The Spontaneous and Glutathione S-Transferase-Mediated Reaction of Chlorambucil with Glutathione

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Abstract
Incubation of 100 μM chlorambucil (CMB) with 1 mM glutathione (GSH) in 0.1 M potassium phosphate buffer yielded a mixture of GSH conjugates and hydrolytic products that were separable by HPLC. The appearance in HPLC analysis of four of these products was GSH-dependent. 35S-Label from 35S-GSH eluted with all four chemical species that also gave U V spectra characteristic of CMB-containing compounds. Mass spectral analysis of three of these compounds gave molecular weights consistent with the following adducts: adduct 2: diglutathionyl CMB; adduct 3: monohydroxy monochloroglutathionyl CMB; adduct 4: monochloro monoglutathionyl CMB. Purified GST α and partially pure GST π and μ. class protein prepared from adult male mouse liver cytosol significantly increased the formation of the monoglutathionyl CMB adduct. At 5 min incubations, this adduct was quantitatively most important and was increased 4.4-fold by the addition of GST α isozymes. At 1 hr incubations, all four adducts were measurable, although enzymes primarily affected the formation of adduct 4. At 1 hr, addition of GST α, π, or μ protein increased the production of monochloro monoglutathionyl CMB 2-fold, 1.5-fold, and 1.7-fold, respectively, demonstrating that CMB is indeed a substrate for GST isozymes in vitro.