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.