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

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 to the conventional enzyme digestion reactions. Also, no temperature annealing should be performed. Due to this limited proteolysis, Cys-Cys disulfide bonds will be kept intact and only surface accessible random coil segments will be cleaved by the enzyme.
5. Dissolve Au(III) chloride (chloroauric acid) in 1 mL of HPLC grade water to a concentration of 750 µM.
6. Into the reaction vial, add the chloroauric acid solution for a resulting BSA:Au molar ratio of 1:10.
7. Stir the mixture for 2 minutes at 37 °C and at 750 rpm using a magnetic stirrer.
8. Add 100 µL of 1 M NaOH to the reaction vial to achieve a pH of 12.5.
   NOTE: The high alkaline conditions of the reaction should induce the formation of the red luminescent complex and quench the enzymatic activity of trypsin.
9. Stir the mixture vigorously at 750 rpm for 2 hours at 37 °C.
   ​NOTE: The final product was used immediately without further purification.

2. Gel electrophoresis of BSA-Au complex fragments by limited proteolysis

1. Rinse a pre-cast 4-12% gradient Bis-Tris gel using deionized water and place in a gel electrophoresis tank.
2. Prepare 500 mL of MES running buffer solution from a concentrated stock solution, diluting with deionized water.
3. Prepare for each well lane by diluting samples to 1 µg of protein/µL in a 20% glycerol solution. This dilution brings the pH from 12.5 to ~8.
   NOTE: No SDS, DTT, or urea is used in the sample buffer. Additionally, temperature annealing of samples should not be performed.
4. Add 10 µL of each sample solution to each lane of the gel.
5. Run the gel for 1 hour at a constant voltage of 150 V.
6. After running the gel, remove the gel from the cast and rinse 3 times for 1 minute each using deionized water to remove running buffer.
7. Store the gel in 200 mL of deionized water and immediately measure the in-gel fluorescence, using a gel imaging system.
8. Prepare a fresh staining solution containing 200 mg of Coomassie Brilliant Blue in 200 mL of the following solution: methanol, acetic acid, and water at a volume ratio of 50:10:40.
9. Wash the gel in 200 mL of staining solution for 30 minutes using gentle rocking.
10. Prepare a fresh de-staining solution by mixing methanol, acetic acid, and water at a volume ratio of methanol:acetic acid:water = 50:10:40.
11. Wash the gel in 100 mL of de-staining solution for 1 hour using gentle stirring.
12. Repeat the above procedure 4 times and finally store the fixed gel deionized water at room temperature.

3. Analysis of BSA-Au complex fragments by limited proteolysis

1. Examine the amino acid sequence of BSA and prepare a table of expected fragments that can be obtained by enzymatic digestion, assuming Cys-Cys bond preservation (limited proteolysis). In the case of trypsin digestion (Table 1), cut locations are C-terminus of Lys and Arg, except followed by Pro. Account for the small errors in fragment molecular weights, arising from the ambiguity in tryptic cut locations.
   NOTE: Analyzing the amino acid sequence of BSA, the expected limited tryptic fragments obtained from this step are: [A] (7.3 kDa, residues 1 - 64); [B] (5.9 kDa, residues 65 - 114); [C] (20.1 ~ 22.4 kDa, residues 115/117 - 294/312); [D] (21.3 ~ 23.4 kDa, residues 295/313 - 499); and [E] (9.5 kDa, residues 500 - 583). Ambiguity in tryptic cut locations result from segments outside the Cys-connected units. For BSA, the residues 107 - 114 (0.9 kDa) and residues 295 - 312 (2.1 kDa) can appear as the N-or C-terminus part of a Cys-Cys bond-connected fragment.
2. Identify location(s) of surface-exposed Cys in these expected fragments. For trypsin-digested BSA, the only surface-exposed Cys34 is in fragment [A].
3. Prepare the list of molecular weights observed as gel electrophoresis bands, below ~66 kDa (molecular weight of BSA).
   NOTE: For trypsin digestion, the observed gel electrophoresis bands are: Band(1) = undigested BSA; Band(2) ~50 kDa; Band(3) ~44 kDa; Band(4) ~42 kDa; Band(5) ~36 kDa; Band(6) ~32 kDa; Band(7) ~26 kDa; Band(8) ~21 kDa; Band(9) ~15 kDa; Band(10) ~12 kDa; Band(11) ~10 kDa; and Band(12) ~8 kDa.
4. Reconstruct the list of the observed molecular weights in the gel, by the sequential additions of the expected BSA fragments. For trypsin, fragment [A] can form [A]-[A] dimer through the surface-exposed Cys residue.

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) ...

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

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