Working with Dansyl Chloride-Labeled Glutathione, Blog Post #1:The Preliminary Production and Testing of Dansyl-Chloride Labeled Glutathione

When Sharon, Olivia, and I walked in for our first week in the laboratory, we were not sure as to what our summer lab sessions would entail. Dr. Landino helped us prepare for the coming weeks, explaining our goals for the summer and clarifying our basic plan of action—all in all, we hope to run protein reactions and gels with the majority of our commonly-used lab proteins, comparing their reactivities under different pH, time, concentration, and temperature conditions. To do this, we would need to purify a lot of our primary protein target: oxidized dansyl-chloride labeled glutathione. So for the first couple of days, we spent our time purifying such samples using C8 columns. We loaded solutions of reacted dansyl chloride and glutathione onto our columns and followed with fractional washes of methanol, deionized water, and acetic acid. (Excess dansyl chloride elutes in 20% methanol, single-labeled oxidized glutathione elutes in 50% methanol, and double-labeled oxidized glutathione elutes in 80% methanol.) Though not exceptionally difficult, the column procedures were time-consuming; but we prepared a good amount of product using the column technique we finally perfected after a semester of trying to get it to work properly. Huzzah!

After that, it was time for us to move on and practice using a plate reader in order to create standard curves, which use absorbance values of diluted chemical solutions in order to calculate their concentrations. (We now consistently use these standard curves to help us measure concentrations of solutions containing our dansyl chloride-labeled glutathione, and it has become a competition for us to see how high we can get our r2 values for our linear concentration plots.) We also ran reduction reactions of oxidized dansyl-labeled and non-labeled glutathione with two reducing agents, dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP), comparing the extents to which the reductions took place under different concentrations of reducing agent and different lengths of time. We decided that letting DTT (which seemed to be the better reducing agent for our purposes) run for at least a day would maximize the amount of reduced dansyl chloride-labeled glutathione (DGSH) we could collect and use.

Of course, this implies that we could purify that DGSH when it had been reduced. Well, fortunately for us, we already had a procedure for such purposes available from when Dr. Landino’s lab was still purifying reduced fluorescein-labeled glutathione. We adapted that procedure and began running C8 column purifications of DGSH, where we removed all of the DTT from the DGSH solutions through fractional washes of water and acetic acid (constantly testing for DTT in the eluted fractions with BCA protein dye assays). We then extracted the DGSH through similar washes of pure methanol with a little bit of acetic acid, drying our samples as soon as possible to prevent the DGSH from re-oxidizing.

With all of this reduced and oxidized dansyl chloride-labeled glutathione, our next step was to observe reactions of the oxidized form with GSH (reduced, unlabeled glutathione) and run it on TLC plates to see if it reduced properly. We also had to find out how long it took for our DGSH to re-oxidize—this would give us an idea as to how much time we would have to work with our reduced samples before they were no longer useful. We found that the oxidized, labeled glutathione did reduce fairly well. And fortunately for us, DGSH does not re-oxidize as easily as we originally thought. However, we are unable to calculate DGSH’s rate of reoxidation, as 1) oxidized impurities were probably still present in our samples, and 2) we do not yet know how to translate fluorescence intensity data from photographing our TLC plates into concentration data. For the moment, though, we have moved on to starting our protein gels, which I will discuss in my next post. Stay tuned!