Spectropotentiometric analysis of metal binding to structural zinc-binding sites: accounting quantitatively for pH and metal ion buffering effects.
Studies of the metal-binding affinity of protein sites are ubiquitous in bioinorganic chemistry and are valuable for the information that they can provide about metal speciation and exchange in biological systems. The potential for error in these studies is high, however, since many competing equilibria are present in solution and must be taken into consideration. Here, we report a new spectropotentiometric titration apparatus that allows pH and UV-vis absorption to be monitored simultaneously on small samples under inert atmosphere. In addition, we explain how data obtained from the complex equilibria can be combined with tabulated information about the protonation and metal-binding constants for common buffers to provide detailed, quantitative information about metal-protein interactions. Application of this approach to the investigation of metal binding to structural zinc-binding domains and common pitfalls encountered when performing these experiments are also discussed. We have used this approach to reevaluate the metal-binding constants of the N-terminal zinc-binding peptide from the HIV-1 nucleocapsid protein (10(-8)M</=K(d)(Co)</=10(-7)M; 10(-11)M</=K(d)(Zn)</=10(-10)M).
Magyar
John S
JS
Department of Chemistry, Northwestern University, 2145 Sheridan Rd., Evanston, IL 60208-3113, USA.
Godwin
Hilary Arnold
HA
D001665:Binding Sites; D001667:Binding, Competitive; D002021:Buffers; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000378:metabolism; D008667:Metalloproteins / Q000737:chemistry / Q000378:metabolism*; D008670:Metals / Q000378:metabolism; D010084:Oxidation-Reduction; D011199:Potentiometry / Q000379:methods*; D011485:Protein Binding; D015203:Reproducibility of Results; D012815:Signal Processing, Computer-Assisted; D013053:Spectrophotometry / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet; D015032:Zinc / Q000378:metabolism*; D016335:Zinc Fingers
D002021:Buffers; D007477:Ions; D008667:Metalloproteins; D008670:Metals; D015032:Zinc