A facile and precise method for quantifying small-large/light-weighted molecular interaction system

A facile and precise method for quantifying small-large/light-weighted molecular interaction system 1 first-author 1 1 1 1 1 1 1 corresp Corresponding Author, Email: fengzhang1978@hotmail.com Corresponding Author, Email: fengzhang1978@hotmail.com fengzhang1978@hotmail.com Agricultural Nanocenter, School of Life Sciences, Inner Mongolia Agricultural University, 306 Zhaowuda Road, Hohhot 010018, China. Agricultural Nanocenter, School of Life Sciences, Inner Mongolia Agricultural University, 306 Zhaowuda Road, Hohhot 010018, China. It is significant to quantify the intermolecular physisorption extent in biomedical field. By taking the advantage of a significant difference from either sizes or weights, we introduced a combination of Scatchard equation and either ultracentrifugation or size exclusion chromatography to obtain both the binding constant and the number of binding sites by using bovine serum albumin and eosin B as models. Compared to the photoluminescence quenching-based methods like Stern-Volmer and Hill equations, the introduced method is not only more precise but also simpler and more straightforward for the operation. Moreover, the protein conformational changes and the corresponding theoretical binding mode with an atomic resolution were also studied by using three-dimensional fluorescence spectroscopy and molecular docking method, respectively. These comparative results could help scientists select right methods to study any interactions between two molecules with significant differences from either sizes or weights. Bovine serum albumin Eosin B Binding constant Ultracentrifugation Scatchard equation Size exclusion chromatography 1 INTRODUCTION 1 The intermolecular interactions play paramount roles in biological activities, and to learn the mechanisms created by Mother Nature needs to understand all the details of the interactions, especially for the binding constant and the number of binding sites, which could reversibly influence labeling, detecting/sensing and therapy efficiencies. A number of methods have been developed to study intermolecular interactions, such as gel electrophoresis (GE) [ 1 1 - 4 4 1-4 ], electrochemistry [ 5 5 ], spectroscopy [ 1 1 , 3 3 , 4 4 , 6 6 - 11 11 6-11 ], quartz crystal microbalance (QCM) [ 12 12 - 14 14 12-14 ], surface plasmon resonance (SPR) [ 15 15 , 16 16 ], and also computer simulations [ 17 17 , 18 18 ] are popularly used in biomedical field. In general, the raw data obtained by the above methods normally needs to be further processed in combination with some classical equations like Stern-Volmer equation, Scatchard equation and Hill equation to get the binding constants, sites and even cooperativities in order to comprehensively understand intermolecular interactions. Let’s take a simple molecular interaction system as an example: R + L ⇌ RL, the binding constant can be described as K a = [RL]/([R] [L]), from which three concentrations must be figured out to calculate the final K a . However, one may only know the concentrations of both [R] and [L] at the beginning of reaction. Alternatively, they could be indirectly obtained by subtracting the final concentration of [RL] from the initial concentration of [L] or [R] at the equilibrium state. Most of the above methods adapt to measuring the concentration of [RL] , such as spectroscopy-, QCM- and SPR-based methods. However, the precision of results could be interfered somehow by the subtle deviations from the calibration curves made before binding. Hence, it will be more precise and reliable to directly measure the concentration of free [L] or [R] at the equilibrium. Inspired by the separation principle of centrifugation and size exclusion chromatography (SEC) [ 19 19 ], we can learn that both these two separation methods are based on the significant intermolecular difference between either weights or sizes. It might not be a coincidence, most of the important interactions in biomedical researches can make best use of this principle, especially for pharmaceutic researches since almost all of designed drugs are much smaller than what they really