Fluorimetric study on the interaction between fluoresceinamine and bovine serum albumin

Fluorimetric study on the interaction between fluoresceinamine and bovine serum albumin 1 first-author 1 1 1 1 1 1 corresp Corresponding author, fengzhang1978@hotmail.com Corresponding author, fengzhang1978@hotmail.com fengzhang1978@hotmail.com School of Life Science, Inner Mongolia Agricultural University, 306 Zhaowuda Road, Hohhot 010018, China. School of Life Science, Inner Mongolia Agricultural University, 306 Zhaowuda Road, Hohhot 010018, China. Fluorescence spectroscopy was employed to investigate the interaction between fluorophore fluoresceinamine (FA) and bovine serum albumin (BSA) under physiological conditions. In the mechanism discussion, it was proved that the fluorescence quenching of BSA by FA is a result of the formation of a BSA-FA complex. Fluorescence quenching constants were determined using the modified Stern-Volmer equation to provide a measure of the binding affinity between FA and BSA. The results of the thermodynamic parameters Δ G , Δ H , and Δ S at different temperatures indicated that several kinds of interactions, except for the electrostatic interactions play cooperative roles in BSA-FA association. Furthermore, the conformation of BSA upon interaction with FA was also studied by synchrotron fluorescence spectroscopy. Bovine serum albumin Fluoresceinamine Fluorescence quenching Binding constant Protein conformation I. INTRODUCTION 1 Fluorophore labeled proteins have been applied to in vitro / vivo research for decades [ 1 1 - 3 3 1-3 ]. Normally for robust labelling, chemical crosslinking has to be involved. For example, the well-known EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) crosslinking chemistry between the primary amines and the carboxylic groups [ 4 4 , 5 5 ], as well as other specific chemical crosslinking pairs between the NHS ester and primary amines, thiol groups, and maleic acid imides (maleimides). Alternatively, the binding constant can also be high between some natural biological conjugating pairs such as enzyme-ligands, biotin-avidin, protein A/G-antibodies, antibody-antigens, aptamer-ligands, and complementary nucleic acids. Compared to chemical labeling, such biological binding pairs have been applied to separation/purification [ 6 6 ] and immobilization [ 7 7 ] for a long time. As a result, th e physical binding is capable of recycling, due to the reversible binding, by altering the incubation conditions, such as pH, ionic strength, solvent polarity, and so on. Hence, developing a novel method to physically label proteins by fluorophores holds potent potential for biomedical applications. In this paper, we have found that bovine serum albumin (BSA) can be stably labeled with fluoresceinamine (FA) via physical binding. To understand the strong interaction between the BSA and the FA molecules, the fluorescence quenching method and synchrotron fluorescence spectroscopy were employed to study the thermodynamic interaction parameters and the conformational changes of BSA, respectively. II. EXPERIMENTS 1 A. Materials 2 BSA (lyophilized powder, > 98%), FA (> 95%), and all other chemical regents were purchased from Sigma-Aldrich Corporation. The deionized water (18.2 MΩ cm) used for all experiments was obtained from a Milli-Q system (Millipore, Bedford, MA). BSA was dissolved in Milli-Q water for a stock solution with a concentration of 1 μm. FA was dissolved in a SB9 (sodium borate 50 mM, pH=9) buffer with a stock concentration of 100 μm. B. Fluorescence measurements 2 Fluorescence spectra were recorded with a fluorescence spectrometer (Fluorolog-MAX 4, Horiba) equipped with a 1.0 cm quartz cell. The excitation wavelength was fixed at 280 nm and the emission spectra were recorded at 300–550 nm. Both excitation and emission slits were set up to 5 nm. For the fluorimetric titration experiment, different solutions with a total volume of 0.9 mL containing different FA/BSA molar ratios with a fixed final BSA concentration of 0.33 μm ( Table 