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. 2019 Dec 9;4(26):22152–22160. doi: 10.1021/acsomega.9b03315

Molecular Interaction of Amino Acid-Based Gemini Surfactant with Human Serum Albumin: Tensiometric, Spectroscopic, and Molecular Docking Study

Jeenat Aslam †,*, Irfan Hussain Lone †, Nagi R E Radwan †, Mohd Faizan Siddiqui ‡, Shazia Parveen †, Rua B Alnoman †, Ruby Aslam §
PMCID: PMC6933778  PMID: 31891097

Abstract

graphic file with name ao9b03315_0007.jpg

Binding effect and interaction of N,N′-dialkyl cystine based gemini surfactant (GS); 2(C12Cys) with human serum albumin (HSA) were systematically investigated by the techniques such as surface tension measurement, UV−visible spectroscopy, fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular docking studies. The surface tension measurement exhibited that HSA shifted the critical micelle concentration of the 2(C12Cys) GS to the higher side that confirms the complex formation among 2(C12Cys) GS and HSA which was also verified by UV–visible, fluorescence, and CD spectroscopy. Increase in the concentration of 2(C12Cys) GS increases the absorption of the HSA protein but has a reverse effect on the fluorescence intensity. The analysis of UV–visible study with the help of a static quenching method showed that the value acquired for the bimolecular quenching constant (kq) quenches the intrinsic fluorescence of the HSA protein. Synchronous fluorescence spectrometry declared that the induced-binding conformational changes in HSA and CD results explained the variations in the secondary arrangement of the protein in presence of 2(C12Cys) GS. The present study revealed that the interaction between 2(C12Cys) GS and HSA is important for the preparation and properties of medicines. Molecular docking study provides insight into the specific binding site of 2(C12Cys) GS into the sites of HSA.

1. Introduction

Proteins are most critical constituents of life that plays an important role in living species and have a tendency to combine with inorganic/organic moieties, for example, fatty acids, hematin, bilirubin, surfactants, metal ions, and drugs.1−7 The interaction of protein–surfactant played a vital role in a broad variety of industrial aspects, for example, biotechnological and biosciences processes, gene delivery, drug delivery, coatings, cosmetics, food industry, and production of pharmaceutical materials such as blood serum, a blend of human serum albumin (HSA) with an integer of compounds, comprising small molecular surface-active molecules. The surface tension of such biological fluids is used as a diagnostic and therapeutic device.8

Proteins are the active components in the area of medicine because they have an affinity to bind various molecules along with the ability to catalyze various biochemical reactions, viz., superoxide dismutase.9 They also form protein–surfactant complexes by adhering to various surfactant molecules, in which the hydrophobic parts of the surfactant molecules tend to bind with the interior of the hydrophobic residues of proteins.10,11 Therefore, an understanding of such phenomena could be helpful in evaluating the effect of surfactants on protein denaturation, solubilization, and renaturation processes.12−14 On the other hand, protein aggregation considered as an important phenomenon, which can be responsible for various human diseases and often viewed as an undesired effect in the field of pharmacy.15−19 The molecular mechanism of protein aggregation is still not interpreted, posing a challenge to researchers.20

Literature reports reveal that there are relatively few scientific reports concerning the interaction of proteins with a novel class of dicationic amphiphilic compounds. They are termed as gemini surfactants (GSs) consisting of two polar groups and two hydrophobic chains. They display higher surface activity, better solubility, and capability of foaming in comparison to conventional surfactants.21−23 The properties of various structural forms of GSs can be adjusted depending on the length of the hydrophobic chains and the distance between the polar groups, in addition to their overall chemical structure. Typically, much lower concentrations of GSs are required to perform the desired function, concluding to their limited impact on the environment.24 In water, GSs form structures similar to the structure of the biological membrane, additionally diminishing the possible toxicity on the human body in medicinal applications.25,26 Hence, these GSs are considered as a motivating group of surfactants because of their other unusual properties, as compared to their conventional surfactant homologs.27,28

HSA consists of 585 amino acid units (in a particular polypeptide chain) and large cysteine content. HSA is a moving protein, having a molecular weight of ∼66 kDa available in blood plasma, playing an important role in binding with several exogenous and endogenous compounds in the human body.29,30 HSA, having a single amino acid unit (tryptophan; Trp 214), was investigated by intrinsic fluorescence. Three α-helical domains (I, II, and III) are present in the HSA structure31 that supports to maintain the colloidal blood pressure and pH of the plasma.32 Numerous ligands bind to serum albumins and therefore, act as model proteins to investigate biophysical and biochemical properties.33

Many researchers report that serum albumins have a similarity to various biologically energetic compounds such as drugs, metabolites, and so forth, which control their metabolism in blood.34,35 The folding and unfolding determines the interaction and binding of HSA with amphiphilic molecules by its concentration and the surfactant concentration that has been used.36,37 It is significant to investigate the binding procedure carefully to find out the binding parameters, number of binding sites and the energetics involved for evaluating the sharing and efficiency of the ligand.

