We report new findings on the red fluorescent (λ = 640 nm) bovine serum albumin (BSA)-gold (Au) compound initially described by Xie et al. (J. Am. Chem. Soc. 2009, 131, 888-889) as Au nanoclusters. The BSA-Au compounds were further reducible to yield nanoparticles, suggesting that these compounds were BSA-cationic Au complexes. We examined the correlations between BSA conformations (pH-induced as well as denatured) and the resulting fluorescence of BSA-Au complexes, to understand the possible cationic Au binding sites. The red fluorescence of the BSA-Au complex was associated with a particular isoform of BSA, the aged form (pH > 10) of the five pH-dependent BSA conformations, while the other conformations, expanded (pH < 2.7), fast (2.7 < pH < 4.3), normal (4.3 < pH < 8), and basic (8 < pH < 10) did not result in red fluorescence. There could be internal energy transfer mechanisms to produce red fluorescence, deduced from excitation-emission map measurements. The ensemble minimum number of Au(III) per BSA to yield red fluorescence was <7. We illustrate the presence of multiple specific Au binding sites in BSA, and present an interpretation of the fluorescence of the BSA-Au complex, alternative to a single-site nucleation of a neutral Au nanocluster.

We revisit the prevailing hypothesis that the red fluorophore (λem = 640 nm) in the bovine serum albumin (BSA)–gold (Au) compound is a Au25 nanocluster. To examine the hypothesis, we investigated the kinetics of Au binding in this compound. In addition to the specific Au­(III) binding sites in BSA, we found a significant degree of nonspecific Au­(III) binding on the BSA surface. Time-course of the emergence of the red fluorescence was measured in detail for a range of pH, temperature, and concentration of Au­(III) with respect to BSA. The red fluorophore formation was a slow yet dynamic process, which was consistent with the pH-induced equilibrium transition in the conformation of BSA. Notably, the kinetic rate of the fluorophore formation was not strongly dependent on the concentration of Au­(III). Incorporating the existence of multiple specific and nonspecific binding sites, we propose a new model of the red fluorophore formation mechanism based on Langmuir-type adsorption of Au to BSA, as an alternative to the single-site nucleation model of Au25 nanoclusters.

We report a valence state-controlled synthesis of vanadium oxide nanocrystalline particles via a non-hydrothermal process using an alcohol and an amine ligand. A non-stoichiometric V3O5 (V4+V3+ 2O5), which is known for its difficulty of formation due to the narrow allowances in the vanadium-to-oxygen ratio, was obtained for the first time as nanocrystals in the anosovite phasea rare phase discovered only recently in bulk form. The time course of the nanocrystal formation revealed a slow seeded growth process, separated from a subsequent fast growth via Ostwald ripening. We highlight the role of vanadium precursor-to-alcohol-to-ligand ratios in precisely controlling the reduction of V5+. Polyvalence of metals, particularly the unusual stability of vanadium(II)–(V), has been considered a negative factor in achieving the targeted oxidation state in nanocrystal syntheses. In the present system, the polyvalence allowed formations of different oxide nanocrystals in a parameter-controlled manner, including anosovite V3O5 (V4+ + 2V3+) and corundum V2O3 (V3+). Such control is unprecedented in metal oxide nanocrystal syntheses.

Serum albumin–gold complexes exhibit UV-excitable red luminescence (λem = 640 nm) with unusual Stokes shifts compared with the innate UV/blue fluorescence arising from the aromatic residues. In order to understand the mechanism of this luminescence, we employed limited proteolysis and molecular cloning techniques and assessed the domain containing the red luminophore in bovine serum albumin (BSA) and human serum albumin (HSA). We identified that the luminophore is localized in a domain of serum albumin, residing within the N-terminus half.

We examined the static and dynamic characters of the red luminescence in the protein–Au­(III) compounds, directly comparing multiple proteins: BSA, OVA, trypsin, and insulin. These four protein–Au­(III) complexes showed a nearly identical excitation–emission pattern, not only the wavelength of luminescence (λem ∼ 640 nm). Lifetimes of the red luminescence shared a common value of ∼300 ns. Kinetics of the luminophore formation was consistently described by a Langmuir-type chemisorption of Au­(III) for these proteins, coinciding with the protein conformation change at pH ∼ 10. These observations and the protein structural analyses support that the red luminophore formation involves Au­(III) coordination to a common motif within these proteins.

The purpose of the presented protocols is to study the process of Au(III) binding to BSA, yielding conformation change-induced red fluorescence (λem = 640 nm) of BSA-Au(III) complexes. The method adjusts the pH to show that the emergence of the red fluorescence is correlated with the pH-induced equilibrium transitions of the BSA conformations. Red fluorescent BSA-Au(III) complexes can only be formed with an adjustment of pH at or above 9.7, which corresponds to the "A-form" conformation of BSA. The protocol to adjust the BSA to Au molar ratio and to monitor the time-course of the process of Au(III) binding is described. The minimum number of Au(III) per BSA, to produce the red fluorescence, is less than seven. We describe the protocol in steps to illustrate the presence of multiple Au(III) binding sites in BSA. First, by adding copper (Cu(II)) or nickel (Ni(II)) cations followed by Au(III), this method reveals a binding site for Au(III) that is not the red fluorophore. Second, by modifying BSA by thiol capping agents, another nonfluorophore-forming Au(III) binding site is revealed. Third, changing the BSA conformation by cleaving and capping of the disulfide bonds, the possible Au(III) binding site(s) are illustrated. The protocol described, to control the BSA conformations and Au(III) binding, can be generally applied to study the interactions of other proteins and metal cations.

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