activity with a highest yield in a short time. The characterization of the purified GAPDH reveals that the native enzyme is a homotetramer of ~150 kDa with an absolute specificity for the oxidized form of nicotinamide adenine dinucleotide (NAD(+)). Western blot analysis using purified monospecific polyclonal antibodies raised against the purified GAPDH showed a single 36 kDa band corresponding to the enzyme subunit. Studies on the effect of temperature and pH on enzyme activity revealed optimal values of about 43 degrees C and 8.5, respectively. The kinetic parameters were also calculated: the Vmax was 4.3 U/mg and the Km values against G3P and NAD(+) were 20.7 and 17.8 muM, respectively. The new protocol described represents a simple, economic, and reproducible tool for the purification of GAPDH and can be used for other proteins.