ANALYSIS OF MONOCHLOROBIMANE AND HUMAN GLUTATHIONE S-TRANSFERASE

Abstract

The means by which tumor cells survive anti-neoplastic chemotherapy is not well understood. Many of the anti-neoplastic drugs may be viewed as electrophilic compounds. The primary means by which cells detoxify electrophiles is through the glutathione-related detoxification scheme. It is important to develop accurate and facile techniques to study the role of glutathione (GSH) in detoxification mechanisms. The electrophile, monochlorobimane (MCB), has been proposed to be an excellent fluorescent probe for the detection of GSH. Initial studies showed that low concentrations of MCB (10-100 μM) were adequate to label GSH in rodent cell lines. However, when the MCB assay was extended to human cell lines higher concentrations of MCB (1000 μM) were required. The MCB assay was found not to be ideal when strictly quantitative results were necessary. However, the MCB assay was found to be adequate for the qualitative analysis of GSH levels between different cell lines. These properties of MCB were exemplified when flow cytometric analysis of GSH levels (using 1000 μM MCB) in seven human and rodent cell lines correlated with data acquired from high pressure liquid chromatography (HPLC) and standard GSH assays. To better define why there is variation in results when using MCB to study rodent and human cells, the differences in MCB affinities and kinetics properties between species-specific glutathione S-transferases (GST) were analyzed. To accomplish this task GST isoenzymes from human liver and rat liver were isolated, purified and characterized by kinetics analysis using MCB as a substrate. The kinetics parameters of MCB and GSH were determined using human GSTs π, ᴪ, B1B2, B2B2, and rat GST 1-2, 3-3 and 3-4. The human acidic GST has a Km of 264 μM for MCB and a Vmax of 1.99 μmole GS-bimane/min-mg protein. The human basic GST B1B2 and B2B2 have Km values of 354 μM and 283 μM, and V max values of 33.3 and 34.6 μmole GS-bimane/min-mg protein. The rat basic GST had a Km of 199 μM and a V max of 35.5 μmole GS-bimane/min-mg protein. The human neutral GST had a Km of 204 μM and a Vmax of 6.5 μmole GS-bimane/min-mg protein. The rat neutral GST had a Km of 2.6 μM and a Vmax of 35.1 μmole GS-bimane/min-mg protein. The kcat/Km ratio showed that the rat liver GSTs were excellent catalysts for the reaction of GSH and MCB. In addition, the GS-bimane adduct was found to act as an inhibitor in the GST-catalyzed reaction of 1-chloro-2,4-dinitrobenzene and GSH (I50 (rat liver GST) = 70 μM; I50 (human placental GST) = 35 μM). This study leads to the conclusion that inadequate labeling of GSH with the MCB assay (< 1000 μM) in human cell lines results from the low affinity of MCB for the human GSTs.