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 (λem = 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.
Bovine serum albumin1,2,3 (BSA)-gold (Au) complexes, obtained by reactions in highly alkaline conditions (pH > 10), are known to exhibit UV-excitable red luminescence (λem = 640 nm)4,5,6,7. Numerous applications of this compound has been proposed and investigated, including sensing,8,9,10 imaging11,12,13, and nanomedicine14,15,16. 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 function17,18, 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.
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 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.
- Dissolve Au(III) chloride (chloroauric acid) in 1 mL of HPLC grade water to a concentration of 750 µM.
- Into the reaction vial, add the chloroauric acid solution for a resulting BSA:Au molar ratio of 1:10.
- Stir the mixture for 2 minutes at 37 °C and at 750 rpm using a magnetic stirrer.
- 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.
- 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
- Rinse a pre-cast 4-12% gradient Bis-Tris gel using deionized water and place in a gel electrophoresis tank.
- Prepare 500 mL of MES running buffer solution from a concentrated stock solution, diluting with deionized water.
- 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.
- Add 10 µL of each sample solution to each lane of the gel.
- Run the gel for 1 hour at a constant voltage of 150 V.
- 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.
- Store the gel in 200 mL of deionized water and immediately measure the in-gel fluorescence, using a gel imaging system.
- 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.
- Wash the gel in 200 mL of staining solution for 30 minutes using gentle rocking.
- 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.
- Wash the gel in 100 mL of de-staining solution for 1 hour using gentle stirring.
- 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
- 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.
- Identify location(s) of surface-exposed Cys in these expected fragments. For trypsin-digested BSA, the only surface-exposed Cys34 is in fragment [A].
- 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.
- 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.
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) = [ABCDE] (undigested); Band(2) = [ABCD] (connected fragments); Band(3) = [BCD]; Band(4) = [CD]; Band(5) = [A]+[ABC]; Band(6) = [ABC]; Band(7) = [BC]; Band(8) = [D]; Band(9) = [A]+[A]; Band(10) = [AB]; Band(11) = [E]; Band(12) = [A] (Figure 2a). Band(5) and Band(9) contain dimerized fragment [A], which are justified by the surface-exposed Cys342. Bands above the molecular weight of BSA ~66 kDa can be excluded from further analyses of BSA fragments.
Smearing of the gel bands was observed in the presence of Au(III). We attribute this smearing to the interaction of the surface-bound Au(III)5 with the gel matrix24,25. This smearing can be minimized, when the protocol is followed. Out of the twelve gel bands of BSA-trypsin, nine bands were observed as [BSA-trypsin]-Au (Figure 2b). Multimer bands (above ~66 kDa) were pronounced, due to aggregation caused by surface-bound Au(III)12.
Five bands exhibited the in-gel red luminescence (Figure 2c). These bands were Band(1) = [ABCDE] (undigested BSA); Band(2) = [ABCD]; Band(3) = [BCD]; Band(6) = [ABC]; Band(7) = [BC]. Luminescence was absent in Band(8) = [D], Band(9) = [A]+ [A], Band(10) = [AB], and Band(12) = [A] (Figure 2d). This in-gel luminescence band pattern can be explained by the presence of a red luminophore in the tryptic fragment [C] (residues 115/117 - 294/312).
The gel band smearing was exacerbated with extended digestion times, in the presence of Au(III). The smearing was minimized when the protocol described above was used. Examples of non-optimized gel electrophoresis are described in Figure 3, Figure 4, Figure 5 and Figure 6, to demonstrate the factors affecting the limited tryptic proteolysis and the gel electrophoresis in the presence of metal cations.
Figure 1. List of Cys-Cys bond-preserving bands of BSA by limited tryptic proteolysis. Tryptic cleaving in the random coil regions can result in 15 (#i-xv) fragments, but Cys-Cys bonds connect the sequences as indicated. This table has been modified from Dixon et al.6. Please click here to view a larger version of this figure.
Figure 2. Gel-electrophoresis of the tryptic fragments of BSA obtained by the limited proteolysis. (a) A total of twelve bands were observed in gel-electrophoresis of BSA by the limited tryptic proteolysis. (b) Tryptic BSA fragments reacted with Au(III). (c) In-gel luminescence of (b). (d) Drawing of the observed in-gel bands. In-gel red luminescence was observed with Band(1) = undigested BSA, Band(2) = [ABCD], Band(3) = [BCD], Band(6) = [ABC], and Band(7) = [BC] (indicated by †). Please click here to view a larger version of this figure.
Figure 3. Optimizing the time of reaction with Au. (A) BSA; (B) BSA-Au (reacted for 2 h); (C) BSA-Au (reacted for 1 h); (D) BSA-Au (reacted for 30 min); (E) BSA-Au (reacted for 5 min). The red luminescence of BSA-Au complexes required at least 30 min at 37 °C, and luminescence was most intense in the gel after reaction for 2 hours. However, the longer reaction time led to more smearing in the gel electrophoresis bands. In order to maximize the luminescence intensity and to minimize the smearing, the optimum reaction time was chosen to be 2 hours. Please click here to view a larger version of this figure.
