Blanchette Brian N, Singh Bal Ram
Department of Chemistry and Biochemistry, School of Marine Science and Technology, University of Massachusetts Dartmouth, 285 Old Westport Road, Dartmouth, MA 02747, United States.
J Biochem Biophys Methods. 2007 Aug 1;70(5):761-5. doi: 10.1016/j.jbbm.2007.04.005. Epub 2007 May 4.
In order to expedite the process of classification of the members of the family of glutathione-S-transferases (GSTs) high performance liquid chromatography with photodiode array detection (HPLC-PDA) was used as a means for measuring enzymatic activity. The GST chosen for the development of the HPLC-PDA technique was from equine liver (E-GST). The characterizing substrates, ethacrynic acid (EA) and bromosulfophthalein (BSP), along with previously gathered characterization data allowed for the distinction of alpha, mu or pi-class enzymes. In an initial characterization of the previously unclassified E-GST it was determined that the enzyme was of the pi-class with specific activities of 0.062, +/-0.0015 micromol min(-1) mg(-1) and 0.0019, +/-0.00064 micromol min(-1) mg(-1) for EA and BSP, respectively. Finally, the activity of the E-GST with the EA and BSP substrates, was measured by HPLC-PDA, and was found to be 0.027, +/-0.003 micromol min(-1) mg(-1) and 0.002, +/-0.0005 micromol min(-1) mg(-1), respectively. While the HPLC-PDA data do not mirror the spectrophotometric results quantitatively the overall response by the E-GST was the same. In general, the E-GSTs were shown to belong to the pi-class when characterized by HPLC-PDA due to an EA specific activity greater than 0.01 micromol min(-1) mg(-1) and a negligible BSP activity (</=0.002 micromol min(-1) mg(-1)).
为了加快谷胱甘肽-S-转移酶（GSTs）家族成员的分类过程，采用高效液相色谱-光电二极管阵列检测法（HPLC-PDA）作为测量酶活性的手段。用于开发HPLC-PDA技术的GST来自马肝脏（E-GST）。特征底物乙磺半胱氨酸（EA）和溴磺酚酞（BSP），以及先前收集的特征数据，有助于区分α、μ或π类酶。在对先前未分类的E-GST进行初步表征时，确定该酶属于π类，对于EA和BSP的比活性分别为0.062±0.0015微摩尔·分钟⁻¹·毫克⁻¹和0.0019±0.00064微摩尔·分钟⁻¹·毫克⁻¹。最后，通过HPLC-PDA测量了E-GST与EA和BSP底物的活性，发现分别为0.027±0.003微摩尔·分钟⁻¹·毫克⁻¹和0.002±0.0005微摩尔·分钟⁻¹·毫克⁻¹。虽然HPLC-PDA数据在定量上与分光光度法结果不一致，但E-GST的总体反应是相同的。一般来说，当通过HPLC-PDA进行表征时，由于EA比活性大于0.01微摩尔·分钟⁻¹·毫克⁻¹且BSP活性可忽略不计（≤0.002微摩尔·分钟⁻¹·毫克⁻¹），E-GSTs被证明属于π类。
