- Nano Express
- Open Access
Evaluating interaction forces between BSA and rabbit anti-BSA in sulphathiazole sodium, tylosin and levofloxacin solution by AFM
© Wang et al; licensee Springer. 2011
- Received: 31 July 2011
- Accepted: 3 November 2011
- Published: 3 November 2011
Protein-protein interactions play crucial roles in numerous biological processes. However, it is still challenging to evaluate the protein-protein interactions, such as antigen and antibody, in the presence of drug molecules in physiological liquid. In this study, the interaction between bovine serum albumin (BSA) and rabbit anti-BSA was investigated using atomic force microscopy (AFM) in the presence of various antimicrobial drugs (sulphathiazole sodium, tylosin and levofloxacin) under physiological condition. The results show that increasing the concentration of tylosin decreased the single-molecule-specific force between BSA and rabbit anti-BSA. As for sulphathiazole sodium, it dramatically decreased the specific force at a certain critical concentration, but increased the nonspecific force as its concentration increasing. In addition, the presence of levofloxacin did not greatly influence either the specific or nonspecific force. Collectively, these results suggest that these three drugs may adopt different mechanisms to affect the interaction force between BSA and rabbit anti-BSA. These findings may enhance our understanding of antigen/antibody binding processes in the presence of drug molecules, and hence indicate that AFM could be helpful in the design and screening of drugs-modulating protein-protein interaction processes.
- Bovine Serum Albumin
- Atomic Force Microscopy
- Adhesion Force
- Specific Force
A molecular level understanding of protein-protein interactions is fundamentally important in the life sciences. A number of human diseases are closely related to the protein-protein association or dissociation events and thus probing and characterizing these interactions have become increasingly significant in the development of novel drugs and medical diagnostics [1–4]. Different solution conditions, such as pH, temperature, ion species, and strength, may influence the protein-protein interactions as previous studies have demonstrated [5–7]. This is particularly important in drug discovery and the computer-aided drug design (CADD) method has identified molecules modifying protein-protein interactions as potential drug candidates [8, 9]. However, the computer studies do not provide more detailed information on forces at nanoscale-to-molecular scale that influence protein-protein interactions, which would allow us to better understanding the factors of drug molecules affecting the interactions. Therefore, it is still challenging to evaluate the protein-protein interactions, such as that between antigen and antibody, in the presence of drug molecules in physiological liquid.
By using an atomic force microscopy (AFM), it has been possible to measure directly the specific and nonspecific force between proteins at molecular scale. AFM is widely applied to characterize biological molecular recognition processes because of its high force sensitivity and the capability of operating under different physiological conditions [15–18]. We have previously testified an experimental method for the characterization of the specific and nonspecific interaction force between human immunoglobulin G (IgG) and rat anti-human IgG in phosphate buffered saline (PBS). Self-assembled monolayer (SAM) method was used for sample preparation and AFM was employed for interaction force measurement . SAM method has been proved to be a facile and effective way to form well-defined and controlled films for AFM sample preparation [20, 21]. In this article, we investigated the interaction between BSA and rabbit anti-BSA when it was measured by AFM in either PBS or PBS solution containing one of the three antimicrobial drugs (sulphathiazole sodium, tylosin, and levofloxacin) under physiological conditions. The results suggest that these three drugs may adopt different mechanisms to affect the interaction force between BSA and rat-anti BSA.
To investigate protein-protein interactions through AFM, we used a thiol-based SAM for protein immobilization because of its effectiveness and simplicity, which is similar to our previous report . In brief, sulphur-containing molecules (thiols, sulphides, and disulphides) have a strong affinity for gold and will interact with it in near covalent manner. Therefore, when gold is immersed into a solution of thiols such as 16-mercaptohexadecanoic acid (MHA), the thiol molecules will spontaneously react with gold and form a SAM of thiols on the gold surface with tightly packed and well-ordered chains. The terminal end of the thiol-based SAM consists of carboxyl tail groups that can be activated by the 1-ethyl-3-(dimethylaminopropyl) carbodi-imide hydrochloride (EDC) and N-hydroxysulphosuccinimide (NHS). The activated SAM can then be soaked into protein solution to form protein layer.
