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

A method has been developed for the determination of interactions of metal ions and protein by using microdialysis sampling technique combined with pr e-column derivation and reversed-phase ion-pair liquid chromatographic (HPL C analysis. Cu(II), Zn(II) and human serum albumin (HSA) were chosen as mod el 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 pe rfusion rate at 1 mul/min and the temperature at 37 degreesC. The free conc entrations of metal ions in microdialysates were assayed by precolumn deriv atization with meso-tetra(4-sulfophenyl)-porphyrin (TPPS4) followed ion-pai r 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) wit h R.S.D. below 3.2%. The primary binding constants and number of binding si te estimated by the Scatchard plot analysis are 5.04 x 10(6) M-1 and 0.85 f or Cu(II), and 9.87 x 10(6) M-1 and 1.10 for Zn(II), respectively. The comp etition of Cu(II) and Zn(II) at the second binding site on HSA was investig ated, 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. (C) 2001 Elsevier Science B.V. All rights reserved.