Chloride ion binding to human plasma albumin from chlorine-35 quadrupole relaxation.
The 35Cl nuclear magnetic quadrupole relaxation enhancement on binding of chloride ions to human plasma albumin (HPA) has been studied under conditions of variable temperature, pH, ionic strength, protein, and sodium dodecyl sulfate concentration. A small number (less than 10) of chloride ions, most of which are bound to the primary detergent binding sites, contribute a major portion of the relaxation enhancement (greater than 80% at neutral pH). A comparison of the pH dependence of the relaxation rate with the hydrogen ion titration curve, which was determined and analyzed, identified ten lysyl and arginyl residues as being involved in the chloride ion binding. These data, in conjuction with NaDodSO4 titrations at different pH values and the amino acid sequence of HPA, suggests that the high-affinity chloride-binding sites are doubly cationic at neutral pH. An irreversible dimerization at acidic pH and 5 x 10(-5) m HPA was detected. The data also indicate the presence of internal modes of motion in the expanded forms of the HPA molecule, probably an independent reorientation of domains. The rate of exchange of chloride ions was shown to be much higher than the corresponding intrinsic relaxation rate in the temperature range 2--26 degrees C and pH values ranging from 4.0 to 10.5. No indications of protein-protein interaction could be found up to the physiological concentration of ca. 6 x 10(-4)m HPA at either neutral or alkaline pH. The mechanistic basis for HPA's exceptional capacity for binding of inorganic anions was discussed.
Chloride ion binding to human plasma albumin from chlorine-35 quadrupole relaxation. The 35Cl nuclear magnetic quadrupole relaxation enhancement on binding of chloride ions to human plasma albumin (HPA) has been studied under conditions of variable temperature, pH, ionic strength, protein, and sodium dodecyl sulfate concentration. A small number (less than 10) of chloride ions, most of which are bound to the primary detergent binding sites, contribute a major portion of the relaxation enhancement (greater than 80% at neutral pH). A comparison of the pH dependence of the relaxation rate with the hydrogen ion titration curve, which was determined and analyzed, identified ten lysyl and arginyl residues as being involved in the chloride ion binding. These data, in conjuction with NaDodSO4 titrations at different pH values and the amino acid sequence of HPA, suggests that the high-affinity chloride-binding sites are doubly cationic at neutral pH. An irreversible dimerization at acidic pH and 5 x 10(-5) m HPA was detected. The data also indicate the presence of internal modes of motion in the expanded forms of the HPA molecule, probably an independent reorientation of domains. The rate of exchange of chloride ions was shown to be much higher than the corresponding intrinsic relaxation rate in the temperature range 2--26 degrees C and pH values ranging from 4.0 to 10.5. No indications of protein-protein interaction could be found up to the physiological concentration of ca. 6 x 10(-4)m HPA at either neutral or alkaline pH. The mechanistic basis for HPA's exceptional capacity for binding of inorganic anions was discussed.