Purification and Characterization of Mutant GAPDH Enzymes

Both mutant forms of the protein were expressed and purified as described previously for wt rGAPDH (Boradia et al., 2016 (link)). Purification was confirmed by western blotting, detection was done after incubation with Mouse anti-His (Sigma) (1:3,000) for 1 h followed by incubation with anti-Mouse IgG HRP (Sigma) (1:8,000) for 1 h. The enzyme activity of wt rGAPDH, rGAPDH(N142S), and rGAPDH(P295L) purified from cytosol fraction was studied by measuring the increase in the absorbance at 340 nm due to oxidative reduction of NAD⁺ to NADH. The reaction mixture containing 200 μl of enzyme assay buffer (50 mM HEPES, 10 mM sodium arsenate, and 5 mM EDTA, pH 8.5), 1 mM NAD⁺ and 2 mM glyceraldehyde-3-phosphate (G-3-P) was added to wells containing 100 ng of each purified enzyme at 25°C. Enzyme activity was measured at 340 nm for 5 min on a Tecan Infinity M200 multimode microplate reader. Negative controls were set up without the specific substrate G-3-P and their values were subtracted from the final absorbance. The experiment was repeated thrice in triplicates. The enzymatic activity of wt rGAPDH was taken as 100% and data was plotted as % residual activity ± SD. Statistical analysis was done using Student's t-test.