interact with in cells. With the deliberate calculation equation invented by an American chemist George Scatchard, we would like to demonstrate that the combination of Scatchard plot and ultracentrifugation/SEC can be a facile and more precise method to solve the binding constant and the number of binding sites for small-large/light-weighted molecular interactions. Eosin B (4’,5’-dibromo-2’,7’-dinitrofuorescein, EB) is a form of eosin dyes which belong to xanthenes class and have been used as histological stains [ 20 20 ], for protein estimations [ 21 21 , 22 22 ], and sonosensitizers [ 23 23 ] for decades, and its analogue eosin Y (EY) has been used for staining proteins on acid-urea polyacrylamide gels [ 24 24 ]. Bovine serum albumin (BSA), also known as "fraction V" which refers to the fifth separation fraction by using the original Edwin Cohn purification methodology, has been considered a model protein because it shares a high sequence homology of 76.52% with human serum albumin (HSA) [ 3 3 ]. Serum albumin is the most abundant drug carrier protein in blood plasma playing an important role in the transportation and distribution for endogenous and exogenous substances. In this work, BSA-EB interaction system has been studied by a comprehensive comparison of different methods since EB and BSA can be considered as the model small/light and large/weight molecules, respectively. Firstly, a combination of Scatchard equation and either ultracentrifugation or SEC for the physisorption of BSA-EB was investigated. Secondly, other methods like combination of photoluminescence (PL) quenching and either Stern-Volmer equation or Hill equation have been introduced to make comparisons. In addition, the conformational changes of BSA upon binding EB has been studied by using three-dimensional fluorescence spectroscopy (3DFS), meanwhile, the binding mode of BSA-EB with atomic resolution was simulated by using molecular docking methods. We hope this study could contribute to the investigation of small-larger molecular interaction system. 2 EXPERIMENTAL 1 2.1 Materials 2 BSA (Fraction V, lyophilized powder > 98%, Mw 66.5 kDa), EB (~ 90%, 590.09) and all other chemical regents were purchased from Sigma-Aldrich Corporation. Deionized water (18.2 MΩ•cm) used for all experiments was made from a Milli-Q system (Millipore, Bedford, MA). Both BSA and EB molecules were dissolved in Milli-Q water for a stock solution with a concentration of 600 μM. 2.2 Spectroscopic measurements 2 Absorption spectra were recorded on a U-2900 UV–Vis spectrometer (Hitachi). PL spectra were recorded with a PL spectrometer (Fluorolog®-MAX 4, Horiba) equipped with a 1.0 cm quartz cell with a fixed excitation wavelength of 280 nm, and both excitation and emission slits were set up to 5 nm. 2.3 Ultracentrifugation, SEC and PL quenching assays 2 The ultracentrifugation-based assays were conducted at 140, 000 rpm for 2 h with an ultracentrifuge (Hitachi, CS150FNX, Japan). The SEC assays were performed on a sephacryl S-300 column equipped with a high performance liquid chromatography (HPLC) system (Agilent 1260) with a flow phase of SB9 (sodium borate 50 mM, pH=9) buffer and the flow rate of 1 mL/min, and the spectra monitored at both 280 nm for BSA and 516 nm for EB by a UV-vis detector. The molar ratios of EB/BSA for both ultracentrifugation and SEC experiments are listed in Table 1 Table 1 , and the BSA’s concentration was fixed to 3 µM. As to the fluorometric titration experiment, 800 µL solutions with different EB/BSA molar ratios were used for the measurement and the final BSA’s concentration was fixed to 0.67 µM. 1 1 Vial number 1 1 BSA 1 1 EB 1 1 1 1 1 1 1 1 2 1 1 2 1 1 1 1 1 4 1 1 3 1 1 1 1 1 6 1 1 4 1 1 1 1 1 8 1 1 5 1 1 1 1 1 10 2.4 Calculation with different equations 2 Scatchard Eq.(1): <math display="block" id="M1"><mrow><mfrac><mtext>ν</mtext><mrow><msub><mtext>C</mtext><mrow><mi>f</mi><mrow><mo>(</mo><mrow><mtext>EB</mtext></mrow><mo>)</mo></mrow></mrow></msub></mrow></mfrac><mo>=</mo><mtext>n</mtext><msub><mi>K</mi><mtext>a</mtext></msub><mo>−</mo><mtext>ν</mtext><msub><mi>K</mi><mtext>a</mtext></msub></mrow></math> In Eq.