2.1. Gold-coated substrate
Gold-coated substrates were prepared by vapor deposition of gold onto freshly cleaved mica in a high vacuum evaporator at approx. 10-7 Torr. Mica substrates were preheated to 325°C for 2 h by a radiator heater before deposition. Evaporation rates were 0.1-0.3 nm/s, and the final thickness of the gold films was approx. 200 nm. A chromium layer was also vapor deposited and sandwiched between the gold and mica to strengthen the adhesion between the surfaces. The gold-coated substrate was then annealed in H2 flame for 1 min before use.
2.2. SAM of thiols on gold surface
The bare gold-coated substrate prepared as above was thoroughly cleaned in hot piranha solution (v/v H2SO4:H2O2 = 3:1) for 30 min. The gold-coated substrate was then immersed into the ethanol solution of 1 mM MHA for 24 h to produce the thiol-based SAM on the gold surface, and unbound thiols were removed by ultrasonication in pure ethanol for 2 min. The prepared SAM was then rinsed sequentially with pure ethanol, ultra pure water, and finally dried in a N2 stream before use.
2.3. Protein immobilization onto the SAM
BSA was covalently immobilized on a gold-coated substrate through the condensation reaction between the amino groups in the protein and the carboxyl groups on the gold-coated substrate . In brief, SAM with carboxylic acid terminal groups was activated by 2 mg/mL NHS and 2 mg/mL EDC in PBS for 1 h, and subsequently rinsed thoroughly with ultra pure water, and dried in N2 stream. The activated SAM was then immersed into 5 μg/mL BSA in PBS at 4°C for 12 h. Finally, the prepared sample of protein layer was kept in PBS at 4°C until use.
2.4. Functionalization of AFM tip
Functionalized AFM tip with rabbit anti-BSA coating was prepared similarly as described above.
2.5. Measurement of antigen-antibody adhesion force by AFM in drug solutions
Adhesion force between BSA and rabbit anti-BSA was measured by AFM using Benyuan CSPM 5000 scanning probe microscope (Benyuan Co., China). The functionalized AFM tip scanned across the well-ordered protein monolayer. At a given location, the tip was moved toward the surface of the monolayer and retracted. When the tip approached the monolayer surface it would deflect because of the antigen-antibody interaction force, which would be detected as a "voltage-displacement" signal and converted into a "force-displacement" curve [24, 25]. Because the tip was considered an elastic cantilever, its deflection was determined by the force (F) exerted on it following Hooke's law, i.e., F = k × d, where d is the deflection, k is the spring constant of the cantilever tip. In general, k should be small for AFM to minimize measurement noise. In this study, commercially available Si3N4 cantilever tip (BudgetSensors®, Innovative Solutions Bulgaria Ltd., Bulgaria) was used of which the spring constant, calibrated by thermal fluctuation method , was 0.2-0.3 N/m. The tip has a pyramidal geometry. Its tip radius is about 25 nm and the thickness of the gold layer is 70 nm.
All force measurements were performed using contact mode AFM at room temperature (25°C). The functionalized AFM tip with rabbit anti-BSA was used to measure the adhesion force between the substrate of BSA and the tip of rabbit anti-BSA in PBS as control experiment. The retraction velocity was estimated to be 0.04 μm/s, and all the measurements were observed under this condition. From the "force-displacement" curve, the adhesion force was calculated. Measurement was repeated about 50-55 times at each of 5 randomly selected locations across the protein monolayer on the gold substrate. To mimic the various antimicrobial drug solution media, the PBS in control experiment was separately changed to sulphathiazole sodium, tylosin, and levofloxacin solution (one of the drugs dissolved in PBS) over a concentration range of 10-70 mM. A complete series of measurements in the control and in each of the drug solutions were conducted using the same functionalized AFM tip. The five selected locations across the protein monolayer in control experiment were measured in the drug solutions.
