Name: Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of Gold(III) (Video) | JoVE Journal
Uploaded: 2018-08-31T15:00:00.000+00:00
Duration: 8 min 26 s
Description: Discover how Au(III) binding induces red fluorescence in bovine serum albumin through pH-dependent conformational changes. This method reveals specific metal binding sites and protein interactions.

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S.E. acknowledges the support from Duke Endowment Special Initiative Fund, Wells Fargo Fund, PhRMA Foundation, as well as Startup Funds from the University of North Carolina, Charlotte.

1. Synthesis of BSA-Au(III) Complex
<ol>
	<li>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.</li>
	<li>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.
	<ol>
		<li>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 ratios of BSA to gold will result in drastically different red fluorescence patterns of the excitation-emission map.</li>
	</ol>
	</li>
	<li>Place the reaction vial of BSA in a 37 &#176;C water bath and vigorously stir at 750 rpm using a magnetic stirrer.</li>
	<li>Immediately after the stirring begins, add 1 mL of chloroauric acid to the solution. The color of the solution should transform from clear to yellow.</li>
	<li>Stir the mixture for 2 min at 37 &#176;C and at 750 rpm using a magnetic stirrer.</li>
	<li>Into the reaction vial, add 100 &#956;L of 1 M NaOH to the solution to bring the pH to 12. 
	NOTE: Immediately after NaOH is added, the solution should darken slightly to a yellow-brown and then turn back to yellow.</li>
	<li>Continue to stir at 750 rpm for 2 h and at 37 &#176;C. The solution should slowly change from yellow to a dark yellow/brown color. This color change indicates the formation of the red fluorescing BSA-Au(III) complex.</li>
	<li>Allow the sample to sit at room temperature for 2 days, and the solution will continue to darken to an amber brown and the fluorescence intensity will increase.
	<ol>
		<li>Alternatively, let the sample continue to stir at 37 &#176;C for 12 more h as the color of the solution evolves to an amber brown.</li>
	</ol>
	</li>
</ol>
2. Synthesis of BSA-Cu(II)-Au(III)
<ol>
	<li>Dissolve 25 mg of BSA in 1 mL of HPLC grade water. The solution should appear clear.</li>
	<li>Dissolve copper (II) chloride dihydrate in HPLC grade water to a concentration of 5 mM. The solution should appear light blue.</li>
	<li>Add 1 mL of the aqueous BSA solution to a 5 mL reaction vial and place in a water bath at 37 &#176;C. Stir the mixture at 750 rpm.</li>
	<li>Immediately add 0.5 mL of the copper (II) chloride dihydrate solution to the reaction vial and mix for 2 min. The solution will remain light blue.</li>
	<li>Add 75 &#956;L of 1 M NaOH to bring the pH to 12 and allow to mix for 2 h. The solution will become purple.</li>
	<li>Dissolve chloroauric acid in HPLC grade water to a concentration of 5 mM.</li>
	<li>Add 0.5 mL of aqueous chloroauric acid to the reaction vial and adjust the pH back to 12 using 1 M NaOH.</li>
	<li>Stir the reaction mixture for 2 h. 
	NOTE: The solution should evolve to a brown color.</li>
</ol>
3. Synthesis of BSA-Ni(II)-Au(III)
<ol>
	<li>Dissolve 25 mg of BSA in 1 mL of HPLC grade water. The solution should appear clear.</li>
	<li>Dissolve nickel (II) chloride hexahydrate in HPLC grade water to a concentration of 5 mM. The solution should appear light green.</li>
	<li>Add 1 mL of the aqueous BSA solution to a 5 mL reaction vial and place in a water bath at 37 oC. Stir the mixture at 750 rpm.</li>
	<li>Immediately add 0.5 mL of the nickel (II) chloride hexahydrate solution to the reaction vial and mix for 2 min. 
	NOTE: The solution will remain light green.</li>
	<li>Add 75 &#956;L of 1 M NaOH to bring the pH to 12 and allow to mix for 2 h. 
	NOTE: The solution will become dark yellow.</li>
	<li>Dissolve chloroauric acid in HPLC grade water to a concentration of 5 mM.</li>
	<li>Add 0.5 mL of the aqueous chloroauric acid to the reaction vial and adjust the pH back to 12.</li>
	<li>Stir the reaction mixture for 2 h. 
	NOTE: The solution should evolve to a brown color.</li>
</ol>
4. Synthesis of [Cys34-capped-BSA]-Au(III)
<ol>
	<li>Dissolve 2 mg of N-ethylmaleimide (NEM) in 1 mL of phosphate buffered saline (PBS, pH 7.4).</li>
	<li>Dissolve 2 mg of BSA in 1 mL of PBS-NEM solution.</li>
	<li>Transfer the solution to a 5 mL reaction vial and stir at 20 &#176;C at 500 rpm for 1 h.</li>
	<li>Dialyze the solution using 12 kDa dialysis tubing in 500 mL of PBS, stirring at 50 rpm with a magnetic stirrer overnight to remove unreacted NEM.</li>
	<li>Dissolve chloroauric acid in PBS to a concentration of 0.4 mM. 
	NOTE: The solution will be a faint yellow.</li>
	<li>Transfer the reaction vial to a water bath at 37 &#176;C. Stir at 750 rpm.</li>
	<li>Immediately add 1 mL of chloroauric acid solution to the reaction vial and allow to mix for 2 min.</li>
	<li>Add 75 &#956;L of 1 M NaOH to the reaction vial to bring the pH to 12 and allow to mix for 2 h.</li>
</ol>
5. Synthesis of [all-thiol-capped-BSA]-Au(III)
<ol>
	<li>Prepare a solution of 2 M urea and 50 mM ammonium bicarbonate (NH4HCO3, pH 8.0) in HPLC grade water.</li>
	<li>Dissolve 3.3 mg of BSA in 1 mL of the above solution and transfer to a 5 mL reaction vial.</li>
	<li>Make a stock solution of 0.25 M tris(2-carboxyethyl)phosphine (TCEP) by dissolving 62.5 mg of TCEP in 1 mL of HPLC water.</li>
	<li>Add the stock solution of TCEP to the reaction vial until the final concentration of TCEP is 8 mM.</li>
	<li>Incubate the solution in a water bath for 1 h at 50 &#176;C. Stir at 500 rpm using a magnetic stirrer.</li>
	<li>Allow the solution to completely cool to room temperature.</li>
	<li>Prepare a stock solution of 100 mM NEM by dissolving 12.5 mg of NEM in 1 mL of HPLC grade water.</li>
	<li>Add the stock solution of NEM to the reaction vial until the final concentration of NEM is 16 mM.</li>
	<li>Allow the solution to combine for 2 h at 20 &#176;C. Stir at 500 rpm.</li>
	<li>Dialyze the solution using a 12 kDa dialysis tubing, stirring the solution at 50 rpm with a magnetic stirrer overnight in 500 mL of 50 mM NH4HCO3 to remove excess TCEP, NEM, and urea.</li>
	<li>Move the reaction vial to a water bath at 37 &#176;C and stir at 750 rpm.</li>
	<li>Dissolve chloroauric acid in HPLC grade water to a concentration of 0.66 mM.</li>
	<li>Immediately add 1 mL of chloroauric acid solution to the reaction vial and allow to mix for 2 min.</li>
	<li>Add 1 M NaOH until the pH of the solution is 12 and allow the solution to continue to mix for 2 h.</li>
</ol>

