Fig. 1 Effect of GSH concentration on the rate of DTNB formation.  Various concentrations of glutathione used to generate a standard curve. These assays were run using the Total Glutathione Assay. The rate of the linear portion of each assay was plotted against the concentration in order to produce a standard curve (Fig. 2). 

Fig. 2  Standard curves of reduced glutathione and reduced glutathione plus 2-vinylpyridine.  This graph shows the amount of contamination, which is in the form of oxidized glutathione, present in a pure sample of reduced glutathione. The 2-VP blocks all existing GSH and only the contaminating GSSG is allowed to enter assay. The pure reduced glutathione is 98.9% pure.

Fig. 3 Standard curves of oxidized glutathione and oxidized glutathione plus 2-vinylpyridine.  This graph shows the amount of conatamination present, in the form of reduced glutathione, that is present in a sample of pure oxidized glutathione. The contamination is blocked by 2-vinylpyridine and only the GSSG is allowed to enter the assay. The pure oxidized glutathione is 98.0% pure. 

Fig. 4  Comparison of oxidized glutathione and reduced glutathione standard curves. The same concentration was used for both compounds. GSSG, in the recycling assay, produces twice as much GSH on a molar basis as the same concentration of GSH. This is shown by the rate of the standard curves for GSSG which is twice the rate for GSH. 

Fig. 5  Comparison of standard curve generated with reduced glutathione using the enzymatic recycling procedure and the DTNB photometric assay. The recycling assay is 25 times more sensitive than using just the DTNB, as shown by the much higher rates for the standard curve of the enzymatic procedure compared to the rate of the DTNB procedure.