Investigation of binding behavior of important metal ions to thioredoxin reductase using mobility-shift affinity capillary electrophoresis: A preliminary insight into the development of new metal-based anticancer drugs

Thioredoxin reductase (TrxR) is a redox enzyme that is found to be elevated in a variety of tumors and its inhibition has been proved to be a new approach for the treatment of cancer. In this study, the binding behaviors of some metal ions on TrxR have been evaluated using mobility-shift affinity electrophoresis. Eighteen medicinally important metal ions from soft, borderline and hard categories were selected for the screening. This study was based on the fact that, overall mass, charge and size of a protein changes after interaction with charged ligands such as metal ions, which affects the electrophoretic movement of the analyte protein under the influence of electroosmotic flow. This alteration in electrophoretic mobility of the analyte is proportional to the strength of interaction between the two species and is utilized to calculate the binding results. The mobility ratio (R) of an EOF marker (injected in same run) and the analyte enzyme was determined and the binding result was expressed as the normalized difference of the mobility ratios of test enzyme with and without metal ion (Delta R/R-f) and its confidence interval. The method showed excellent precision, as the % RSD of mobility ratios of replicate runs (n = 6) for most of the enzyme-metal ion pair was found to be < 1%. Six out of eighteen tested metal ions including Ir3+, Ag +, Os3+, Cr3+, Fe2+ and Rh3+ showed significant binding interactions (Delta R/R-f >= +/- 0.01), while for others, it was found to be weak or insignificant. Among all the tested metal ions, Ir3+ (Delta R/R-f = -0.27065 +/- 0.00818) showed the strongest interaction followed by Ag+ (Delta R/R-f = -0.09821 +/- 0.00783), Fe2+ (Delta R/R-f = -0.0482 +/- 0.00701), Os3+ (Delta R/R-f = -0.04382 +/- 0.00751), Cr2+ (Delta R/R-f = -0.04093 +/- 0.01206) and Rh3+ (Delta R/R-f = -0.01455 +/- 0.00857). The values of Delta R/R-f obtained for most of the enzyme-metal ion pair were negative, suggesting the multifaceted complexation behavior of metal ions, as they get coordinated with surrounding anions after initial binding with the enzyme. As a result, the overall charge on the enzyme becomes more negative. Significant interactions of the enzyme with some metal ions suggested that the drugs based on these metal ions may prove to be potential inhibitor of the enzyme, however, it would be interesting to evaluate that how these interactions would affect the function and stability of the enzyme.