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The authors have nothing to disclose.

The BSA-Au(III) compounds prepared at pH 12 exhibit red fluorescence at an emission wavelength of &#955;em= 640 nm when excited with ultraviolet (UV) light &#955;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 &#176;C will yield the optimum results, though higher temperature can be used to produce the red fluorescence faster. I...

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 sensing2,3,4, imaging5,6,7, or nanomedicine8,9,10,11,12,13. This compound has been studied extensively by many researchers in the field of nano-science in recent years14,15,16. The BSA-Au compound has been interpreted as Au25 nanoclusters. The goal of the presented method is to examine this compound in detail and to understand the origin of the red fluorescence. By following the presented approach, the presence of multiple Au binding sites, and the origin of fluorescence, alternative to the single-site nucleation of Au25 nanoclusters, can be illustrated. The same approach can be employed to study how other proteins17,18,19 complexed with Au(III) can change their intrinsic fluorescent properties.
The synthesis of the red-fluorescent BSA-Au compound requires a narrow control of the molar ratios of BSA to Au (BSA:Au) to maximize the intensity of the fluorescence and the location of the peaks in the excitation-emission map (EEM)20. It can be shown that multiple binding sites exist for Au(III) to bind, including the Asparagine fragment (or Asp fragment, the first four amino acid residues at the N-terminus of BSA)21,22. The 34th amino acid of BSA (Cys-34) is also shown to coordinate Au(III) and to be involved in the mechanism of the red fluorescence([Cys34-capped-BSA]-Au(III))20. Upon cleaving all Cys-Cys disulfide bonds and capping all thiols, red fluorescence is not produced ([all-thiol-capped-BSA]-Au(III)). This indicates the necessity of Cys-Cys disulfide bonds as the Au(III) binding site to produce the red fluorescence.
Protein chemistry techniques have not been widely used to study the BSA-Au(III) complexes in the nano-science community. However, it would be valuable to employ these techniques to understand certain aspects of these complexes, as well as to gain detailed understanding of the Au(III) binding sites in BSA. This article is intended to show some of these techniques.

