Mutation of histidine 53 antagonizes heme binding in vitro.
A, heme binding to human GAPDH WT, H57A, and H53A measured using nanoscale thermophoresis on a label-free Nanotemper device. Values represent mean ± S.E. (H53A and H57A, n = 4, and WT, n = 7). Dashed lines represent 95% confidence interval. B, koff measurement of WT and H57A GAPDH using heme binding to apo-myoglobin as measured at 410 nm. Difference in koff values is not statistically significant. C, koff measurement of H53A GAPDH using heme binding to apomyoglobin as measured at 410 nm using stopped-flow.