Limited Proteolysis and Gel Electrophoresis in the Presence of Metal Cations: Au(III)-binding Luminescent Domain in Serum Albumins

Summary

We present a protocol for studying the binding domain of Au(III) in bovine serum albumin (BSA).

Abstract

The purpose of the presented protocols is to determine the domain of Au(III) binding in BSA. The BSA-Au(III) compound exhibits ultraviolet (UV)-excitable red luminescence (λ_em = 640 nm), with unusual Stokes shifts compared to the innate UV/blue fluorescence arising from the aromatic residues. Red-luminescent complexes are formed in highly alkaline conditions above pH 10 and require a conformation change within the protein to occur. In addition, preservation of Cys-Cys disulfide bonds in BSA is necessary to obtain this red luminescence. In order to understand the mechanism of this luminescence, elucidation of the luminophore-forming Au(III) binding site is essential. A facile way to assess the luminophore-forming site would be to (1) predictably fragment the protein by enzymatic digestion, (2) react the obtained fragments with Au(III), then (3) perform gel electrophoresis to observe the well-separated fragment bands and analyze the in-gel red luminescence. However, due to the alkaline conditions and the reaction with metal cations, new limited proteolysis techniques and gel electrophoresis conditions must be applied. Particularly, the presence of metal cations in gel electrophoresis can make the band separations technically difficult. We describe this new protocol in steps to identify the red-luminophore-forming metal binding domain in BSA. This protocol can thus be applied for analyzing protein fragments that must remain in a non-denatured or a partially denatured state, in the presence of metal cations. Because the majority of proteins need metal cations to function, analyses of metal-bound proteins are often desired, which have relied on x-ray crystallography in the literature. This method, on the other hand, could be used in supplement to study the interactions of proteins with metal cations without requiring the protein crystallization and at a desired pH condition.

Introduction

Bovine serum albumin^1,2,3 (BSA)-gold (Au) complexes, obtained by reactions in highly alkaline conditions (pH > 10), are known to exhibit UV-excitable red luminescence (λ_em = 640 nm)^4,5,6,7. Numerous applications of this compound has been proposed and investigated, including sensing,^8,9,10 imaging^11,<....

Protocol

1. Synthesis of BSA-Au complex fragments

1. Dissolve 5 mg of BSA in 1 mL of HPLC grade water containing 50 mM Tris-HCl and 50 mM NaCl with a pH of 8.0 in a 5 mL vial.
2. Dissolve 2 mg of trypsin in 1 mL of a freshly prepared solution of HPLC water containing 50 mM Tris-HCl and 50 mM NaCl with a pH of 8.0.
3. Place the reaction vial of BSA in a 37 °C water bath and stir vigorously at 750 rpm using a magnetic stirrer.
4. Immediately after stirring begins, add 50 µL of the freshly prepared trypsin to the solution.
   NOTE: No sodium dodecyl sulfate (SDS), dithiothreitol (DDT), or urea should be added to the solution, as opposed....

Results

The observed twelve gel bands were uniquely reconstructed from the five expected BSA fragments [A] - [E] (Figure 1). The results were consistent with the literature, in which the secondary structures including α-helices and β-strands are preserved^{19,20,21,22,23}. Band(1) = [ABC.......

Discussion

The purpose of the present protocol was to identify the red-luminophore-forming domain in BSA-Au complexes. We employed limited tryptic proteolysis to obtain the BSA fragments, while preserving the Cys-Cys bonds that are necessary to produce the red luminescence. We optimized the conditions for proteolysis and electrophoresis in the presence of Au(III). The same principles can be broadly applied to the gel analyses of fragmented proteins in the presence of metal cations.

We performed multiple .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ammonium bicarbonate, 99.5%	Sigma-Aldrich	9830
Azure Biosystems C400 gel imaging system	Azure Biosystems	C400
Bovine Serum Albumin (BSA), 96%	Sigma-Aldrich	A5611
Glycerol, >99.0%	Sigma-Aldrich	G5516
gold (III) chloride trihydrate, 99.9%	Sigma-Aldrich	520918
NuPAGE 4-12% Bis-Tris Mini Protein Gel	Thermo Fisher	NP0321BOX
NuPAGE MES Running Buffer (20X)	Thermo Fisher	NP0002
Sodium Chloride (NaCl), >99.5%	Sigma-Aldrich	S7653
Sodium hydroxide, >98.0%	Sigma-Aldrich	S8045
Tris Hydrochloride (Tris-HCl)	Sigma-Aldrich	10812846001
Trypsin from Bovine Pancreas (>10,000 BAEE units/mg)	Sigma-Aldrich	T1426