Toribio F, Alhama J, López-Barea J
Departmento de Bioquímica y Biologia Molecuiar, Facultad de Veterinaria, Universidad de Córdobo, Spain.
J Chromatogr B Biomed Appl. 1996 Sep 20;684(1-2):1-23. doi: 10.1016/0378-4347(95)00453-x.
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.
本文综述了吡啶核苷酸依赖性酶纯化方案中描述的不同技术，主要涵盖过去六年发表的论文。100%的综述论文都报道了色谱法，在色谱技术中，亲和色谱法使用最为频繁（约92%），其次是离子交换色谱法（约79%）、尺寸排阻色谱法（约64%）和疏水色谱法（约24%）。其他色谱技术使用较少。每种色谱技术都有不同的比容量和化学选择性，因此，选择顺序应基于对样品性质及其所含目标酶量的精确了解。约95%的综述论文使用了分析电泳，其中变性聚丙烯酰胺凝胶电泳（PAGE）应用最为广泛（约92%），其次是天然PAGE（约48%）。14%的论文报道了等电聚焦的使用，而制备性凝胶电泳仅在8%的情况下使用。仅有少数论文报道了其他电泳技术的使用。大多数论文采用了连续酶活性测定方法（分光光度法），而基于高效液相色谱的方法（间断测定）仅在11%的综述文章中被报道。
