Noncovalent associations of glutathione S-transferase and ligands: A studyusing electrospray quadrupole/time-of-flight mass spectrometry

Human glutathione S-transferase A1-1 was observed predominantly as dimeric ions (51 kDa) during electrospray mass spectrometric analysis from aqueous solution at pH 7.4, in keeping with the known dimeric structure in solution . When analyses were performed on solutions of the enzyme containing glutat hione (GSH), noncovalent adducts of protein dimer and one or two ligand mol ecules were observed; each mass increment, which exceeded the mass of GSH a lone, was provisionally interpreted to indicate concomitant association of two water molecules per bound GSH. Noncovalent adducts of ligand and protei n dimer were similarly observed for oxidized glutathione and for two glutat hione inhibitors, both incorporating substituted thiol structures. In these instances, the mass increments exactly matched the ligand masses, suggesti ng that the apparent concomitant binding of water was associated with the p resence in the ligand of a free thiol group. Collisionally activated decomp osition during tandem mass spectrometry analyses of noncovalent adducts inc orporating protein dimer and ligands yielded initially the denuded dimer; a t higher collision energies the monomer and a protein fragment were formed. (J Am Soc Mass Spectrom 2000, 11, 606-614) (C) 2000 American Society for M ass Spectrometry.