Cloning and biochemical characterization of three glucose‑6‑phosphate dehydrogenase mutants presents in the Mexican population

Effects of Single and Double Mutants in Human Glucose-6-Phosphate Dehydrogenase Variants Present in the Mexican Population: Biochemical and Structural Analysis

“…The kinetic parameters of the recombinant enzymes G6PD A+ (Asn126Asp), G6PD San Luis Potosi (Asn126Tyr), G6PD Guadalajara (Arg387Cys), and G6PD Mount Sinai (Asn126Tyr/Arg387Cys) were determined spectrophotometrically at 25 • C when monitoring the reduction of the NADP+ substrate into an absorbance of 340 nm [11,15,19,25,33]. Standard activity assay was performed in a final volume of 1 mL of the reaction in buffer T (Tris-HCl 0.1 mM, pH 8.0, and MgCl 2 3 mM).…”

“…Far UV-CD spectra (120-250 nm) of each G6PD protein were collected with 1 nm intervals on a path-length quartz cuvette of 1 nm. The far UV-CD spectra of the blank was subtracted from those containing the respective protein [11,15,25,33]. The experiments were performed in triplicate at 25 • C.…”

“…To determine the effects of the four mutations on the tertiary structure of the proteins, intrinsic fluorescence assays and 8-anilinonaphthalene-1-sulfonate (ANS) binding assays were performed to monitor the conformations on the tertiary structure. Both assays were performed on a Perkin-Elmer LS-55 fluorescence spectrophotometer (Perkin Elmer, Wellesley, MA, USA) equipped with a quartz cell and a step length of 1 cm [11,15,25,33]. The intrinsic fluorescence assay of WT G6PD and the four variants was performed using a final protein concentration of 0.1 mg/mL variant in 50 mM phosphate buffer at pH 7.35 at 25 • C. The excitation wavelength used for the samples was 295 nm and fluorescence emission was obtained from 300-500 nm with 4.5 and 3.7 nm slits for excitation and emission, respectively.…”

“…The unfolding of the WT G6PD and variants were determined following the changes in molar ellipticity at 222 nm as the temperature was varied from 20-75 • C at a rate of 1 • C/2.5 min. Before the assay, the structural NADP + of the proteins was removed as previously reported [15,19,33], then the WT G6PD and four variants were adjusted at 0.2 mg/mL. Melting temperature (T m ) was defined as the fraction at which 50% of the protein was folded/unfolded and was calculated as previously reported [11,15,28].…”

“…Thermal inactivation assays are used as an indicator of the stability of the G6PD enzyme based on the residual activity when they are exposed to different temperatures and NADP+ concentrations as a stabilizing agent. To perform this assay, we removed the structural NADP + molecule as previously reported [15,19,33]. The WT G6PD and the four variants were adjusted at 0.2 mg/mL and incubated with three concentrations of NADP + (10, 100, and 500 µM) in a thermocycler (Biorad) for 20 min to different temperatures (37 to 60 • C), after the samples were cooled to 4 • C in 5 min increments.…”