(1), C f( EB ) is the concentration of free ligand which is the unbound EB’s concentration, and n is the number of binding sites per protein molecule. K a is the association/binding constant/affinity of EB for BSA. The real bound ratio ν can be obtained by Eq.(2). <math display="block" id="M2"><mrow><mtext>ν</mtext><mo>=</mo><mrow><mo>(</mo><mrow><msub><mi>C</mi><mrow><mi>t</mi><mrow><mo>(</mo><mrow><mtext>EB</mtext></mrow><mo>)</mo></mrow></mrow></msub><mo>−</mo><msub><mi>C</mi><mrow><mi>f</mi><mrow><mo>(</mo><mrow><mtext>EB</mtext></mrow><mo>)</mo></mrow></mrow></msub></mrow><mo>)</mo></mrow><mo>/</mo><msub><mi>C</mi><mrow><mi>t</mi><mrow><mo>(</mo><mrow><mtext>BSA</mtext></mrow><mo>)</mo></mrow></mrow></msub></mrow></math> C t( EB ) is the concentration of total EB which is known before the binding assay. C f( EB ) is the free or unbound EB’s concentration which can be obtained by either SED or SEC measurement. C t( BSA ) is the concentration of total BSA for the binding assay, which is known and constant in the current assay. 2.5 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.do). The Auto Dock Tools 1.5.6 package (http://mgltools.scripps.edu) was employed to generate the docking input files. The important docking parameters were recorded as followings: The center x, y, and z coordinates of the search grid of BSA were 97.127, 24.933, and 20.919, respectively. And all of the x , y , and z dimension sizes were set as 15. The default parameters were used if it was not mentioned, and the best-scoring pose judged by the Vina docking score is chosen and visually analyzed using PyMOL software (http://www.pymol.org/). 3 RESULTS AND DISCUSSION 1 The absorption spectra comparison of BSA at 280 nm before and after ultracentrifugation have proved that the current ultracentrifugation condition is enough for a perfect separation of bound EB and free EB ( Fig.1a Fig.1a ), and this has largely simplified both measurements and calculations: on one hand, normally an ultracentrifuge can contain at least six vials and this will be enough for the plot; on the other hand, one can just stop the centrifuge and take about no more than half supernatant from the vials for the concentration measurement simply with a lab pipette. With an absorption maximum dependent concentration calibration curve ( Fig.1b Fig.1b ), the free EB’s concentration can be calculated from the maximum absorption of supernatants ( Fig.1c Fig.1c ) and thus the corresponding Scatchard plot curve can be obtained ( Fig.1d Fig.1d ). With the slope and intercept from the best linear fit of the Scatchard plot, the K a and number of binding sites were determined to 0.624 µM -1 (6.24 × 10 5 M -1 ) and 3.11, respectively. By using SEC, EB molecules can be also well repeatedly, precisely eluted during 20 to 40 min. One can see there are shoulder peaks at 24 min ( Fig. 2a Fig. 2a ) of the elution spectra, which could be assigned to some micelle-like nanostructures formed by EB molecules due to the π- π interaction since it can be removed in the diluted solutions (lower than its critical micelle concentration). A calibration curve was then made by plotting the integrated eluted peaks against the elution time ( Fig. 2b Fig. 2b ), which can facilitate the calculation of the free EB’s concentration. With a control sample of free BSA, it can be clearly seen that BSA and BSA-EB cannot separated with the current SEC columns ( Fig.2c Fig.2c ). Similar to the ultracentrifugation method, the free EB’s concentrations can be directly calculated from the SEC results by running the mixtures with the varied the molar ratios of EB/BSA (as shown in Table 1 Table 1 ) on HPLC. With calculations, another Scatchard equation can be plotted ( Fig.2d Fig.2d ) and from which the binding constant K a and the number of binding sites n were obtained as 0.57 µM -1 (5.7 × 10 5 M -1 ) and 2.93, respectively. In contrast to the Scatchard method, all of Stern-Volmer equation (Eq.(3)), modified Stern-Volmer equation (Eq.(4)) and Hill equation (Eq.