Isolation, Purification And Characterization Of G6PD From B.subtilis

Glucose 6-phosphate dehydrogenase (G6PD) was isolated and purified from Bacillus subtilis BF by a procedure including cell sonicatioru salting out with (NH4) 2SO4, ion-exchange chromatographing with DEAE-Sepharose FF, affinity chromatographing with Blue Sepharose CL-6B and gel filtration with Sephadex G-200. The purified G6PD showed a single protein band on PAGE and SDS-PAGE determination respectively. The purified enzyme had a specific activity of 1. 7375IU/mg, a 72. 7-fold purification was obtained with a recovery of 13.6%. The apparent molecular weight of G6PD, determined by gel filtration, was 220KD; The subunit molecular weight was 50. 5KD as determined by SDS-PAGE, suggesting that the native enzyme was a tetramer consisting of four identical subunits. The result of isoelectrofocusing showed that the isoelectric point of G6PD was 6. 3. The optimal pH of G6PD was 8. 5 and the optimal temperature was 37℃.The Km value of G6P on G6PD was 0. 177mmol/L.The effect of some metal ions and EDTA on enzyme activity, ultraviolet absorbance spectra and fluorescent spectra were all investigated: the G6PD activity was stimulated by Mg2+ while inhibited by Ag+ and Fe2+;the G6PD activity was activated by Zn2+ in lowconcentration condition while inhibited by Zn2+in high; EDTA and Mn2+ had no influence on G6PD activity. Some metal ions made G6PD conformation changed and caused the micro-environment of chromophores varied, which made the ultraviolet absorbance peak of G6PD in 276nm wider and flatter. Ag+ and Fe2+ caused the fluorescence of Trp residues in G6PD quenched ;Mg2+ and EDTA made fluorescence intensity of G6PD increased, this indicates that they caused Trp residues wrapped and came to the inner core and located in the hydrophobic area; while Zn2+ or Mn2+ made fluorescence intensity of G6PD decreased, this indicates that they made the conformation of G6PD relaxed and chromophores exposed to polarity environments.In native condition and in the far circular dichroic (CD) region, G6PD exhibited two characteristic negative band centered at 208nm and 222nm respectively, thus it is estimated to contain about 41.2% a -helix, 20.6% β-pleated sheet and 38.2% random coil and turn.I used acrylamide (Acr) and KI to probe the micro-environments of Trp residues in the G6PD molecule to show that the Trp residues fluorescence of G6PD were quenched by the two quenching reagents, 100% and 78% was quenched by Acr and KI respectively. I deduced that some Trp residues of G6PD were located in the inner core and the others located in the polarity surface.