Methods for chromatographic and electrophoretic separation and assay of NADP oxidoreductases.
The different techniques described in purification protocols for pyridine nucleotide-dependent enzymes have been reviewed, covering mainly the papers published in the past six years. Chromatography was reported in 100% of reviewed papers and among the chromatographic techniques, affinity chromatography was the most used (ca. 92%), followed by ion-exchange chromatography (ca. 79%), size-exclusion chromatography (ca. 64%) and hydrophobic chromatography (ca. 24%). Other chromatographic techniques were used infrequently. Each chromatographic technique has a different specific capacity and chemical selectivity and, therefore, the order of selection should be based on a precise knowledge of the nature of the sample and the amount of the target enzyme that it contains. Analytical electrophoresis was used in about 95% of the reviewed papers, with denaturing polyacrylamide gel electrophoresis (PAGE) being the most widely used mode (ca. 92%), followed by native PAGE (ca. 48%). The use of isoelectric focusing was reported in 14% of the papers, while preparative gel electrophoresis was used in only 8% of the cases. The use of other electrophoretic techniques was reported in only a few papers. The use of continuous enzymatic activity assay methods (spectrophotometric) was found in most papers, while high-performance liquid chromatography-based methods (discontinuous assays) were reported in only 11% of the reviewed articles.
Toribio
F
F
Departmento de Bioquímica y Biologia Molecuiar, Facultad de Veterinaria, Universidad de Córdobo, Spain.
Alhama
J
J
López-Barea
J
J
D002845:Chromatography / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D009249:NADP / Q000378:metabolism*; D010088:Oxidoreductases / Q000032:analysis* / Q000302:isolation & purification
D009249:NADP; D010088:Oxidoreductases