Metal binding to ligands: cadmium complexes with glutathione revisited.
We studied the interaction of gamma-L-glutamyl-L-cysteinyl-glycine (glutathione, GSH) with cadmium ions (Cd(2+)) by first performing classical potentiometric pH titration measurements and then turning to additional spectroscopic methods. To estimate the residual concentrations of free cadmium, we studied the competition of glutathione with a Cd(2+)-sensitive dye, either an absorbing dye (murexide) or a fluorescent one (FluoZin-1), and consistent results were obtained with the two dyes. In KCl-containing Tes, Mops, or Tris buffer at pH 7.0 to 7.1 and 37 degrees C (and at a total Cd(2+) concentration of 0.01 mM), results suggest that free cadmium concentration is halved when the concentration of glutathione is approximately 0.05 mM; this mainly reflects the combined apparent dissociation constant for the Cd(glutathione) 1:1 complex under these conditions. To identify the other complexes formed, we used far-UV spectroscopy of the ligand-to-metal charge transfer absorption bands. The Cd(glutathione)(2) 1:2 complex predominated over the 1:1 complex only at high millimolar concentrations of total glutathione and not at low submillimolar concentrations of total glutathione. The apparent conditional constants derived from these spectroscopy results made it possible to discriminate between sets of absolute constants that would otherwise have simulated the pH titration data similarly well in this complicated system. Related experiments showed that although the Cl(-) ions in our media competed (modestly) with glutathione for binding to Cd(2+), the buffers we had chosen did not bind Cd(2+) significantly under our conditions. Our experiments also revealed that Cd(2+) may be adsorbed onto quartz or glass vessel walls, reducing the accuracy of theoretical predictions of the concentrations of species in solution. Lastly, the experiments confirmed the rapid kinetics of formation and dissociation of the UV-absorbing Cd(glutathione)(2) 1:2 complexes. The methods described here may be useful for biochemists needing to determine conditional binding constants for charge transfer metal-ligand complexes under their own conditions.
Leverrier
Pauline
P
CNRS, URA 2096 (Protéines Membranaires Transductrices d'Energie), and CEA, iBiTec-S, SB(2)SM, Laboratoire des protéines membranaires, CEA Saclay, F-91191 Gif-sur-Yvette, France.
Montigny
Cédric
C
Garrigos
Manuel
M
Champeil
Philippe
P
D000085:Acetates / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002021:Buffers; D002104:Cadmium / Q000737:chemistry* / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D009114:Murexide / Q000737:chemistry / Q000378:metabolism; D011199:Potentiometry; D011522:Protons; D012996:Solutions / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D014966:Xanthenes / Q000737:chemistry / Q000378:metabolism
D000085:Acetates; D002021:Buffers; C484689:FluoZin-1; D008024:Ligands; D011522:Protons; D012996:Solutions; D014966:Xanthenes; D002104:Cadmium; D009114:Murexide; D005978:Glutathione