A COLORIMETRIC MICRO-METHOD FOR THE DETERMINATION OF GLUTATHIONE

Abstract

1. A rapid colorimetric and apparently specific micromethod for the determination of total glutathione in small amounts of tissue is described. Generally, less than 30mg. of tissue is sufficient and this is homogenized in ice-cold 3% metaphosphoric acid. The product is filtered through sintered glass and neutralized or diluted before being added to a cuvette containing phosphate buffer, pH7.1, 5,5'-dithiobis-(2-nitrobenzoic acid), EDTA and glutathione reductase. Addition of NADPH(2) to the system initiates a progressive reduction of 5,5'-dithiobis-(2-nitrobenzoic acid) by catalytic amounts of GSH, and this causes a colour increase at 412mmu. The rate of this change, calculated over 5min., is proportional to the total amount of glutathione present, and consequently unknown concentrations may be determined by reference to standards. 2. A preparation (based on that of Racker, 1955) of a suitable sample of glutathione reductase from yeast is described. 3. A less specific and less sensitive determination of extracted thiol groups with 5,5'-dithiobis-(2-nitrobenzoic acid) at pH8.0, based on observations of Ellman (1959) and Jocelyn (1962), is also described. 4. Although the precise nature of the reaction is not known, evidence is put forward to support a process of cyclo-reduction. GSSG is reduced enzymically to GSH, which reacts with 5,5'-dithiobis-(2-nitrobenzoic acid) to produce a coloured ion: [Formula: see text] (E(max.) 412mmu) and a mixed disulphide. This disulphide reacts with further quantities of GSH to liberate another ion and GSSG, which then re-enters the cycle.