In recent years, Gemini surfactants have emerged as promising surface active agents. A Gemini surfactant is made up of two hydrophobic chains and two polar head groups covalently linked through a spacer group.1 In comparison to conventional single-chain surfactants, Gemini surfactants usually exhibit much lower critical micelle concentration (CMC), strong dependence on spacer structure, special aggregate morphology, and strong hydrophobic microdomain.2 Because of these characteristics, Gemini surfactants have been widely exploited in many areas of research, such as new materials synthesis, pharmaceuticals (drug vehicles), and biochemistry (gene therapy).3
Bovine serum albumin (BSA), a type of globular protein having 583 amino acid residues with a single polypeptide chain, function biologically as carriers of fatty acid anions and other amphiphiles in the bloodstream.4 Protein is also a type of polyampholyte with a special structure—due to the hydrophobic and hydrophilic properties of the amino acids, proteins exhibit dualism, which can make small amphiphilic molecules interact with them.5 For this reason, the interaction between protein and Gemini surfactant is suggested to be much more unique and complicated.
The interaction of surfactants with proteins is important in a wide variety of industrial, biological, pharmaceutical, and cosmetic applications.6 When drug delivery, protein purification, and biological enzyme catalysis occur in microemulsion solution, they are all involved in the interaction between protein and surfactant. Thus, studying the interaction can further our understanding of the intrinsic mechanism and tell us how Gemini surfactants can be used.
The conformational changes induced in the protein, when the surfactant binds to it, result in changes in polarity and protein stability.7 The mechanism of protein unfolding after the addition of the surfactant has been studied using several techniques, i.e., circular dichroism (CD),8fluorescence spectroscopy, nuclear magnetic resonance (NMR),9 microcalorimetry,10light scattering,11 and small-angle neutron scattering (SANS).12 Among them, fluorescence is the most frequently used, since the changes in the intensity of the emission arising from the tryptophan residues allow the surfactant binding efficiency to be determined and the protein structure to be elucidated.13
Figure 1 - Structure of Gemini surfactant G12-3-12.
The aim of the present work was to investigate the interaction between BSA and Gemini surfactant G12-3-12 (as shown in Figure 1). The effects of BSA on the micellization and microenvironment of the Gemini surfactant G12-3-12 were also analyzed. The physiochemical properties and details of the interaction were mainly evaluated using steady-state fluorescence methods.
Experimental
Materials
All reagents used for synthesis were of analytical grade unless otherwise stated. Double- distilled water was used throughout the work. The Gemini surfactant propane-1,3-bis(dodecyldimethyl ammonium bromide) (G12-3-12) was synthesized as described in Ref. 14.
The working concentration of G12-3-12 (synthesized in the authors’ laboratory) was 0.01 mol/L. The stock solution of BSA (purchased from Wuhan Tianyuan Biotechnology Co. Ltd., Wuhan, China) was prepared by directly dissolving protein in double-distilled water and storing at 1–4 °C. Pyrene (Py) (Alfa Aesar, Beijing, China), used as a fluorescence probe, was dissolved in methanol. Methanol was also chosen as a solvent in benzophenine (Bp) solution (Tianjin BoDi Chemical Co., Ltd., Tianjin, China), which can quench the fluorescence of Py. The prepared concentrations of Py and Bp were 1.0 × 10–3 mol/L and 5 × 10–3 mol/L, respectively.
Apparatus and methods
Steady-state fluorescence measurements and titration curves were performed on an RF-540 spectrofluorophotometer (Shimadzu, Tokyo, Japan) equipped with 1.0- cm quartz cells. During the experiments, the fluorescence spectra of BSA were recorded from 300 to 500 nm at an excitation wavelength of 280 nm. The synchronous fluorescence spectra were obtained when Δλ was 15 nm and 60 nm, respectively. The width of both the excitation slit and the emission slit was set to 10 nm.
Py stock solution (20 μL) was added to the samples prepared by diluting the different G12-3-12 stock solutions. Py spectra were collected in the 360–450 nm range when the fixed excitation was 335 nm. The excitation and emission slit widths were 10 nm and 2 nm, respectively. The intensities of I1 and I3 were taken from emission intensities at 375 nm and 385 nm, respectively.
Py was used as the probe to determine the microenvironmental polarity by observing its fluorescence fine structure. The critical micelle concentration (CMC) of G12-3-12 can be obtained using the intensity ratio I1/I3 of Py.15–17 The fluorescence quenching studies were carried out using Py as the probe and Bp as the quencher in order to investigate the micellar aggregation numbers.
Results and discussion
Intrinsic fluorescence studies
Figure 2 - Effect of G12-3-12 on fluorescence spectrum of BSA. CG12-3-12 from (a) to (e): 0, 0.02, 0.06, 0.1, and 0.2 × 10–4 mol/L, respectively; CBSA: 4.00 × 10–7 mol/L.
Figure 3 - Stern-Volmer plots for the quenching of BSA by G12-3-12 at different temperatures. CBSA: 3.99 × 10–7 mol/L.
- Fluorescence spectra. Variations in the fluorescence intensity and the wavelength of emission maximum, parameters sensitive to protein conformation, can be used effectively to probe protein folding and unfolding.18 The interaction of G12-3-12 with BSA was evaluated by monitoring the fluorescence intensity changes of BSA upon the addition of G12-3-12 solution. The fluorescence spectrum of BSA at different concentrations of G12-3-12 solutions is shown in Figure 2. BSA had a strong fluorescence emission that peaked at 350 nm upon excitation at 280 nm. In addition, it was found that the BSA fluorescence intensity was quenched with an obvious blue shift of maximum emission wavelength when the surfactant concentration was increased. This phenomenon indicated the existence of an interaction between G12-3-12 and BSA, and the conformation of BSA had been changed.
- Fluorescence quenching investigation. As shown, the fluorescence of BSA was quenched by G12-3-12. The quenching mechanism and interaction properties were then studied.
Quenching can occur by different means, which are usually classified as dynamic quenching and static quenching. In general, these two quenching mechanisms can be distinguished by their dependence on temperature. The fluorescence quenching data are analyzed by the Stern-Volmer equation19:
F0/F = 1 + Ksv[Q] (1)
Table 1 - Quenching constants and thermodynamic parameters of G12-3-12/BSA interaction at different temperatures
where F0 and F are the fluorescence intensities of BSA in the absence and presence of the quencher, respectively. Ksv is the Stern-Volmer quenching constant, and [Q] is the concentration of quencher (G12-3-12). Eq. (1) was applied to determine Ksv by linear regression of a plot of F0/F against [Q]. The Stern-Volmer plots at different temperatures (295K, 303K, and 311K) are shown in Figure 3; the data are listed in Table 1. The plots were found to be linear at the experimental concentration range, and the slopes increased with increasing temperature, which was consistent with the dynamic type of quenching mechanism. Dynamic quenching depends upon diffusion, since higher temperatures result in larger diffusion coefficients.20