Figure 4. Optimizing the timings of the addition of trypsin for digestion, and Au for reaction. The reaction time with Au was explored in the range of 2 to 24 h. (A) BSA; (B) [BSA-trypsin]-Au (after 2 h); (C) [BSA-trypsin]-Au (after 4 h); (D) [BSA-trypsin]-Au (after 8 h); (E) [BSA-trypsin]-Au (after 24 h). Longer reaction time resulted in more smearing of the gel bands. Please click here to view a larger version of this figure.
Figure 5. Effects of metal cations and the gel band smearing. The smearing of the gel bands by the additions of and reactions with Au(III) were similarly observed with Cu(II) and Ni(II). (A) BSA; (B) BSA-Au; (C) [BSA-trypsin]-Au (2-hour digestion); (D) [BSA-trypsin]-Au (overnight digestion); (E) [BSA-trypsin]-Cu (2-hour digestion); (F) [BSA-trypsin]-Cu (overnight digestion); (G) [BSA-trypsin]-Ni (2-hour digestion); (H) [BSA-trypsin]-Ni (overnight digestion). The red luminescence was not observed upon reactions with Cu(III) or Ni(II). Please click here to view a larger version of this figure.
Figure 6. Effects of pH and the order of the additions of trypsin and Au. The fragmented compounds were prepared by reacting Au with digested BSA ([BSA-trypsin]-Au), or by digesting the pre-formed BSA-Au ([BSA-Au]-trypsin), and then run in gel as is or after adjusting the pH to 7 in sample buffer. (A) BSA; (B) BSA-Au (as is); (C) BSA-Au (pH=7); (D) BSA-trypsin; (E) [BSA-trypsin]-Au (as is); (F) [BSA-Au]-trypsin (as is); (G) [BSA-trypsin]-Au (pH=7); (H) [BSA-Au]-trypsin (pH=7). The presence of the red luminescence was not affected by the order of the additions of trypsin and Au, however the smearing of the gel bands was more pronounced for [BSA-Au]-trypsin. The adjustment of pH to neutral in the sample buffer did not significantly improve the gel band smearing, however increased the aggregation of fragments as indicated by the gel bands above ~66 kDa. Please click here to view a larger version of this figure.
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 optimizations to enable the analysis of BSA-Au fragments and their in-gel luminescence. The red luminescence of BSA-Au complexes and [BSA-trypsin]-Au fragments required at least 30 min of reaction time at 37 °C to be observable in gels, and luminescence was most intense in the gel after reaction for 2 hours (Figure 3, Figure 4). However, the longer reaction time led to more smearing in the gel electrophoresis bands. In order to maximize the luminescence intensity and to minimize the smearing, the optimum reaction time was chosen to be 2 hours.
The presence of metal cations appears to be the major cause of the gel band smearing. The smearing of the gel bands were observed by the additions of and reactions with Au(III), as well as with Cu(II)24,25 and Ni(II) (Figure 5). We also assessed the effects of the order of the addition of trypsin and Au, and the sample loading buffer pH (Figure 6). We observed the in-gel red luminescence regardless of the order of the additions of trypsin and Au. However, smearing of the gel bands was suppressed for [BSA-trypsin]-Au, namely by digesting BSA first and then reacting the obtained fragments with Au. The adjustment of pH to neutral in the sample loading buffer for the gel electrophoresis did not significantly improve the gel band smearing, rather, it increased the aggregation of fragments as indicated the gel bands above the molecular weight of 66.4 kDa. Other important factors that we note to optimize the gel-band analysis are the ratio of BSA:Au (maximum intensity was obtained for the ratio of 1:255, but we optimized it to 1:10 to minimize the metal cation-induced gel smearing, while maximizing the red luminescence intensity) and the gel loading (10 µg of protein per lane, to maximize the in-gel band luminescence intensity while avoiding the overloading). For gel-based proteomics, a further improvement in the smearing may be necessary.
The majority of proteins need metal cations to function, and at a particular physiological pH. For a complete understanding of these proteins, x-ray crystallography is ultimately required for the structural analyses and the identification of metal coordination. However, protein crystallization is often difficult and is not always possible at desired (physiologically functional) pH. Taking the experiments presented herein as an example, the structures of BSA, and their fragments, are not known for the non-neutral pH conformation that is required to produce the red luminescence. Yet we were able to obtain critical functional information of the metal-bound BSA fragments at the desired pH. This method could therefore be used in supplement to study the interactions of proteins with metal cations in a facile manner without requiring the protein crystallization.
Molecular cloning can be technically difficult in some cases, due to low solubility of the product fragment or low expression in E. coli, and is usually time-consuming6. The facile method based on the limited proteolysis presented here can complement the molecular cloning approach.
The authors have nothing to disclose.
S.E. acknowledges support from PhRMA Foundation, Leukemia Research Foundation, and National Institutes of Health (NIH R15GM129678).
|Ammonium bicarbonate, 99.5%||Sigma-Aldrich||9830|
|Azure Biosystems C400 gel imaging system||Azure Biosystems||C400|
|Bovine Serum Albumin (BSA), 96%||Sigma-Aldrich||A5611|
|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|
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