Binding of metal ions with protein studied by a combined technique of microdialysis with liquid chromatography

Abstract

A method has been developed for the determination of interactions of metal ions and protein by using microdialysis sampling technique combined with pre-column derivation and reversed-phase ion-pair liquid chromatographic (HPLC) analysis. Cu(II), Zn(II) and human serum albumin (HSA) were chosen as model metal ions and protein, respectively. The mixed solutions of metal ions and HSA with different molar ratios buffered with 0.1 M Tris–HCl containing 0.1 M NaCl at pH 7.43 were sampled with a mirodialysis probe by keeping perfusion rate at 1 μl/min and the temperature at 37°C. The free concentrations of metal ions in microdialysates were assayed by precolumn derivatization with meso-tetra(4-sulfophenyl)-porphyrin (TPPS 4) followed ion-pair HPLC analysis. The recovery ( R) of microdialysis sampling was measured in vitro under similar conditions as 65.74% for Cu(II), 70.45% for Zn(II) with R.S.D. below 3.2%. The primary binding constants and number of binding site estimated by the Scatchard plot analysis are 5.04×10 6 M −1 and 0.85 for Cu(II), and 9.87×10 6 M −1 and 1.10 for Zn(II), respectively. The competition of Cu(II) and Zn(II) at the second binding site on HSA was investigated, and it was observed that there is a second site on HSA to bind Cu(II) and Zn(II), the affinity of Cu(II) is stronger than that of Zn(II) to this second site of HSA.