1 Table 1 ) were prepared and incubated at different temperatures (298, 304 and 310 K) for 30 min before measurements. The results were analyzed by using the (modified) Stern-Volmer equation. a Here FA’s concentration is 33 μM. 1 1 Vial number 1 1 1 1 1 2 1 1 3 1 1 4 1 1 5 1 1 6 1 1 7 1 1 8 1 1 9 1 1 BSA 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 FA 1 1 0 1 1 1.56 1 1 3.13 1 1 6.25 1 1 12.5 1 1 25 1 1 50 1 1 100 1 1 – a a C. UV-vis absorption spectroscopic measurements 2 UV-vis absorption spectra were recorded on a U-2900 UV-Vis spectrometer (Hitachi). III. RESULTS AND DISCUSSION 1 A. Fluorescence characteristics of BSA-FA 2 Fluorescence quenching is a sensitive method used to investigate the interaction between a protein and molecules when the environment and the protein’s structure are changed [ 8 8 , 9 9 ]. The mechanism of fluorescence quenching is usually classified as either dynamic or static. Dynamic quenching normally results from diffusive encounters between the fluorophore and the quencher during the lifetime of the excited state, which is a time-dependent process. In contrast, static quenching occurs as a result of the formation of a non-fluorescent ground-state complex between the fluorophore and the quencher. Dynamic and static quenching can be distinguished by their dependence on temperature [ 10 10 ]. Because higher temperatures result in larger diffusion coefficients, the bimolecular quenching constants are expected to rise with temperature. In contrast, the increasing temperature is likely to result in a decrease in the stability of complexes, thus lowering the values of the static quenching constants [ 1 1 ]. When BSA was excited at 280 nm it showed a strong fluorescence emission at 340 nm due to two types of amino acid residues, tyrosine and tryptophan. The effect of FA (structure shown in Fig. 1(a) Fig. 1(a) ) on the fluorescence spectra of BSA was explored and the changes are shown in Fig. 1(b) Fig. 1(b) . Upon addition of FA to the BSA solution, the fluorescence intensity showed a progressive decrease around 340 nm. The decrease in the fluorescence intensity of BSA can be observed regularly and acutely with an increasing concentration of FA, which confirms that FA quenches the intrinsic fluorescence of BSA and the quenching effects depend on the concentration of FA molecules. To clarify the fluorescence quenching, we performed fluorescence tests at different temperatures (298, 304 and 310 K), which can be described using the well-known Stern-Volmer equation (Eq. (1)) <math display="block" id="M1"><mrow><mfrac><mrow><msub><mi>F</mi><mn>0</mn></msub></mrow><mi>F</mi></mfrac><mo>=</mo><mn>1</mn><mo>+</mo><msub><mi>K</mi><mrow><mtext>SV</mtext></mrow></msub><mtext>[FA]</mtext><mo>=</mo><mn>1</mn><mo>+</mo><msub><mi>k</mi><mtext>q</mtext></msub><msub><mi>τ</mi><mn>0</mn></msub><mtext>[FA]</mtext><mn>,</mn></mrow></math> where, F 0 and F are the fluorescence intensities in the absence and presence of the quencher (FA), respectively. The Stern-Volmer quenching constant is given by K SV = k q τ 0 , where k q is the quenching constant, τ 0 is the lifetime of the fluorophore in the absence of a quencher, and [FA] is the concentration of the quencher FA. In this work, the concentration of the BSA solution was fixed at 0.33 μm and the curve i in Fig. 1 Fig. 1 is the emission spectrum of FA alone, which indicates that FA does not significantly emit in this wavelength range, thus can be negligible at the current excitation wavelength (280 nm). However, the standard Stern-Volmer plots (the inset of Fig. 1(b) Fig. 1(b) ) show a downward curvature (concave towards x -axis), indicating that the chromophoric residues are not all accessible to the quencher molecules, which might be caused by the static quenching due to the BSA-FA complex formation [ 10 10 ]. B. Binding parameters 2 In this case, the PL data was further examined using the modified Stern-Volmer equation (Eq. (2)) <math