Analysis of secondary structure of the recombinant human WT G6PD and the mutants enzymes was analyzed by CD in a spectropolarimeter (Jasco J-810®) equipped with a peltier thermostated cell holder [19 (link),23 (link),24 (link)]. Far-UV CD spectral scans at 25 °C were performed from 200 to 260 nm at 1 nm intervals in a quartz cell with a path length of 0.1 cm. The assays were conducted with a protein concentration of 0.2 mg/mL in 50 mM phosphate buffer at pH 7.35. Furthermore, CD measurements of recombinant human WT G6PD and the mutant’s enzymes were performed with 0.25 M of Gdn-HCl to evaluate if the loss activity in the time-curse inactivation assay was due to a wider structural disruption or local effect in the secondary structure. For both trials, the spectra of blanks were subtracted from those that contained the protein.
Analysis of conformational changes in the tertiary structure of recombinant human WT G6PD enzymes and mutants were evaluated by intrinsic fluorescence and their capacity to bind 8-anilinonaphthalene-1-sulfonate (ANS) assays. Both assays were performed in a Perkin-Elmer LS-55 fluorescence spectrometer (Perkin Elmer, Wellesley, MA, USA) as formerly reported [19 (link),23 (link)]. The fluorescence emission spectra from 310 to 500 nm were recorded after excitation at 295 nm, with an excitation and emission slits of 4.5 and 3.7 nm, respectively. All the assays of intrinsic fluorescence were conducted with a protein concentration of 0.1 mg/mL. ANS assays were performed in 25 mM phosphate buffer, pH 7.4 at 25 °C, using an excitation wavelength of 395 nm and recording emission spectra from 400 to 600 nm with an excitation and emission slits of 10 and 10 nm, respectively. The final concentrations of ANS and the recombinant human G6PD enzymes were the same as previously reported [19 (link),23 (link)]. For both spectroscopic assays, the spectra of blanks without protein were subtracted from the experimental samples that contained the respective protein.
Analysis of conformational changes in the tertiary structure of recombinant human WT G6PD enzymes and mutants were evaluated by intrinsic fluorescence and their capacity to bind 8-anilinonaphthalene-1-sulfonate (ANS) assays. Both assays were performed in a Perkin-Elmer LS-55 fluorescence spectrometer (Perkin Elmer, Wellesley, MA, USA) as formerly reported [19 (link),23 (link)]. The fluorescence emission spectra from 310 to 500 nm were recorded after excitation at 295 nm, with an excitation and emission slits of 4.5 and 3.7 nm, respectively. All the assays of intrinsic fluorescence were conducted with a protein concentration of 0.1 mg/mL. ANS assays were performed in 25 mM phosphate buffer, pH 7.4 at 25 °C, using an excitation wavelength of 395 nm and recording emission spectra from 400 to 600 nm with an excitation and emission slits of 10 and 10 nm, respectively. The final concentrations of ANS and the recombinant human G6PD enzymes were the same as previously reported [19 (link),23 (link)]. For both spectroscopic assays, the spectra of blanks without protein were subtracted from the experimental samples that contained the respective protein.
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