(5)) have been employed to analyze the binding constant as well, moreover, Hill equation can figure out the cooperativity in the binding procedure. <math display="block" id="M4"><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><mrow><mo>[</mo><mtext>Q</mtext><mo>]</mo></mrow><mo>=</mo><mn>1</mn><mo>+</mo><msub><mi>k</mi><mtext>q</mtext></msub><msub><mtext>τ</mtext><mn>0</mn></msub><mrow><mo>[</mo><mtext>Q</mtext><mo>]</mo></mrow><mo>,</mo></mrow></math> <math display="block" id="M5"><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><mrow><mo>[</mo><mtext>Q</mtext><mo>]</mo></mrow></mrow></mfrac><mo>+</mo><mfrac><mn>1</mn><mrow><msub><mi>f</mi><mtext>a</mtext></msub></mrow></mfrac><mo>,</mo></mrow></math> <math display="block" id="M6"><mrow><mtext>log</mtext><mfrac><mrow><msub><mi>F</mi><mn>0</mn></msub><mo>−</mo><mi>F</mi></mrow><mrow><mi>F</mi><mo>−</mo><msub><mi>F</mi><mrow><mtext>sat</mtext></mrow></msub></mrow></mfrac><mo>=</mo><mi>log</mi><msub><mi>K</mi><mtext>a</mtext></msub><mo>+</mo><mtext> </mtext><mi>n</mi><mtext> log</mtext><mrow><mo>[</mo><mtext>Q</mtext><mo>]</mo></mrow><mo>,</mo></mrow></math> where F 0 , F and F sat are the PL intensities in the absence, presence and saturated state of quencher, 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 quencher, f a , K a , n and [Q] are the fraction of accessible fluorophores, the effective quenching constant or the binding constant for the accessible fluorophores, number of binding sites, and the concentration of the quencher, respectively ( Table 2 Table 2 ). 1 1 Principle 2 1 Ultracentrifugation 2 1 FRET/PL quenching 1 1 Equation K a (M -1 ) 1 1 Scatchard 6.24 × 10 5 1 1 SEC 5.70 × 10 5 1 1 Modified Stern-Volmer 5.33 × 10 6 1 1 Hill 5.72 × 10 4 1 1 n 1 1 3.11 1 1 2.93 1 1 - 1 1 0.68 1 1 f a 1 1 - 1 1 - 1 1 0.94 1 1 - However, both Stern-Volmer and Hill equations based methods require PL variations, for an example the PL quenching upon binding. Owning to spectra overlap between the BSA’s PL and EB’s absorption ( Fig.3a Fig.3a ), the FRET can made best use to realize these two methods. To this end, the PL spectra were recorded for the mixtures with varied EB/BSA molar ratios ( Fig. 3b Fig. 3b ), and the EB’s PL range didn’t show up because of the strong interruption from the second harmonic peak (@ 560 nm which is very close to EB’s PL @ 548 nm) of current excitation wavelength (280 nm), which has no real reference value since the measured samples were not purified. In practice, the Stern-Volmer plot showed a downwards bent curve (Fig. 3c inset), indicating a complicated binding procedure which most of time involves both static and dynamic quenching or even conformational changes upon binding [ 25 25 ]. Hence, a modified Stern-Volmer equation was plotted again with better linear results ( Fig.3c Fig.3c ), and K a and f a was determined to 5.33 × 10 6 M -1 and 0.94, respectively. Similarly, by applying Hill equation to the corresponding data, a double logarithmic plot and its best linear fit ( Fig.3d Fig.3d ) can give out 5.72 × 10 4 M -1 and 0.68 as an additional K a and an important parameter for cooperativity n , respectively. With comparison of several references’ report, we think that both Scatchard-based ultracentrifugation and SEC results should approach the truth. However, in contrast to the Scatchard-based methods, the binding constant K a deduced from FRET-based methods are not well stable, because the difference of K a values deduced from modified Stern-Volmer and Hill equations was around two orders of magnitude, though that f a is less 1 makes much sense (the accessible fraction of EB to BSA cannot be more than 1). We attribute this huge deviation to the unstable PL of BSA due to the confirmation changes of BSA upon binding with EB. To further study the conformational information of BSA, we have employed 3DFS to see whether BSA conformation changes. 