Hi Xiaopeng To add to Artem's comments: Does the presumed gsh make a mixed disulfide in the active site?i.e. is it covalently bonded to the active site via a s-s bond? If yes then MS on your purified sample should easily give you the answer. If a mixed s-s is indeed the scenario then purifying the enzyme in the presence of reducing agents and playing with the pH a bit should yield a gsh free preparation.
Best regards Savvas On 16 Mar 2011, at 02:19, Artem Evdokimov <artem.evdoki...@gmail.com> wrote: > 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 >
