Rhonda Moeller: Quantification of reduced Glutathione and Glutathione Disulfide using Glutathione Reductase and 2-vinylpyridine.  The reactions underlying the described assay involve a truncated electron transport chain that utilizes the cyclical regeneration of GSH from GSSG by GSH reductase.  GSH reductase in the reaction is maintained in the reduced state by exogenous NADPH introduced into the assay mixture.  For a given concentration of reductase, NADPH and DTNB, the initial velocity of the colorometric reaction follows first order kinetics at a rate that is directly proportional to the amount of GSSG and GSH in the reaction.       To quantitatively differentiate between the oxidized and reduced forms of the compound, the assay blocks pre-existing GSH from entering the reaction by the addition of a derivitization agent that prevents reaction of the functional groups with DTNB(Griffith, 1980).  The present studies (Fig. 2 in results section) show that the incubation of GSH with 2-VP causes a 98.7% reduction in the rate of the reaction.  The remaining activity presumably results from the presence of contaminating GSSG.       In addition to efficient blockage of GSH, another critical aspect of this derivatization process is that the rate constants for the reaction must be substantially slower than those for both the reductase and the colorometric reduction of the DTNB.  Failure to meet these kinetic criteria would result in the blockage of GSH generated from GSSG via the reductase, causing inhibition of the reaction and the need to quantitatively recover excess 2-VP from the reaction.  Another important feature of the derivitization agent is that it must not inhibit the reductase (Griffith, 1980).  The current data shows minimal inhibition of the enzyme by 2-VP.  Evidence for this is given by the standard curves produced from purified GSSG (Fig. 3).  Thus, the addition of reductase to GSSG pre-incubated for one hour with 2-VP produced a rate of reduction that was twice that produced by an equi-molar concentration of GSH.  This is in accordance with the expected ratios of GSH produced from the reductive cleavage of GSSG.       Finally, one of the major virtues of the procedure described above is the sensitivity of the assay.  By using an enzymatic process in which the GSH substrate is regenerated, the spectroscopic factors are substantially higher than assays where the GSSG is not recycled.   This is clearly shown in figure 5 which compares the sensitivity of the reductase procedure against the conventional DTNB methodology.  Although accurate, the DTNB method was 25 times less sensitive for the analysis of GSH.  Over more, because the reductase is specific for GSSG it offers a degree of selectivity for the GSH substrate.  Conversely, the DTNB reaction assays for total thiol content and will include measurements from other species such as free cysteine and –SH rich proteins.       Due to DTNB having a large affinity for proteins with a sulfhydrl group, the initial reaction with reduced DTNB can be caused by a variety of other compounds.  A more specific assay for GSH quantification was tested utilizing glutathione-S-transferase and the colorometric substrate 1-chloro-2,4-dinitrobenzene (CDNB).   By combining this method with the initial steps of the enzymatic recycling assay it should be possible to develop a procedure that capitalizes on the benefits of both the specificity of GST and CDNB and the high sensitivity of the recycling procedure.  Using this protocol, attempts were made to quantify reduced glutathione, glutathione reductase and glutathione-S-transferase in Helix aspersa cytosol.  As seen from Table 1, various amounts of different reactants were added to the reaction and the reaction rates calculated.    From run F, where GSH was added to the sample and no GST was added, activity was seen giving rise to the conclusion that GST must be present in the Helix cytosol.  In run C, where GSH is produced from the GSSG reduction and no GST is added, no activity occurs.  But in run D, where GSH is produced form the reduced GSSG and mammalian GST is added, activity occurs.   These reactions were repeated and similar results were observed in reactions I, J, and K.  The only conclusions that can be drawn from the table are as follows:   · Glutathione-S-Tranferase is present in the cytosol of  Helix Aspersa.  · The GST that is present in the cytosol will react with added GSH, but will not react with GSH produced from reducing GSSG.  · Mammalian GST that is added to the sample will react with GSH produced from reducing GSSG.  We currently have no rationale explanation for the apparent paradox in the observed data.  Is there a significant difference between molluscan GST and mammalian GST, where the latter can use the GSH produced from GSSG and the former can not, or is the difference somehow in the GSH that is cleaved from GSSG?