A facile method for studying interaction of rhodamine B and bovine serum albumin: Towards physical-binding mediated fluorescence labeling of proteins

A facile method for studying interaction of rhodamine B and bovine serum albumin: Towards physical-binding mediated fluorescence labeling of proteins 1 first-author 1 1 1 1 1 1 1 1 corresp Corresponding author, fengzhang1978@hotmail.com Corresponding author, fengzhang1978@hotmail.com fengzhang1978@hotmail.com Agricultural Nanocenter, School of Life Sciences, Inner Mongolia Agricultural University, Hohhot 010018, China Agricultural Nanocenter, School of Life Sciences, Inner Mongolia Agricultural University, Hohhot 010018, China Strategies for labeling proteins with fluorophores are always important for biotechnology. Here we take a model protein (bovine serum albumin) and a typical fluorophore (rhodamine B) to demonstrate a direct labeling method just by physical adsorption. In combination with size exclusion chromatography and the Scartchard equation, we have developed a facile analysis method for calculating the binding constant and binding sites. The molecular docking method has been used to study the binding site in amino acid level. Bovine serum albumin Rhodamine B Binding sites Scatchard equation Size exclusion chromatography I. INTRODUCTION 1 Fluorescence labeling of proteins, especially antibodies, has been widely used in current biotechnology applications. Generally, there are two requirements for a successful bio-labeling: 1) both original functionality of bio-molecules and original fluorescence property of the markers must be kept intact after labeling; and 2) the labeling should be robust enough in subsequent applications. Protein labeling kits are commercially available from Invitrogen, Sigma-Aldrich, etc. The labeling technologies can be classified as chemical conjugation and physical binding. The former includes carbodiimide mediated crosslinking of carboxylic groups and primary amino groups, and the azide and alkyne mediated click chemistry, while the latter includes bio-melocular pair-mediated labeling using DNA, biotin-avidin, aptamer-target, protein A/G-antibody, ligand-receptor, and antigen-antibody. Although fluorescent nanoparticles like semiconductor quantum dots [ 1 1 - 4 4 1-4 ], graphene quantum dots [ 5 5 - 7 7 5-7 ], nanodiamonds [ 8 8 - 10 10 8-10 ], fluorescence carbon dots [ 11 11 , 12 12 ] have attracted increasing interest for both labeling/imaging and sensing due to their perfect photostability, organic dyes are still playing main labeling role with the concern of toxicities and size limitations. Rhodamine B (RB), as a dye or a dye laser gain medium [ 13 13 , 14 14 ], is often used as a tracer dye within water to determine the rate and direction of flow and transport. Due to its good solubility in water ( ∼ 15 g/L), high quantum yield (about 94%), and the fact that its fluorescence can be easily and inexpensively detected by a fluorometer, RB has been extensively used as a biomaker in biotechnological labeling and imaging in fluorescence microscopy, flow cytometry, fluorescence correlation spectroscopy (FCS) and enzyme-linked immunosorbent assay (ELISA). Bovine serum albumin (BSA), also known as "fraction V", which refers to albumin being the fifth fraction of the original Edwin Cohn purification methodology that made use of differential solubility characteristics of plasma proteins [ 15 15 ], is a serum albumin protein derived from cows. And due to its stability, low-cost and clear structural information, BSA has been used as a model protein for many research aims, for example it can be used as a protein concentration standard in lab experiments, and its amphiphilic property has also made itself as a good carrier for both natural and artificial drugs like vitamins and paclitaxel. The mature BSA protein contains 583 amino acids (Mw 66463 Da, 66.5 kDa), which is a product from twice enzymatic cleavages from a 607-amino-acid full-length BSA precursor protein [ 16 16 ]. In this paper, we take the conventional fluorophore RB and the model protein BSA as objects for an interaction study towards a direct labeling method. With an idea to develop a new facile analysis approach based on the size exclusion chromatography (SEC), the association constant/binding affinity ( K a ) and the maximum binding sites of RB to BSA have been determined by employing the well-known Scartchard