3DFS is a facile and powerful method to provide more detailed information about the conformational and micro-environmental changes of protein that combined with molecules [ 1 1 , 7 7 , 10 10 ]. The principle of 3DFS is based on that the intrinsic fluorophores (like Trp residues) in proteins, whose PL can be strongly influenced by the polarity of the local environment. For example, the PL of a protein containing a single Trp in its hydrophobic core will be red-shifted upon exposure this Trp to an aqueous environment [ 26 26 ]. From Figure 4 Figure 4 , two typical PL peaks can be clearly observed in the 3DFS of BSA (peak 1: λ ex / λ em = 276 nm / 346 nm, peak 2: λ ex / λ em = 244 nm / 350 nm). Refers to the previous report [ 27 27 ], peak 1 mainly reveals the spectral characteristics of Trp and Tyr residues, is the primary PL peak, while peak 2 may mainly exhibit the PL characteristic of polypeptide backbone structures therefore its PL intensity can be correlated with the secondary structure of proteins. The corresponding spectral parameters were summarized in Table 3 Table 3 . Combining Figure 4 Figure 4 with Table 3 Table 3 , it is clear to see that both PL intensities of peak 1 and peak 2 of BSA decrease with the gradual addition of EB, whereas the Rayleigh scattering peak is almost unchanged. Analyzing from the intensity changes of both peak 1 and peak 2, the quenching results indicated that the interaction of EB with BSA induced a slight unfolding of the protein polypeptides, resulting in a conformational change of the protein, this could increase the exposure region of some hydrophobic regions that have previously been buried. Moreover, we realized that the maximum of PL of BSA occurred a blue shift due to water Ramon peak, which might be the one of the main reasons that cause the huge deviation of results obtained from two PL quenching-based methods. * I F is the PL intensity from the 3DFS. 1 2 EB/BSA molar ratios 3 1 Peak 1 3 1 Peak 2 1 1 Peak position λ ex /λ em (nm/nm) 1 1 Stokes shift ∆λ (nm) 1 1 I F * 1 1 Peak position λ ex /λ em (nm/nm) 1 1 Stokes shift∆λ (nm) 1 1 I F * 1 1 0 1 1 276/345 1 1 69 1 1 1.88 × 10 6 1 1 236/351 1 1 115 1 1 4.38 × 10 5 1 1 2 1 1 276/346 1 1 70 1 1 1.66 × 10 6 1 1 244/346 1 1 102 1 1 3.78 × 10 5 1 1 4 1 1 276/346 1 1 70 1 1 1.40 × 10 6 1 1 244/350 1 1 106 1 1 3.21 × 10 5 1 1 8 1 1 276/344 1 1 68 1 1 9.65 × 10 5 1 1 248/346 1 1 98 1 1 2.76 × 10 5 With the aim to get the detailed binding site information at the atomic level, the molecular docking method [ 19 19 ] was employed by using Auto dock software package. As shown in Fig. 5 Fig. 5 , EB can be perfectly docked into the binding pocket of the BSA. The benzoic acid ring of EB fits into bottom of the binding pocket of BSA, surrounded by the residues Trp 213 , Arg 217 , Arg 194 and Arg 198 . Detailed analysis shows that carboxy group forms a hydrogen bond (2.9 Å) with residue Arg 198 . Furthermore, carbonyl oxygen forms two hydrogen bonds (2.3 and 3.6 Å) with residue Arg 198 , while the nitro group forms two hydrogen bonds (3.1 and 3.3 Å) with residue Asp 450 and Arg 194 , respectively, which could be the main binding affinity between EB and BSA. 4 Conclusion 1 The BSA-EB interaction has been quantified by successfully introducing a combinational method to quantify the interaction between two molecules by taking the advantages of significant differences of either sizes or weights. In comparison with PL quenching-based methods, the current introduced methods are simpler to operate and lead to results that are more precise. 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Interaction of human serum albumin with 10-hydroxycamptothecin: spectroscopic and molecular modeling studies 10.1007/s41365-016-0123-5 This work was supported by the National Natural Science Foundation of China (Nos. 21171086 and 81160213), the Inner Mongolia Autonomous Region science and Technology Department (No. 211-202077), the Inner Mongolia Grassland Talent (No. 108-108038), the Natural Science Foundation of Inner Mongolia Autonomous Region of China (Nos. 2013MS1121, 2015ms0806 and 2016MS0211) and the Inner Mongolia Agricultural University (Nos. 109-108040, 211-109003 and 211-206038) . 2016-06-24 2016-08-04 2016-08-12 2016-08-25 2016-10-20 2021-04-11 © China Science Publishing & Media Ltd. (Science Press), Shanghai Institute of Applied Physics, the Chinese Academy of Sciences, ChineseNuclear Society and Springer Nature Singapore Pte Ltd. 2016 This is a post-peer-review, pre-copyedit version of an article published in Nuclear Science and Techniques. The final authenticated version is available online at: http://dx.doi.org/10.1007/s41365-016-0123-5 http://dx.doi.org/10.1007/s41365-016-0123-5 . 2016 Nuclear Science and Techniques 05 27