Characterization of the interaction of galectin-1 with sodium arsenite.

We previously showed that galectin-1 (GAL1) is an arsenic-binding protein. In the current study, we further characterize the interaction of GAL1 with sodium arsenite (As(III)). The GALl-As(III) complex was prepared from the cell extracts of GAL1-transfected Escherichia coli (E. coli) that were pretreated with As(III). The results of the circular dichroism (CD) spectrum of GAL1-As(III) exhibited a negative signal at around 205-210 nm, whereas that of GAL1 showed a negative signal at around 215-220 nm. This shift in the CD spectrum is indicative of a substantial change in the secondary structure arising from the binding of As(III) to the GAL1 protein. The UV absorptive spectrum of the GAL1-As(III) complex was significantly lower than that of GAL1 itself. A mobility shift binding assay showed that the GAL1-As(III) complex migrated closer than GAL1 toward the anode. Capillary electrophoretic analysis also showed that As(III) binding decreased the mobility of GAL1. These results further confirmed the structural change of the GAL1 complex with As(III). Furthermore, isothermal titration microcalometric studies showed that As(III) titration into the GAL1 protein solution was an endothermic process with absorption enthalpy (DeltaH(abs)) around 8-10 kJ/mol As(III). The affinity constant (K(d)) of As(III) toward GAL1 was around 8.239 +/- 2.627 microM as estimated by tryptophan (Trp) fluorescence quenching. However, the binding of As(III) did not significantly affect the biological activity of GAL1, since the GAL1-As(III) complex only partially lost its lectin activity. In addition, we show that GAL1-transfected KB cells accumulated more arsenic than did the parental cells. Taken together, these results suggest that GAL1 might serve as a target protein of As(III) in vivo, and the binding of GAL1 with As(III) could interfere with the excretion of As(III).

Application of cyclodextrin-mediated capillary electrophoresis to simultaneously determine the apparent binding constants and thermodynamic parameters of naphthalenesulfonate derivatives.

Application of capillary electrophoresis (CE) to simultaneously determine the apparent binding constants and thermodynamic parameters for six positional and structural naphthalenesulfonate derivatives with beta-cyclodextrin (beta-CD) is presented. The change in electrophoresis mobilities was used to assess the binding constants by non-linear regression and three different linear plots methods (named double reciprocal, x-reciprocal and y-reciprocal). The substituent group(s) attached to the naphthalene ring considerably affected the inclusion behaviors of these naphthalenesulfonate derivatives. The binding constant varies over almost one order of magnitude and a highly selective sequence is obtained between these guest model compounds. Naphthalenesulfonates with the substituent(s) at the 2-position(s) displayed stronger interaction with beta-CD, and gave well compatible results by these four plot methods. While at least one substituent was substituted into the 1-position of naphthalene showed the weak interaction or no interaction with beta-CD. Comparison to three linear regression methods, the non-linear regression method proves to be the most suitable for these determinations. Additionally, apparent binding constants for each structural isomer with beta-CD at several temperature, and thermodynamic parameters for binding were also calculated and discussed.

Application of cyclodextrin-mediated capillary electrophoresis to determine the apparent binding constants and thermodynamic parameters of the alkylnaphthalene derivatives.

The separation and migration behavior of seven positional and structural neutral alkylnaphthalene derivatives in cyclodextrin-mediated capillary electrophoresis were systematically investigated. The effective separation conditions were to use 10 mM phosphate buffer with negatively charged carboxymethyl-beta-cyclodextrin (CM-beta-CD) at pH 6.0. The guest-host interactions with 1:1 or both 1:1 and 1:2 binding stoichiometries for various derivatives were evaluated by comparing their apparent binding constants. The results reveal that the substituent group(s) attached to the naphthalene ring significantly affected the inclusion stoichiometric behaviors. Alkylnaphthalene derivatives with the substituent(s) at the 1-position(s), such as 1-ethylnaphthalene, 1,4-dimethylnaphthalene, may undergo complexation with one and two CM-beta-CD molecules. The binding constants of these derivatives were consistent with the data obtained by a spectrophotometric method. The thermodynamic parameters were also calculated in order to improve our understanding of the interaction between the neutral alkylnaphthalene derivatives and CM-beta-CD at various temperatures. The positive entropy (deltaS degrees) values of the alkylnaphthalenes with the substituent(s) at the 2-position(s) indicate that the inclusion of the guest molecule into the cavity of CM-beta-CD is favored at all temperatures.