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^11,12,13, and nanomedicine^14,15,16. However, the mechanism of the luminescence is not fully understood. Identifying the location of Au(III) binding and the luminophore formation in BSA is an important step.

It has been recently elucidated that pH-controlled dynamic conformation change of BSA, followed by a Au(III) binding to a Cys-Cys disulfide bond, is necessary for yielding the red luminescence⁴. In order to gain further insights into 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 is to fragment the BSA-Au compound by enzymatic digestion, and to analyze each fragment for the luminescence. However, due to the alkaline conditions and the presence of metal cations, new proteolysis and gel electrophoresis protocols are needed.

We employed limited enzymatic proteolysis as the method of protein fragment preparations, while preserving the Cys-Cys disulfide bonds. In the conventional proteolysis, cleaving of all disulfide bonds and linearization of a protein (by denaturing agents such as dithiothreitol and urea, as well as heat) is necessary. Herein, we demonstrate a Cys-Cys bond-preserving proteolysis and evaluate the obtained fragments and their luminescence after the reaction with Au(III). We use trypsin for the digestive enzyme, as a concrete example.

The protocol generally describes the gel electrophoresis of proteins and fragments in the presence of metal cations. Because the majority of proteins need metal cations to function^17,18, analyses of metal-bound proteins are often desired, which have relied on x-ray crystallography in the literature. Structures of BSA, and their fragments, are not known for non-neutral pH conformations including at pH > 10. Therefore, the structural details of the Au(III) coordination cannot be analyzed by gel electrophoresis alone. 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, which may not be possible at a desired functional pH condition. The presence of metal cations can cause significant "smearing" of the gel bands. The focus of this paper is to overcome this technical difficulty and to present a protocol to minimize the metal-induced band smearing.

Protocol

1. Synthesis of BSA-Au complex fragments 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. 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. Place the reaction vial of BSA in a 37 °C water bath and stir vigorously at 750 rpm using a magnetic stirrer. Immediately after stirring begins, add 50 µL of the freshly pre…

Representative 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 preserved19,20,21,22,23. Band(1) …

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

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