<table><tbody><tr><td>Bovine Serum Albumin (BSA), 96%</td><td>Sigma-Aldrich</td><td>A5611</td><td></td></tr><tr><td>gold (III) chloride trihydrate, 99.9%</td><td>Sigma-Aldrich</td><td>520918</td><td></td></tr><tr><td>Copper (II) chloride dihydrate, 99.999%</td><td>Sigma-Aldrich</td><td>459097</td><td></td></tr><tr><td>Nickel (II) chloride hexahydrate, 99.9%</td><td>Sigma-Aldrich</td><td>654507</td><td></td></tr><tr><td>N-Ethylmaleimide (NEM), &gt;99.0%</td><td>Sigma-Aldrich</td><td>4259</td><td></td></tr><tr><td>Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), &gt;98.0%</td><td>Sigma-Aldrich</td><td>C4706</td><td></td></tr><tr><td>Sodium hydroxide, &gt;98.0%</td><td>Sigma-Aldrich</td><td>S8045</td><td></td></tr><tr><td>Urea, 99.5%</td><td>Chem-Implex Int&#039;l</td><td>30142</td><td></td></tr><tr><td>Phospate buffered saline (PBS)</td><td>Corning</td><td>MT21040CV</td><td></td></tr><tr><td>Ammonium bicarbonate, 99.5%</td><td>Sigma-Aldrich</td><td>9830</td><td></td></tr></tbody></table>

luminophore formation in various conformations of bovine serum albumin by binding of gold(iii)

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

The purpose of the presented protocols is to study the process of Au(III) binding to BSA, yielding conformation change-induced red fluorescence (&#955;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 &#34;A-form&#34; 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.

From the fluorescence of the BSA-Au(III) complex, it has been observed that the conversion of the intrinsic blue fluorescence of BSA (λem = 400 nm) to red fluorescence (λem = 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...

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.

The purpose of the presented protocols is to study the process of Au(III) binding to BSA, yielding conformation change-induced red fluorescence (&#955;em ...

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In this video, we demonstrate gold immunostaining of biomarkers localized within the lumen region of ultrathin exosomal sections. This technique enables early diagnosis of diseases by visualization of target molecules within the exosomes secreted by the diseased cells.

Gold Immunostaining of Exosomal Sections: A Technique to Visualize Diagnostic Markers in Exosomal Lumen

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

To obtain basic information on the sorption and recycling of gold from aqueous systems the interaction of Au(III) and Au(0) nanoparticles on S-layer proteins were investigated. The sorption of protein polymers was investigated by ICP-MS and that of proteinaceous monolayers by QCM-D. Subsequent AFM enables the imaging of the nanostructures.

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

Nanoparticle-based optical probes have been designed as a vehicle for detecting antigens using Raman and UV-Vis spectroscopy. Here we describe a protocol for preparing such probes for a UV-Vis/Raman spectroscopy immunoassay in such a way to incorporate future multiplexing capabilities.

Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

A reliable and easily reproducible method for preparation of functionalizable, near-infrared emitting photoluminescent gold nanoclusters and their direct detection inside HeLa cells by flow cytometry and confocal laser scanning microscopy is described.

Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications

Here, we present a systematic method describing the quasi-reversible self-assembly of the amyloid beta 1-40 (Aβ1-40)-coated 20 nm gold aggregates. The nano-size-dependent quasi-reversible networking between peptides was correlated with specific amino acids or sections of the Aβ1-40 monomer.

Characterization of pH-Dependent Reversible Self-Assembly of Amyloid Beta 1-40-Coated Gold Colloids

Ice binding proteins (IBPs), also known as antifreeze proteins, inhibit ice growth and are a promising additive for use in the cryopreservation of tissues. The main tool used to investigate IBPs is the nanoliter osmometer. We developed a home-designed cooling stage mounted on an optical microscope and controlled using a custom-built LabVIEW routine. The nanoliter osmometer described here manipulated the sample temperature in an ultra-sensitive manner.

LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

Discover how Au(III) binding induces red fluorescence in bovine serum albumin through pH-dependent conformational changes. This method reveals specific metal binding sites and protein interactions.

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

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