Characterization of the Interaction of Galectin-1 with Sodium Arsenite Chen-Huan Lin, † Chi-Fei Huang, † Wen-Yih Chen, ‡ Yu-Ying Chang, † Wang-Hsien Ding, § Ming-Shen Lin, ‡ Szu-Hsun Wu, § and Rong-Nan Huang* ,† Department of Life Science, College of Science, National Central UniVersity & UniVersity System of Taiwan, Chung-Li, Taoyuan, Taiwan 32054, ROC, and Departments of Chemical & Materials Engineering and Chemistry, National Central UniVersity, Chung-Li, Taoyuan, Taiwan 32054, ROC ReceiVed December 1, 2005 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 (ΔH abs ) around 8-10 kJ/mol As(III). The affinity constant (K d ) of As(III) toward GAL1 was around 8.239 ( 2.627 µM 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). Introduction Arsenite (As(III)) is a well-documented human carcinogen. Environmental exposure to arsenic from copper smelters and drinking water has been associated with a number of pathologi- cal problems, such as skin, bladder, and lung cancers (1, 2). Arsenic exposure has also been related to the etiology of diabetes and cardiovascular disease (e.g., hypertension) (3-6). The exact biological targets of arsenic inside cells are not known. However, the binding of arsenic to tissue proteins has been proposed as the first step in the metabolism of arsenic (7). The adverse effects of arsenic on biological systems have also been proposed by its reaction with closely spaced cysteine residues on critical cellular proteins (8). Therefore, the binding of arsenic to proteins is an important determinant of arsenic toxicity (9). Galectin-1 (GAL1) is a sugar-binding protein specific for -galectosides. It exists as a soluble protein which forms a noncovalent homodimer and is expressed with a broad tissue distribution (10, 11). GAL1 is found mainly in the cytosol of most cells in which it is synthesized (12) and then externalized to bind to -galactosy-containing glycoconjugates on the cell surface and in the extracellular matrix (13-15). Since this lectin lacks an identifiable signal sequence, it does not appear to be secreted through normal secretory pathways (16, 17). Arsenic has been shown to exert at least some of its toxic effects through interactions with thiol groups of proteins, thereby modulating the activities of key regulatory proteins (18). GAL1 has six sulfhydryl groups which are not involved in the formation of the GAL1 dimer, but the free sulfhydryl groups are critical for its lectin-binding activity (19-21). GAL1, therefore, is an ideal model for probing the interaction of arsenic with proteins. We previously demonstrated that GAL1 is an arsenic-binding protein (22). In this study, GAL1 and GAL1- As(III) proteins were purified from GAL1-transfected Escheri- chia coli (E. coli) without or with As(III) treatment, and both proteins were used to probe the interactive mechanism of GAL1 with As(III). Experimental Procedures Cell Growth Assay and Arsenic Measurements. KB cells (oral epidermoid carcinoma cells) were obtained from American Type Culture Collection (Rockville, MD) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat- inactivated fetal calf serum and antibiotics as described previously (23). The cultures were maintained at 37 °C in a humidified gaseous phase of 95% air and 5% CO 2 . Sodium arsenite (NaAsO 2 ; As(III)) was purchased from Sigma (Sigma, St. Louis, MO) and freshly prepared by dissolving it in double-distilled water. After being treated with As(III), KB cells were washed with PBS three times and subjected to survival assay or arsenic content determination * Corresponding author. E-mail: lsrong@cc.ncu.edu.tw. † Department of Life Science, National Central University & University System of Taiwan. ‡ Department of Chemical & Materials Engineering, National Central University. § Department of Chemistry, National Central University. 469 Chem. Res. Toxicol. 2006, 19, 469-474 10.1021/tx0503348 CCC: $33.50 © 2006 American Chemical Society Published on Web 02/25/2006