Tightly bound ligands commonly survive purification :) Several unexpected discoveries have been made this way!
If you think your stuff is GSH, soak some 'real' GSH or co-crystallize with it, and see if density shape changes from what you had before. This is not guaranteed to work because sometimes the ligand may be bound so tightly/exchange slowly that the original ligand just won't budge. This was a significant issue with some of the kinase inhibitors and nuclear hormone receptor antagonists that I've had a chance to work with - the binding was almost 'one way' in real-time situation, and (partial) denaturation was required to get the ligands out. If your ligand is indeed GSH, in equimolar amount with the protein, then you could also try MS as detection technique. The ligand should come off when protein is subjected to the normal MS environment (typically 0.1% TFA or formic acid, in a mixture of water and acetonitrile, etc.). To detect it, don't forget to expand the mass window to get the mass, and set the ionization mode to positive - you should see a clear 308.3 Da peak, with a lovely isotope splitting pattern (assuming you have access to MS). In negative mode the mass will be 1/2 of the already low m.w. of 307, since GSH has two negative charges. Notably, GSH should also accept e.g. an iodoacetamide group on the -SH, meaning that you should be able to treat crystals with iodoacetamide and observe the addition of -CH2CONH2 to the sulfur. Naturally, the S atom should be pretty prominent in the density anyway. Ditto mercurials, but they may wreck the crystal. Since your enzyme may be a GST (assumption on my part here) it also may present GSH to be reactive with whatever substrates that yor GST is targeting, so you may be able to identify conjugation products. Artem 2011/3/14 Xiaopeng Hu <huxp...@mail.sysu.edu.cn> > Dear all, > > Sorry for this off topic question. > > We are working on protein/inhibitor complex structure although we can not > get our inhibitors in. However, we did find a strange density at the active > site, it looks really like GSH, the natural co-enzyme of thiis protein.We > tried to use very simple solution to get crystal then exclude the possiblity > of buffer moleculors,but that density is aways there. > > I am wondering how this ligand (if it is GSH) can survive all purification > steps and want to indentify it. Are there any methodes to do this work? > Let's say to pick up a crystal and do some analysis? > > Many many thanks!!! > > > Xiaopeng >
