Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of Gold(III)

Summary

The protocols for studying the binding of gold cations (Au(III)) to various conformations of bovine serum albumin (BSA) as well as for characterizing the conformational dependent unique BSA-Au fluorescence are presented.

Abstract

The purpose of the presented protocols is to study the process of Au(III) binding to BSA, yielding conformation change-induced red fluorescence (λ_em = 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.

Introduction

A BSA-Au compound exhibiting an ultraviolet (UV)-excitable red fluorescence, with remarkable stokes shift, has been originally synthesized by Xie et al.¹. The unique and stable red fluorescence can find various applications in fields such as sensing^2,3,4, imaging^5,6,7, or nanomedicine^{8,9,10,11,12....}

Protocol

1. Synthesis of BSA-Au(III) Complex

1. Dissolve 25 mg of BSA in 1 mL of high-performance liquid chromatography (HPLC) grade water in a 5 mL reaction vial.
   NOTE: The solution should appear clear.
2. Dissolve gold (III) chloride trihydrate (chloroauric acid) to a concentration of 5 mM in HPLC grade water.
   NOTE: The solution should appear yellow. Chloroauric acid solution prepared at this concentration will result in a BSA to Au ratio of 1:13.
   1. Alternatively, prepare a solution of chloroauric acid with a concentration of anywhere between 0.38 mM (BSA:Au = 1:1) to 20 mM (BSA:Au = 1 : 50) in HPLC grade water.
      NOTE: Different....

Results

From the fluorescence of the BSA-Au(III) complex, it has been observed that the conversion of the intrinsic blue fluorescence of BSA (λ_em = 400 nm) to red fluorescence (λ_em = 640 nm) occurs at about pH 9.7 through an equilibrium transition (Figure 1). EEM of BSA-Au(III) at different BSA to Au molar ratios is shown in Figure 2, and this data shows how altering the molar ratios yields the sam.......

Discussion

The BSA-Au(III) compounds prepared at pH 12 exhibit red fluorescence at an emission wavelength of λ_em= 640 nm when excited with ultraviolet (UV) light λ_ex= 365 nm (Figure 1A). The emergence of red fluorescence is a slow process and will take a few days at room temperature to increase to a maximum intensity. Running the reaction at 37 °C will yield the optimum results, though higher temperature can be used to produce the red fluorescence faster. I.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bovine Serum Albumin (BSA), 96%	Sigma-Aldrich	A5611
gold (III) chloride trihydrate, 99.9%	Sigma-Aldrich	520918
Copper (II) chloride dihydrate, 99.999%	Sigma-Aldrich	459097
Nickel (II) chloride hexahydrate, 99.9%	Sigma-Aldrich	654507
N-Ethylmaleimide (NEM), >99.0%	Sigma-Aldrich	4259
Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), >98.0%	Sigma-Aldrich	C4706
Sodium hydroxide, >98.0%	Sigma-Aldrich	S8045
Urea, 99.5%	Chem-Implex Int'l	30142
Phospate buffered saline (PBS)	Corning	MT21040CV
Ammonium bicarbonate, 99.5%	Sigma-Aldrich	9830