equation, and the detailed interaction information is further studied by using molecular docking. II. EXPERIMENTAL SECTION 1 A. Materials 2 BSA (lyophilized powder > 98%), RB (> 99%) and all other chemical regents were purchased from Sigma-Aldrich Corporation. Deionized water (18.2 MΩ cm) from a Milli-Q system (Millipore, Bedford, MA) was used for all experiments. BSA was dissolved in Milli-Q water for a stock solution with a concentration of 600 μm. RB was dissolved in Milli-Q water with a stock concentration of 600 μm. B. Spectroscopic measurements 2 UV-vis absorption spectra were recorded on a U-2900 UV-vis spectrometer (Hitachi). C. SEC assays 2 The SEC assays were performed on a sephacryl S-300 column equipped with a high performance liquid chromatography (HPLC) system (Hitachi L2000 or Agilent 1260) with a flow phase of SB9 (sodium borate 50 mM, pH = 9) buffer and the flow rate of 1 mL/min. The spectra were monitored at 280 nm for UV detector and excitation (Ex): 555 nm / emission (Em): 575 nm for the fluorescence detector. For the fluorometric titration experiment, 995 μL solutions containing different RB/BSA ratios of 64/1, 32/1, 16/1, 8/1 and 4/1 with a fixed final BSA concentration of 2.29 nM was prepared and incubated at 298 K for 2 h. D. Molecular docking 2 The three-dimensional (3D) structure of the BSA (PDB ID: 4JK4) was downloaded from Protein Data Bank (http://www.rcsb.org/pdb/home/home.dohttp://www.rcsb.org/pdb/home/home.do). The Auto Dock Tools 1.5.6 package (http://mgltools.scripps.eduhttp://mgltools.scripps.edu) was employed to generate the docking input files. The search grid of BSA was identified as center x : 97.127, center y : 24.933, and center z : 20.919 with dimensions size x : 15, size y : 15, and size z : 15. For Vina docking, the default parameters were used if it was not mentioned. The best-scoring pose as judged by the Vina docking score is chosen and visually analyzed using PyMOL software (http://www.pymol.org/http://www.pymol.org/). III. RESULTS AND DISCUSSION 1 A. Binding constant and binding sites 2 A number of methods can be used to calculate/measure the binding constant and binding sites. Some authors like using fluorescence quenching [ 17 17 - 19 19 17-19 ] to study interactions between proteins and other molecules. This method is facile and easily accessible, but it is limited when the molecules are not quenchers to proteins’ fluorescence. Other popular methods include quartz crystal microbalance (QCM)-based [ 20 20 ], and surface plasmon resonance (SPR)-based [ 21 21 ] techniques, but they cost quite a lot by consuming specific chips. The raw data obtained with these methods normally need further processing in combination with some classical equations, like Stern-Volmer equation, Scatchard equation and Hill equation, to get the binding constants, sites and even cooperativity. SEC is a powerful tool for both analysis and separation. Because proteins are several times larger than the labeling fluorophores which are normally less than 1 nm, so we can readily separate the bound fluorophores and free ones, which is exactly the data required by the Scatchard equation, <math display="block" id="M1"><mrow><mi>ν</mi><mo>/</mo><msub><mi>C</mi><mrow><mtext>f(RB)</mtext></mrow></msub><mo>=</mo><mo stretchy="false">(</mo><mi>n</mi><mo>−</mo><mi>ν</mi><mo stretchy="false">)</mo><msub><mi>K</mi><mtext>a</mtext></msub><mn>,</mn></mrow></math> where C f is concentration of the free ligand, which in our case is the unbound RB’s concentration; n is the number of binding sites per protein molecule; K a is the association/binding constant/affinity of RB for BSA; and ν is the real bound ratio defined as, <math display="block" id="M2"><mrow><mi>ν</mi><mo>=</mo><mo stretchy="false">(</mo><msub><mi>C</mi><mrow><mtext>t(RB)</mtext></mrow></msub><mo>−</mo><msub><mi>C</mi><mrow><mtext>f(RB)</mtext></mrow></msub><mo stretchy="false">)</mo><mo>/</mo><msub><mi>C</mi><mrow><mtext>t(BSA)</mtext></mrow></msub><mn>,</mn></mrow></math> where C t(RB) is concentration of total RB which is known before the binding assay; C f(RB) is the free or unbound RB’s concentration which can be obtained by SEC measurement; and C t(BSA) is concentration of total BSA for the binding assay, which is known and constant in the current assay. We took RB and BSA for an example to justify the use of SEC in combination with Scatchard equation to calculate the