Wheatley J B, Montali J A, Schmidt D E
Terrapin Technologies, Inc., South San Francisco, CA 94080.
J Chromatogr A. 1994 Jul 29;676(1):65-79. doi: 10.1016/0021-9673(94)80456-7.
HPLC affinity and reversed-phase modes were coupled for the direct measurement of glutathione S-transferases (GSTs) in cytosol extracts. Two coupling designs were examined. In the sequential configuration the affinity column served to extract the isoenzymes which were then eluted directly onto the reversed-phase column as a single fraction. Subsequent separation in the reversed-phase mode provided a GST profile based on the subunit composition of the isoenzymes as a whole. In the second configuration (rapid sampling configuration), gradient elution was performed in the affinity mode resulting in resolution of the intact isoenzymes. The eluate from the affinity separation was sampled in continuous, repetitive intervals and automatically subjected to ongoing reversed-phase analysis. This multidimensional approach provided information on the GST subunit content and also gave information about the distribution of the subunits among individual isoenzymes, thereby forming a basis for the determination of the actual isoenzymatic composition of the GSTs. In both configurations, events were automated and co-ordinated through the use of computer and multiport switching valves. Examples of GST separations from these procedures are shown for human lung and liver tissues. A comparison of the GST subunit analyses from normal and cancer lung tissue excised from the same patient showed substantial elevations of GSTs in the cancer sample. Two-dimensional affinity-reversed-phase analysis of a human liver sample illustrates the utility of the technique for determining the isoenzymatic organization of GST subunits. The criteria for extending two-dimensional analysis to more complex GST mixtures are discussed.
高效液相色谱（HPLC）亲和模式和反相模式相结合，用于直接测定细胞溶质提取物中的谷胱甘肽S-转移酶（GSTs）。研究了两种联用设计。在顺序配置中，亲和柱用于提取同工酶，然后将其作为单一馏分直接洗脱到反相柱上。随后在反相模式下进行分离，根据整个同工酶的亚基组成提供GST图谱。在第二种配置（快速采样配置）中，在亲和模式下进行梯度洗脱，从而分离完整的同工酶。亲和分离的洗脱液以连续、重复的间隔进行采样，并自动进行反相分析。这种多维方法提供了关于GST亚基含量的信息，还给出了亚基在各个同工酶中的分布信息，从而为确定GSTs的实际同工酶组成奠定了基础。在这两种配置中，通过使用计算机和多端口切换阀实现了事件自动化和协调。展示了从这些方法中分离人肺和肝组织中GSTs的示例。对同一患者切除的正常和癌肺组织的GST亚基分析比较显示，癌样本中的GSTs大幅升高。对人肝样本的二维亲和-反相分析说明了该技术在确定GST亚基同工酶组织方面的实用性。讨论了将二维分析扩展到更复杂的GST混合物的标准。
