Identifying the Red-Luminophore-Forming Domain in Serum Albumin–Gold Complexes

Abstract

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.

Advancement of Fluorescent and Structural Properties of Bovine Serum Albumin-Gold Bioconjugates in Normal and Heavy Water with pH Conditioning and Ageing

“…The same authors concluded that cysteine, 34 of which form disulfide bonds in BSA, is the binding site of Au(III) but not the location of the red-emitting fluorophore. Recently, the same research group identified the Au(III) binding domain of BSA and localized the origin of red fluorescence within the N-terminal domain using limited proteolysis and molecular cloning techniques based on luminescence measurements [ 11 ]. However, the changes and evolution in fluorescent properties are primarily connected to global changes in BSA–gold complexes dependent on the pH value and accompanied by significant structural changes on the atomic level as observed by small-angle X-ray scattering and infrared spectroscopy [ 12 ].…”

Common Motif at the Red Luminophore in Bovine Serum Albumin–, Ovalbumin–, Trypsin–, and Insulin–Gold Complexes

“…7 The domain of this Au(III) binding was recently identified. 8 Other than for bovine and human serum albumins, 8 the red luminescence has been reported for ovalbumin, 9 trypsin, 10 insulin, 11 lactotransferrin, 12 pepsin, 13 horseradish peroxidase, 14 papain, 15 and lysozyme 16 (Supporting Information, Figure S1). For example, BSA is a 66.4 kDa transport protein; ovalbumin (OVA) is a 42.7 kDa protein constituting most of egg white; trypsin is a 23.3 kDa protease; insulin is a 5.7 kDa hormone (Figure S2).…”

“…For BSA, we analyzed within the previously identified Au(III)-binding domain. 8 We expanded the analysis to all proteins in the literature for the red luminescence, 12−16 as well as to proteins (α-2-macroglobulin and trypsin inhibitor) that did not yield the red luminescence by the Standard protocol (Figure S14).…”

“…Many applications of the BSA–Au compound exists, including sensing, imaging, and nanomedicine. − Two possible mechanisms of this luminescence have been suggested: One scenario assumes BSA as a “static cage”, in which Au­(III) are reduced by Tyr residues (p K a = 10.5) and nucleate into a neutral Au 25 nanocluster; another scenario embraces the dynamic character of BSA. , For the latter, it was shown that Langmuir-type chemisorption of a Au­(III) at a Cys–Cys disulfide bond site, but not nucleation of Au(0), reproduces the kinetics of the red luminophore formation . The domain of this Au­(III) binding was recently identified …”

“…For BSA–Au­(III) complex, our prior work − illustrated that Cys is necessary for the formation of red luminophore. The same result was obtained for OVA, trypsin, and insulin (Figure S5).…”