binding constant and sites between molecules and proteins. To be more precisely, we made a pure dye’s SEC to get the SEC-based calibration curve of concentration ( Fig. 1 Fig. 1 ) by plotting the integrated elution peaks against the concentrations. From Fig. 2 Fig. 2 , it can be seen that, with the molar ratios of RB/BSA in Section II C, the RB’s fluorescence intensity decreases linearly with increasing BSA/RB ratios at a fixed RB molar concentration. The binding constant and binding sites were calculated at 2.2×10 5 M -1 and 1.3, respectively. Cai et al . reported that the binding constant and binding sites were 4.8×10 4 M -1 and 1.2, respectively. However, their further analysis by time-resolved fluorescence indicated at least two binding sites existed [ 22 22 ]. The real binding sites can be highly resolved by crystallographic studies of the complexes of proteins and molecules, for example Sekula B. and colleagues found there are four binding sites of 3,5-diiodosalicylic acid to BSA [ 23 23 ]. The binding affinity obtained by the current method seems much higher than Ref. [ 22 22 ]. Thus, we believe that different buffers and sensitivities of the instruments might be the culprit, and that the fluorescence quenching method can produce huge deviations for the binding affinity evaluation [ 24 24 ]. So, it would be helpful to compare different methods in the same conditions. It is not difficult to find the intermolecular interaction studies by searching the scientific publication bank/library, but it may be difficult to figure out how those scientists get necessary values for the Scatchard plot. By using SEC assisted with an HPLC system, one can clearly learn how to do this plot step-by-step. With the well-known reputation of the repeatability of a commercial HPLC system such as the Agilent used in this paper, one can directly calculate both binding constant and binding sites just by simply preparing the mixture solution. In addition, the concentration calibration curve of free dye alone obtained by the same configuration constants of HPLC system such as the column, flow rates and loading volumes, is necessary in that it minimizes the measurement error. B. Molecular docking of RB and BSA 2 To study the binding mode of RB to BSA, molecular docking was performed by using the Auto dock. As shown in Fig. 3 Fig. 3 , the RB molecule is docked into the binding pocket of the BSA. One of diethylamino group of RB fits into bottom of the binding pocket of BSA, surrounded by the residues Trp 213 , Arg 194 and Arg 198 , while the other diethylamino group located at the entrance of the pocket, interacted with the hydrophobic residues Val 342 , Ala 341 and Pro 446 . Importantly, the carboxyl group forms two key hydrogen bonds (2.0 and 3.3 Å) with the residue Arg 194 , which is important for the affinity between RB and BSA. In summary, the computational approach helps in better understanding of inhibitors binding to the protein active site, which provides valuable information for further study of interaction between RB and BSA. Limited by the docking software which does not support the multi-molecular docking, we didn’t try more docking sites. IV. CONCLUSION 1 Compared with different chemical labeling strategies, the direct labeling method with such a high binding affinity will hold great promise for future biotechnology. The facile method in combination with SEC and Scatchard equation has shown the feasibility to get the binding constant and sites of BSA-RB interaction, which might find more applications in other proteins and small molecules or any two substances with large size differences. 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DOI: 10.1021/la402920f http://doi.org/10.1021/la402920f Nanoparticle-protein interactions: a thermodynamic and kinetic study of the adsorption of bovine serum albumin to gold nanoparticle surfaces 10.13538/j.1001-8042/nst.26.060502 Supported by the National Natural Science Foundation of China (Nos. 21171086 and 81160213), Inner Mongolia Autonomous Region science and Technology Department (No. 211-202077), Inner Mongolia Grassland Talent (No. 108-108038), Natural Science Foundation of Inner Mongolia Autonomous Region of China (No. 2013MS1121) and Inner Mongolia Agricultural University (Nos. 109-108040, 211-109003 and 211-206038) 2015-06-08 2015-08-02 2015-12-20 2015-12-20 2021-04-11 2015 Nuclear Science and Techniques 6 26