METHODS FOR CHROMATOGRAPHIC AND ELECTROPHORETIC SEPARATION AND ASSAY OF NADP(+) OXIDOREDUCTASES

The different techniques described in purification protocols for pyrid ine nucleotide-dependent enzymes have been reviewed, covering mainly t he 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-e xchange chromatography (ca. 79%), size-exclusion chromatography (ca. 6 4%) and hydrophobic chromatography (ca. 24%). Other chromatographic te chniques were used infrequently. Each chromatographic technique has a different specific capacity and chemical selectivity and, therefore, t he order of selection should be based on a precise knowledge of the na ture of the sample and the amount of the target enzyme that it contain s. Analytical electrophoresis was used in about 95% of the reviewed pa pers, 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, w hile preparative gel electrophoresis was used in only 8% of the cases, The use of other electrophoretic techniques was reported in only a fe w papers. The use of continuous enzymatic activity assay methods (spec trophotometric) was found in most papers, while high-performance liqui d chromatography-based methods (discontinuous assays) were reported in only 11% of the reviewed articles.