Rapid isolation of glucose-6-phosphate dehydrogenase from human erythrocytes by combined affinity and anion-exchange chromatography for biochemical characterization of variants.
A new method is presented for the purification of human glucose-6-phosphate dehydrogenase (G6PD) using affinity chromatography with 2'5'-ADP-Sepharose 4B followed by automated ion-exchange chromatography with DEAE 5PW. This rapid method allows a high recovery of enzyme activity from even a small amount of blood; the yield is about 90% after the first purification step and 70% at the end of the procedure. There is an excellent reproducibility of the kinetic parameters and optimal biochemical characterization of G6PD is achieved even for variants associated with severe enzyme deficiency (e.g. G6PD Mediterranean).
Pittalis
S
S
Istituto di Scienze Biomediche San Gerardo, Università di Milano, Monza, Italy.
Martinez di Montemuros
F
F
Tavazzi
D
D
Fiorelli
G
G
D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000201:enzymology*; D005954:Glucosephosphate Dehydrogenase / Q000097:blood / Q000302:isolation & purification*; D005955:Glucosephosphate Dehydrogenase Deficiency / Q000097:blood / Q000201:enzymology; D006801:Humans
D005954:Glucosephosphate Dehydrogenase