2.6. AFM imaging
All images were acquired using Benyuan CSPM 5000 scanning probe microscope (Benyuan Co., China) equipped with a 1.6-μm E scanner. Commercial Si3N4 cantilevers (BudgetSensors) with resonant frequency of 200 kHz were used. AFM worked with tapping mode in PBS and drug solutions at typical scanning rate of 2.0 Hz and scanning size of 1000 nm × 1000 nm. The roughness of surfaces in different solutions was analyzed by CSPM Image 4.62 software program (provided by the manufacturer).
16-MHA, 1-ethyl-3-(dimethylaminopropyl) carbodi-imide hydrochloride (EDC), NHS, sulphathiazole sodium, tylosin, and levofloxacin were purchased from Sigma Aldrich Chemical Co. and used as-received. PBS (140 mM NaCl, 3 mM KCl, pH 7.4) and ethanol (guaranteed grade) were purchased from Merck Co., and ultra pure water (resistivity of 18.2 MΩ cm) was obtained by Millpore purification system. BSA and rabbit anti-BSA were purchased from Biosun Co. (China).
Adhesion forces between BSA and rabbit anti-BSA measured at five different locations on BSA substrate in PBS, sulphathiazole sodium, tylosin and levofloxacin solution (10 mM)
Mean force μ m (pN)
Variance of force σ m 2 (×104 pN2)
Number of measurement (n)
PBS (control experiment)
PBS+ sulphathiazole sodium
The largest nonspecific force observed in sulphathiazole sodium solution (Figure 4b) could be attributed to the effect of increasing solution ionic strength (IS). Both BSA and rabbit anti-BSA are negatively charged when immersed in solution (pH 7.4), as the isoelectric points of BSA and rabbit anti-BSA are 4.7, 4.8-5.2, respectively . Increasing the solution IS compressed the thickness of the electrostatic double layer surrounding proteins, and finally resulted in an increase in nonspecific adhesion. This phenomenon is qualitatively consistent with predictions based on DLVO (Derjaguin, Landau, Verwey, Overbeek) theory as an increase in the solution IS will reduce the range of electrostatic repulsion between two negatively charged surfaces [36, 37]. Similar effect was reported by Javid et al. . They observed that the positive charge on the lysozyme molecule was screened by the salt anions as the salt concentration increased, hence diminishing the strong repulsive protein-protein interactions. In addition, increasing the solution IS may disrupt the hydration shell coating on protein surfaces and thus reduce repulsive interactions between the two interacting surfaces . Benítez et al.  studied the effect of IS on the stability of apple juice particles which are mainly composed of proteins and carbohydrates. They concluded that increasing IS resulted in reduction of surface charge and hydration constant, and led to an increase in adhesion. Compared with tylosin molecule, the spatial structure of sulphathiazole sodium salt in solution is smaller and sulphathiazole sodium molecules may not cover available binding sites and weaken the specific adhesion between antigen and antibody. In levofloxacin solution, the specific adhesion force and nonspecific force are almost equal to the force values in PBS. This suggests that levofloxacin as a small nonionic drug may not affect the interactions of BSA and rabbit anti-BSA because of neither bigger spatial structure of levofloxacin molecule nor increasing IS in solution.
The interaction between BSA and rabbit anti-BSA was investigated by AFM in PBS and three antimicrobial drug (sulphathiazole sodium, tylosin and levofloxacin) solutions under physiological conditions. The results suggest that increasing the concentration of tylosin solution decreased the single-molecule-specific force, demonstrating the important contribution of tylosin molecules spatially covering available binding sites to decreased specific adhesion force. At a certain critical concentration of sulphathiazole sodium, the single-molecule-specific force decreased dramatically because of the change in the initial conformation of the BSA monolayer. The nonspecific force increased as the concentration of sulphathiazole sodium increased, suggesting that sulphathiazole sodium as an ionic drug increasing solution IS was the dominant mechanism of nonspecific force. The presence of levofloxacin as a small nonionic drug did not significantly affect the interactions of BSA and rabbit anti-BSA in the solution. These findings may enhance our understanding of antigen/antibody binding processes in the presence of drug molecules, and hence indicate the AFM could be helpful in the design and screening of drugs modulating protein-protein interaction processes.
This study was supported by the National Natural Science Foundation of China (No. 30670496, 30770529) and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (2006-331) and the Natural Science Foundation Project of CQ CSTC (2006BB5017).
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