Hello Ambra.

Ellman's reagent (DTNB) reacts with any molecule that has a free thiol group (-SH), generating TNB (increasing yellow color), with a gain in absorbance that can be read at 412 nm. Due to the non-specificity of DTNB with the different thiols in your sample extracts (cysteine, GSH, coenzyme A), it is necessary to evaluate glutathione concentrations using an exogenous GR. Depending on the concentration of glutathione in your sample extract, you will obtain a proportional slope (delta A/delta T) on the spectrophotometer. Therefore, in addition to analyzing your samples, it will also be important for you to perform a calibration curve using exogenous glutathione (analytical standard), so that you can later interpolate your data obtained with biological samples using this calibration curve. Answering your questions, the reason why NADPH is the last reagent to be added is precisely because before it is added, all the GSH in your sample will be in the form of GSSG (as it has already been completely oxidized by DTNB). When adding NADPH, GR will begin to convert it into GSH, which then reacts with DTNB until all this NADPH is exhausted, thus generating a slope proportional to the concentrations of its glutathione, as mentioned previously. To be able to differentiate the initial GSH from the initial GSSG, you must perform a new test using vinylpyridine. Vinylpyridine has the ability to complex with the initial GSH, leaving only the GSSG available for you to carry out the cyclic reaction with GR, NADPH and DTNB again. This way, by calculating the concentrations of total glutathione and GSSG, you can determine the initial GSH concentration: Total glutathione = GSH + GSSG. It is important to remember that each GSSG is equivalent to 2 GSH. If you have any specific questions, you can ask them in the comments below.

Best Regards.