Partial purification of glucose 6-phosphate dehydrogenase and phosphofructokinase from rat erythrocyte haemolysate by partitioning in aqueous two-phase systems.
Glucose 6-phosphate dehydrogenase shows a high partition coefficient in poly-(ethylene glycol)-dextran aqueous two-phase systems in comparison with those for 6-phosphogluconate dehydrogenase, phosphofructokinase and the bulk of proteins present in rat erythrocyte haemolysates. As a consequence, fractions highly enriched in glucose 6-phosphate dehydrogenase can be obtained after multiple partitions in the above systems with a counter-current distribution procedure. Phosphofructokinase shows a high affinity for Cibacron Blue and, as a result, the enzyme can be extracted in the top phase of poly(ethylene glycol)-dextran systems containing Cibacron Blue-poly(ethylene glycol) (affinity systems). The efficiency for the purification of the enzymes by partitioning is increased up to 10-fold when enzyme-rich fractions, obtained by precipitation with poly(ethylene glycol), are used instead of original haemolysate. The recovery of enzyme activities is near 100% in both instances.
Delgado
C
C
Departamento de Bioquimica y Biologia Molecular, Universidad de Alcala de Henares, Madrid, Spain.
Tejedor
M C
MC
Luque
J
J
D000818:Animals; D004396:Coloring Agents; D004912:Erythrocytes / Q000201:enzymology*; D005954:Glucosephosphate Dehydrogenase / Q000097:blood / Q000302:isolation & purification*; D006461:Hemolysis; D008297:Male; D010732:Phosphofructokinase-1 / Q000097:blood / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains; D014227:Triazines
D004396:Coloring Agents; D014227:Triazines; C012842:Cibacron Blue F 3GA; D005954:Glucosephosphate Dehydrogenase; D010732:Phosphofructokinase-1