Name: luminophore formation in various conformations of bovine serum albumin by binding of gold(iii)
Uploaded: 2018-08-31T15:00:00
Description: 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 ...

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 ch...

<ol>
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A Comparative Study of Dog, Bovine, and Human Albumin.</a> Journal of Biological Chemistry. 269, 25557-25561 (1994).</li><li>Takeda, K., Wada, A., Yamamoto, K., Moriyama, Y., Aoki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+Change+of+Bovine+Serum+Albumin+by+Heat+Treatment.">Conformational Change of Bovine Serum Albumin by Heat Treatment.</a> Journal of Protein Chemistry. 8 (5), 653-659 (1989).</li><li>Klotz, I. M., Curme, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Thermodynamics+of+Metallo-protein+Combinations.+Copper+with+Bovine+Serum+Albumin.">The Thermodynamics of Metallo-protein Combinations. Copper with Bovine Serum Albumin.</a> Journal of the American Chemical Society. 70, 939-943 (1948).</li><li>Fiess, H. A., Klotz, I. 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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), &#62;99.0%</td>
			<td>Sigma-Aldrich</td>
			<td>4259</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), &#62;98.0%</td>
			<td>Sigma-Aldrich</td>
			<td>C4706</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide, &#62;98.0%</td>
			<td>Sigma-Aldrich</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea, 99.5%</td>
			<td>Chem-Implex Int&#39;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)

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 (&#955;em = 400 nm) to red fluorescence (&#955;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 yiel...

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.

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 ...

This method can help answer key questions in the nanoscience field, such as fluorophore formation and middle protein complexes. The main advantage of this technique is that it helps to illustrate specific metal binding sites and proteins. First, dissolve 25 milligrams of BSA in one milliliter of HPLC grade water in a five milliliter reaction vial containing a magnetic stir bar.Next, prepare a five millimolar solution of chloroauric acid and HPLC grade water. Place the reaction vial containing the BSA solution in a 37 degree Celsius water bath and stir at 750 RPM using a magnetic stirrer. Immediately after the stirring begins, add one milliliter of the chloroauric acid solution to the BSA solution.After stirring for two minutes, add 100 microliters of one molar sodium hydroxide to the solution to bring the PH to 12. Dissolve 25 milligrams of BSA in one milliliter of HPLC grade water in a reaction vial. Next, prepare a five millimolar solution of copper chloride dihydrate in HPLC grade water.Add one milliliter of the BSA solution to a five milliliter reaction vial containing a magnetic stir bar, and place it in a water bath at 37 degrees Celsius. Stir the solution at 750 RPM. Immediately add 0.5 milliliters of the copper chloride dihydrate solution to the reaction vial.After stirring for two minutes, add 75 microliters of one molar sodium hydroxide to the solution to bring the PH to 12 and stir for two hours. Following this, prepare a five millimolar solution of chloroauric acid in HPLC grade water. Add 0.5 milliliters of the chloroauric acid solution to the reaction vial.Then, adjust the PH to 12 using one molar sodium hydroxide and stir the solution for two hours. Add one milliliter of BSA solution to a five milliliter reaction vial containing a magnetic stir bar and place it in a water bath at 37 degrees Celsius. Stir the solution at 750 RPM.Immediately add 0.5 milliliters of a previously prepared five millimolar nickel chloride hexahydrate solution to the reaction vial. After stirring for two minutes, add 75 microliters of one molar sodium hydroxide to bring the PH to 12 and stir for two hours. Add 0.5 milliliters of five millimolar chloroauric acid solution to the reaction vial.Then, adjust the PH to 12 using one molar sodium hydroxide and stir the reaction mixture for two hours. Dissolve two milligrams of N-Ethylmaleimide in one milliliter of PBS. Then, dissolve two milligrams of BSA in one milliliter of the maleimide solution.Transfer the solution to a five milliliter reaction vial containing a magnetic stir bar and stir at 20 degree Celsius and 500 RPM for one hour. After one hour, dialyze the solution using 12 kilodalton dialysis tubing in 500 milliliters of PBS. Stirring at 50 RPM with a magnetic stirrer overnight to remove unreacted and ethylmaleimide.On the following day, transfer the reaction vial containing the maleimide solution to a water bath at 37 degrees Celsius and stir at 750 RPM. Immediately, add a previously prepared 0.4 millimolar chloroauric acid solution to the reaction vial. After stirring for two minutes, add 75 microliters of one molar sodium hydroxide to the solution to bring the PH to 12 and stir for two hours.Prepare a solution of two molar urea and 50 millimolar ammonium bicarbonate in HPLC grade water. Then, dissolve 3.3 milligrams of BSA in one milliliter of the urea solution and transfer to a five milliliter reaction vial containing a magnetic stir bar. Next, prepare a stock solution of 0.25 molar TCEP by dissolving 62.5 milligrams of TCEP and one milliliter of HPLC grade water.Add the stock solution to the reaction vial until the final concentration of TCEP is eight millimolar. Then, incubate the solution in a water bath for one hour at 50 degree Celsius with stirring at 500 RPM. In the meantime, prepare a 100 nanomolar stock solution of N-ethylmaleimide by dissolving 12.5 milligrams of N-ethylmaleimide in one milliliter of HPLC grade water.Add the stock solution to the cooled reaction vial until the final concentration of N-ethylmaleimide is 16 millimolar. Then, stir at 20 degrees Celsius and 500 RPM for two hours. Dialyze the solution using 12 kelodalton dialysis tubing in 500 milliliter of 50 millimolar ammonium bicarbonate, stirring at 50 RPM with a magnetic stirrer overnight to remove excess reagents.On the following day, transfer the reaction vial to a water bath at 37 degrees Celsius and stir at 750 RPM. Immediately, add one milliliter of previously prepared 0.66 millimolar chloroauric acid solution to the reaction vial. After stirring for two minutes, add one molar sodium hydroxide to the solution until the PH is 12 and stir for two hours.From the fluorescence of BSA gold three complex, it has been observed that the conversion of the intrinsic blue fluorescence of BSA to red fluorescence occurs at about PH 9.7 through an equilibrium transition. Excitation-emission maps of BSA gold three at different BSA to gold molar ratios show how altering the molar ratios yields the same emission wavelength at different excitation wavelengths. Copper, Nickel, and Gold competitively bind to a known site in BSA.The cysteine 34 capped BSA shows a change in excitation-emission map peak patterns upon gold binding and these results show how altering of specific binding sites alters fluorescence patterns. The all-thiol-capped-BSA shows no red fluorescence and reveals cysteine-cysteine disulfide bonds as possible binding sites to produce the red fluorophore. While attempting this procedure, it is important to remember to adjust the PH as described or to use the correct buffer because fluorophore formation is dependent on the PH-induced change of protein confirmations.

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