display="block" id="M2"><mrow><mfrac><mrow><msub><mi>F</mi><mn>0</mn></msub></mrow><mrow><msub><mi>F</mi><mn>0</mn></msub><mo>−</mo><mi>F</mi></mrow></mfrac><mo>=</mo><mfrac><mn>1</mn><mrow><msub><mi>f</mi><mtext>a</mtext></msub><msub><mi>K</mi><mtext>a</mtext></msub></mrow></mfrac><mfrac><mn>1</mn><mrow><mtext>[FA]</mtext></mrow></mfrac><mo>+</mo><mfrac><mn>1</mn><mrow><msub><mi>f</mi><mtext>a</mtext></msub></mrow></mfrac><mn>,</mn></mrow></math> where, f a is the fraction of accessible fluorophores and K a is the effective quenching constant for the accessible fluorophores, which are analogous to the associative coefficient of the quencher-acceptor system. The dependence of F 0 /( F 0 - F ) on the reciprocal value of the quencher concentration, [FA] -1 , should be linear with the slope of ( f a K a ) -1 , whereas the value <math id="M3"><mrow><msubsup><mi>f</mi><mtext>a</mtext><mrow><mo>−</mo><mn>1</mn></mrow></msubsup></mrow></math> is fixed on the ordinate. Therefore, the constant K a is a quotient of <math id="M4"><mrow><msubsup><mi>f</mi><mtext>a</mtext><mrow><mo>−</mo><mn>1</mn></mrow></msubsup></mrow></math> and the slope ( f a K a ) -1 . The corresponding results shown in Fig. 2(a) Fig. 2(a) at different temperatures are listed in Table 2 Table 2 . The decreasing trend of binding constants with increasing temperature indicates that BSA-FA adsorption is initiated by the conjugate/complex formation (static quenching). The results summarized in Table 2 Table 2 also show that the binding constant between FA and BSA is moderate and the effect of temperature can not be neglected, in that the interaction between BSA and FA can be accelerated by increasing temperature. Hence, the stability of FA labeled BSA should be considered when handled at higher temperatures. 1 2 T (K) 4 1 Modified Stern-Volmer equation 1 2 Δ H 0 (kJ/mol) 1 2 Δ G 0 (kJ/mol) 1 2 Δ S 0 (kJ/(mol K)) 1 1 K a (/M) 1 1 f a 1 1 R 1 1 SD 1 1 298 1 1 1.33×10 5 1 1 0.94 1 1 0.995 1 1 0.06 1 1 -96.88 1 1 -29.53 1 1 -0.23 1 1 304 1 1 7.04×10 4 1 1 0.96 1 1 0.997 1 1 0.08 1 1 — 1 1 -28.17 1 1 — 1 1 310 1 1 5.37×10 4 1 1 0.97 1 1 0.999 1 1 0.07 1 1 — 1 1 -26.82 1 1 — C. Thermodynamic parameters 2 All chemical, physical, and biological processes are accompanied by a change in thermodynamic parameters (Δ G 0 , Δ H 0 , and Δ S 0 ), which can be further studied by employing the van’t Hoff equation <math display="block" id="M5"><mrow><mi>ln</mi><msub><mi>K</mi><mtext>a</mtext></msub><mo>=</mo><mfrac><mrow><mo>−</mo><mi>Δ</mi><msup><mi>H</mi><mn>0</mn></msup></mrow><mrow><mi>R</mi><mi>T</mi></mrow></mfrac><mo>+</mo><mfrac><mrow><mi>Δ</mi><msup><mi>S</mi><mn>0</mn></msup></mrow><mi>R</mi></mfrac><mn>,</mn></mrow></math> where K a is the binding/association constant and R , T , Δ H 0 , and Δ S 0 are the gas constant (8.314 J/(mol K)), absolute temperature, standard enthalpy, and standard entropy, respectively. By plotting "ln K a " against "1/ T ", as shown in Fig. 2(b) Fig. 2(b) , both values of Δ H 0 and Δ S 0 can be determined from the slope and intercept, respectively. The value of free energy change, Δ G 0 , can be determined by the Gibbs-Helmholtz equation <math display="block" id="M6"><mrow><mi>Δ</mi><msup><mi>G</mi><mn>0</mn></msup><mo>=</mo><mi>Δ</mi><msup><mi>H</mi><mn>0</mn></msup><mo>−</mo><mi>T</mi><mi>Δ</mi><msup><mi>S</mi><mn>0</mn></msup><mn>.</mn></mrow></math> The interaction processes of the current study were found to be spontaneous, as evidenced by negative Δ G 0 values ( Table 2 Table 2 ). On the other hand, both the negative Δ H 0 (-96.88 kJ/mol) and the positive Δ S 0 (-0.23 kJ/(mol K)) values indicated that the BSA-FA interaction can be spontaneous because the physical adsorption of FA-BSA releases heat to environment/surroundings and, in fact, the Δ S 0 of the whole system, including the surroundings and the FA-BSA reaction system should be positive, which is consistent with the second law of thermodynamics. In addition, the sign and magnitude of the thermodynamic parameters for protein reactions are usually used to identify the main forces contributing to ligand-protein stability [ 11 11 ]. Normally, the interactions between molecules and proteins can be classified into 5 catalogues: 1) hydrophobic interactions, 2) hydrogen mediated interactions, 3) coulombic/electrostatic interactions, 4) van der Waals forces, and 5) π- π stacking interactions. According to the thermodynamic study of protein association reactions [ 12 12 ], in the case of Δ H 0 < 0 and Δ S 0 < 0, only the coulombic/electrostatic interactions were ruled out (both FA and BSA in a pH 9 buffer are negatively charged by measuring their zeta potentials) and all the other interactions could be involved in the formation of BSA-FA conjugates/complexes. D. Confirmation investigation 2 Spectroscopy is an ideal tool to observe conformational changes in proteins, since it allows for non-intrusive measurements of substances in a low concentration under physiological conditions. Synchronous fluorescence spectroscopy (SFS) is a multidimensional fluorescence technique, which has become an increasingly active subject of research ever since its introduction by Lloyd [ 13 13 ]. The constant wavelength SFS technique involves simultaneous scanning of the excitation and emission monochromators, while maintaining a constant wavelength interval between them. The SFS gives information about the molecular environment in the vicinity of the chromosphere molecules [ 14 14 ]. When the D -value (Δ λ ) between the excitation wavelength and emission wavelength is stabilized at 15 nm or 60 nm, the synchronous fluorescence gives the characteristic information of tyrosine residues or tryptophan residues [ 15 15 ]. The effect of FA on BSA synchronous fluorescence spectroscopy is shown in Fig. 3 Fig. 3 . It is apparent from Fig. 3 Fig. 3 that the maximum emission wavelength blue shifts (from 302 to 299 nm) at the investigated concentration range when Δ λ = 15 nm ( Fig. 3(a) Fig. 3(a) ) and the maximum emission wavelength blue shifts (from 340 to 338 nm) when Δ λ = 60 nm ( Fig. 3(b) Fig. 3(b) ). The blue shift of the emission maximum suggests a less polar (or more hydrophobic) environment of chromophore (tyrosine and tryptophan) residues in BSA. IV. CONCLUSION 1 In this paper, the interaction between FA and BSA was studied by spectroscopic methods. The experimental results indicate that the interaction between BSA and FA involves hydrophobic, π-π stacking, van der Waals forces and hydrogen-bond mediated interactions. The static quenching mechanism can be used to understand that there is BSA-FA complex formed spontaneously during the interaction. Furthermore, the protein’s conformation was also studied by SFS. The biological significance of this work is that the BSA-FA conjugates formed merely by physical adsorption might apply to bio-labeling and reversible bio-sensors. References R B Zhong , Y S Liu , P Zhang , et al . Discrete nanoparticle-BSA conjugates manipulated by hydrophobic interaction . Acs Appl Mater Inter , 2014 , 6 : 19465 - 19470 . DOI: 10.1021/am506497s http://doi.org/10.1021/am506497s Discrete nanoparticle-BSA conjugates manipulated by hydrophobic interaction C Uttamapinant , K A White , H Baruah , et al . A fluorophore ligase for site-specific protein labeling inside living cells . P Natl Acad Sci USA , 2010 , 107 : 10914 - 10919 . DOI: 10.1073/pnas.0914067107 http://doi.org/10.1073/pnas.0914067107 A fluorophore ligase for site-specific protein labeling inside living cells S H Kim , M Jeyakumar , J A Katzenellenbogen , et al . Dual-mode fluorophore-doped nickel nitrilotriacetic acid-modified silica nanoparticles combine histidine-tagged protein purification with site-specific fluorophore labeling . J Am Chem Soc , 2007 , 129 : 13254 - 13264 . DOI: 10.1021/ja074443f http://doi.org/10.1021/ja074443f Dual-mode fluorophore-doped nickel nitrilotriacetic acid-modified silica nanoparticles combine histidine-tagged protein purification with site-specific fluorophore labeling F Zhang , E Lees , F Amin , et al . Polymer-coated nanoparticles: a universal tool for biolabelling experiments . Small , 2011 , 7 : 3113 - 3127 . DOI: 10.1002/smll.201100608 http://doi.org/10.1002/smll.201100608 Polymer-coated nanoparticles: a universal tool for biolabelling experiments A V Yakovlev , F Zhang , A Zulqurnain , et al . Wrapping nanocrystals with an amphiphilic polymer preloaded with fixed amounts of fluorophore generates FRET-based nanoprobes with a controlled donor/acceptor ratio . Langmuir , 2009 , 25 : 3232 - 3239 . DOI: 10.1021/la8038347 http://doi.org/10.1021/la8038347 Wrapping nanocrystals with an amphiphilic polymer preloaded with fixed amounts of fluorophore generates FRET-based nanoprobes with a controlled donor/acceptor ratio E Sada , S Katoh , A Kondo , et al . Characterization of the immunointeraction and its application to bioaffinity separation . Ann NY Acad Sci , 1990 , 613 : 874 - 877 . DOI: 10.1111/j.1749-6632.1990.tb18280.x http://doi.org/10.1111/j.1749-6632.1990.tb18280.x Characterization of the immunointeraction and its application to bioaffinity separation V Singh , M Sardar and M N Gupta . Immobilization of enzymes by bioaffinity layering . Method Mol Biol , 2013 , 1051 : 129 - 137 . DOI: 10.1007/978-1-62703-550-7_9 http://doi.org/10.1007/978-1-62703-550-7_9 Immobilization of enzymes by bioaffinity layering J Jayabharathi , K Jayamoorthy , V Thanikachalam , et al . Fluorescence quenching of bovine serum albumin by NNMB . Spectrochim Acta A , 2013 , 108 : 146 - 150 . DOI: 10.1016/j.saa.2013.01.092 http://doi.org/10.1016/j.saa.2013.01.092 Fluorescence quenching of bovine serum albumin by NNMB R Mi , Y J Hu , X Y Fan , et al . Exploring the site-selective binding of jatrorrhizine to human serum albumin: Spectroscopic and molecular modeling approaches . Spectrochim Acta A , 2014 , 117 : 163 - 169 . DOI: 10.1016/j.saa.2013.08.013 http://doi.org/10.1016/j.saa.2013.08.013 Exploring the site-selective binding of jatrorrhizine to human serum albumin: Spectroscopic and molecular modeling approaches J R Lakowicz . Principles of fluorescence spectroscopy . New York (USA) : Springer US , 2006 . DOI: 10.1007/978-0-387-46312-4 http://doi.org/10.1007/978-0-387-46312-4 Principles of fluorescence spectroscopy I M Klotz . Physiochemical aspects of drug-protein interactions: a general perspective . Ann NY Acad Sci , 1973 , 226 : 18 - 35 . DOI: 10.1111/j.1749-6632.1973.tb20465.x http://doi.org/10.1111/j.1749-6632.1973.tb20465.x Physiochemical aspects of drug-protein interactions: a general perspective P D Ross and S Subramanian . Thermodynamics of protein association reactions: forces contributing to stability . Biochemistry , 1981 , 20 : 3096 - 3102 . DOI: 10.1021/bi00514a017 http://doi.org/10.1021/bi00514a017 Thermodynamics of protein association reactions: forces contributing to stability J B F Lloyd . Synchronized excitation of fluorescence emission spectra . Nature , 1971 , 231 : 64 - 65 . DOI: 10.1038/physci231064a0 http://doi.org/10.1038/physci231064a0 Synchronized excitation of fluorescence emission spectra Y J Hu , Y Liu , R M Zhao , et al . Spectroscopic studies on the interaction between methylene blue and bovine serum albumin . J Photoch Photobio A , 2006 , 179 : 324 - 329 . DOI: 10.1016/j.jphotochem.2005.08.037 http://doi.org/10.1016/j.jphotochem.2005.08.037 Spectroscopic studies on the interaction between methylene blue and bovine serum albumin J N Miller . Recent advances in molecular luminescence analysis . Proc Anal Div Chem Soc , 1979 , 16 : 203 - 208 . DOI: 10.1039/AD9791600199 http://doi.org/10.1039/AD9791600199 Recent advances in molecular luminescence analysis We thank Mr. GAO Hai-Yang and Ms. LI Wan-Rong for their help and valuable discussion. 10.13538/j.1001-8042/nst.26.030505 Supported by National Natural Science Foundation of China (Nos. 21171086 and 81160213), the Inner Mongolia Grassland Talent (No. 108-108038), the Inner Mongolia Autonomous Region Natural Science Foundation (No. 2013MS1121), and the Inner Mongolia Agricultural University (Nos. 211-109003 and 211-206038) 2015-01-09 2015-03-23 2015-06-20 2015-06-20 2021-04-11 2015 Nuclear Science and Techniques 3 26