Unravelling mechanism of tetrathiomolybdate (TTM) mediated copper sequestration and copper-TTM complex formation in bovine serum albumin.

In pathological conditions, elevated Cu levels in serum albumin are treated with the potent copper chelator, ammonium tetrathiomolybdate (ATTM, [NH4]2[MoS4]), however, the mechanism of Cu sequestration and Cu/TTM complex formation remains unclear. Here, bovine serum albumin (BSA) containing ATCUN motif (Amino terminal copper nickel binding), drug-binding sites (Sites I-III), and fluorescence probes, (Trp-213/Trp-134) was used as a model to investigate potential interaction sites of Cu and TTM, and to examine the effect of mixing order among BSA, Cu, and TTM on Cu sequestration and Cu/TTM complex formation. UV/vis spectra showed decreased peak intensity with a shift from 465 nm to 500 nm, indicating Cu/TTM complex formation. The transformation was rapid when TTM was added first, but relatively slower when Cu(II) was pre-bound to the ATCUN site of BSA. In contrast, the spectrum of preformed Cu/TTM remained unchanged in the presence of BSA. After desalting, in-situ formed Cu/TTM complex remained associated with BSA, whereas the preformed Cu/TTM complex did not, indicating weak binding with BSA. Emission quenching and wavelength shifting identified potential binding sites for Cu(II), TTM, and Cu/TTM complex. EPR spectroscopy showed that Cu(II) signal of Cu(II)-ATCUN-BSA was partially reduced upon TTM addition, despite the high affinity, but the Cu(II) signal was more reduced when TTM was added before Cu(II). TTM efficiently extracted Cu from overloaded Cu(II)-BSA, as evidenced by distinct UV/vis and EPR signatures.