Spectroscopic determination of the thermodynamics of lead, zinc, and cobalt interactions with GATA proteins

Detailed metal-binding studies monitored by absorption spectroscopy were used to determine the affinity of zinc and lead for GATA proteins. The affinities of zinc for the C-terminal domain of chicken GATA-1 (CF) and the double finger domain of human GATA-1 (DF) were determined through a series of competition experiments in which Co2+ was used as a spectroscopic probe. For CF, the affinity of Co2+ and Zn2+ binding was determined to be K_bCo = 1.0 (±1.3) × 107 M−1 and K_bZn = 2.0 (±1.3) × 10 10 M−1. For DF, the affinity of Co2+ and Zn2+ binding was determined to be β₂ Co = 2.5 (±1.6) × 1014 M−2 and β₂Zn = 6.3 (±2.5) × 1020 M−2. These experiments suggest that the individual NF and CF domains of the DF are independent metal-binding domains that have roughly the same affinity for Zn2+ as that measured for the CF domain.; The stoichiometries and affinities of Pb2+ binding for CF and DF were determined through spectroscopically-monitored direct Pb 2+ and Pb2+/Zn2+ competition titrations. These studies reveal that Pb2+ binds in a 1:1 stoichiometnc ratio to CF and in a 2:1 stoichiometric ratio to DF with a high affinity given by K_bPb = 6.4 (±2.0) × 109 M−1 for CF and β₂ = 6.3 (±6.3) × 1019 M−2 for DF. Furthermore, Zn2+/Pb2+ competition experiments demonstrated that Pb2+ is able to displace Zn2+ from GATA sites in minutes at 37°C, suggesting that metal occupancy of these sites should be dictated by the thermodynamics of metal binding (rather than site accessibility) under physiological conditions.; To resolve remaining questions regarding metal binding to GATA proteins, the full length protein and the NF, CF, DF domains of murine GATAs 1, 2, and 3 were subcloned into the pGEX-PKT vector for future expression and purification as glutathione-S-transferase fusion proteins for use in experiments that characterize the interaction of metals with GATA proteins.; These studies constitute the first quantitative studies of metal binding to the GATA family of transcription factors and suggest that these proteins are potential targets for lead. These results point to the need for detailed studies to test whether lead affects GATA protein activity in vivo . (Abstract shortened by UMI.)...