The ultrafiltration and ESR study of Cd2+ binding to human erythrocyte spectrin.
The Cd2+ binding to human erythrocyte spectrin tetramer (SPT) was studied by the ultrafiltration method. The results indicate that the Cd2+ binding depends on Cd2+ concentration in a biphasic feature. Thus, it is different from the Tb(3+)-SPT reaction. The biphasic feature is in accordance with the results of fluorescence and circular dichroism (CD) studies on Cd(2+)-SPT complex. The data were analyzed by the Scatchard method. At the first stage (the mole ratio of Cd2+/SPD < 45), there are two types of Cd2+ binding sites, with the binding sites and corresponding binding constants determined as: n1 = 6, K1 = 3.3 x 10(5) M-1; n2 = 8, K2 = 9.1 x 10(4) M-1. At the second state (Cd2+/SPD > 45), the Cd2+ binding manifests a positive cooperative effect. The interaction between Cd2+ and maleimide spin labeled (MSL) SPT was studied by the ESR method. The results were fitted with the multiple equilibrium model, and one Cd2+ high-affinitive binding site with association constant 9.61(7) x 10(5) M-1 was obtained. The conjugation of maleimide with thiol group results in the decrease of Cd2+ high-affinitive binding sites from 6 to 1. This demonstrated that the thiol groups of SPT were involved in the high binding sites. The increase of mobile fraction of MSL in low Cd2+ concentration revealed that Cd2+ binding to SPT induces a significant conformation change of MSL-SPT. The rotation correlation time of MSL attached to SPT varied from 4.4 x 10(-9) to 6.5 x 10(-9) sec.
Department of Inorganic Chemistry, School of Pharmaceutical Sciences, Beijing Medical University, China.
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D001665:Binding Sites; D002104:Cadmium / Q000378:metabolism* / Q000494:pharmacology; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D004912:Erythrocytes / Q000737:chemistry*; D005453:Fluorescence; D006801:Humans; D011485:Protein Binding; D011487:Protein Conformation; D013049:Spectrin / Q000378:metabolism*; D013113:Spin Labels / Q000138:chemical synthesis; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014462:Ultrafiltration
D013113:Spin Labels; D013438:Sulfhydryl Compounds; D002104:Cadmium; D013049:Spectrin