The aim of our work was to find out the substantial molecular interaction between an amino acid-based GS derived from cystine, N,N′-dialkyl cystine 2(C12Cys) GS with HSA, which form protein–surfactant complexes. This particular HSA protein was chosen because of its pharmaceutical and physiological properties. Based on the surveys undertaken, the present study is an endeavor to describe the effect of hydrophobicity on the protein conformation and stability.

2. Results and Discussion

2.1. cmc and Interfacial Adsorption

Surface tensiometry is considered an easy and effectual measurement to expose the macromolecule/surfactant interactions. The representative surface tension versus log surfactant concentration plotted for 2(C12Cys) GS in the absence and presence of HSA are shown in Figure 1. At small concentrations, the molecules of the surfactant adsorb at the air/liquid interface until the liquid surface is fully occupied. Additionally, when the excess molecules tend to self-associate in the solution to form micelles, the surface tension becomes constant. In the presence of phosphate buffer, the critical micelle concentration (cmc) value of 2(C12Cys) GS is smaller than the cmc (0.00109 mM) value acquired in the aqueous medium38 and is because of the high ionic environment exerted by the phosphate buffer. It has been found (Figure 1) that in the presence of HSA, the smaller value of cmc of 2(C12Cys) GS specify the interaction among HSA and 2(C12Cys) GS. It may be because of the presence of overall negative charge on the HSA protein at a particular pH of 7.4 and this negative charge controls the electrostatic repulsions between the head groups of 2(C12Cys) monomers, which in turn support aggregation of surfactant molecules. Furthermore, cation−π interactions (among protein aromatic parts and head groups of surfactants) and hydrophilic/hydrophobic contributions have also been reported for cmc decrease.39

Figure 1.

Figure 1

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Surface tension profile of 2(C12Cys) GS in the absence and presence of 4 μM HSA in buffer solution (pH 7.4).

To give more insights into 2(C12Cys) GS + HSA interaction, interfacial parameters at the air/liquid interface and the resulted parameters are given in Table 1. ∏cmc (surface pressure) were calculated by employing the equation

2.1. 1

where γ0 and γcmc stand for the solvent and the mixture surface tension at the cmc, respectively.

Table 1. Surface Properties (cmc, ∏cmc, Γmax, Amin, ΔGm°, and ΔGads°) for 2 (C12Cys) GS in the Absence and Presence of 4 μM HSA (Aqueous Solution, pH 7.4) at 25 °C.

sample cmc (mM) γcmc (mN/m−1) ∏cmc (mN/m−1) Γmax (107 × mol/m−2) Amin (Å2) ΔGmic° (kJ/mol−1) ΔGads° (kJ/mol−1)
2(C12Cys) 0.0011 35.14 35.86 18.01 92.18 –27.28 –29.27
2(C12Cys) + HSA 0.00087 41.08 29.92 20.26 81.93 –27.87 –29.35
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The reducing and rising values of γcmc and ∏cmc in mixed systems specify that the efficacy of the system rises as shown in Table 1. The Γmax (Gibbs surface excess concentration) was evaluated by applying the equation40

2.1. 2

where R and T are the universal gas constant (8.314 J mol–1 K–1) and temperature in kelvin, respectively. The prefactor n is the species number at the air/liquid interface and n = 1 (because of swamping quantity of the electrolyte).41,42 It is found from Table 1 that in the presence of HSA, the concentration of surface excess is enhanced. This means that many 2(C12Cys) molecules get adsorbed at the surface interface. The minimum values of the surface area per molecule (Amin) calculated by employing the equation

2.1. 3

where NA is the Avogadro’s number (6.023 × 1023 mol–1) were established to reduce in the presence of HSA, which supports the concept that sites of negatively charged or negatively charged amino acid residues on HSA possess more electrostatic repulsions between the head groups. This implies the efficiency and denseness of 2(C12Cys) monomers to occupy the interface. The micellization and adsorption Gibbs free energy change were calculated by using equations.43,44

2.1. 4
2.1. 5

where Xcmc is the cmc of the mixture of the two components at a given mole fraction.

The standard free energies (ΔGmic° and ΔGads) are commonly used to examine whether the adsorption at the air/liquid interface and the micellization in solution is enhanced. The ΔGmic° and ΔGads values for 2(C12Cys) GS + HSA and alone 2(C12Cys) GS are given in Table 1. The negative values of both ΔGmic° and ΔGads indicate that the absolute values for 2(C12Cys) GS + HSA are higher than those for 2(C12Cys) GS alone. In both the cases, the values for adsorption are also significantly higher than those for micellization (Table 1). It could be inferred that adsorption at the air/liquid interface is more than the micellization in solution and 2(C12Cys) GS + HSA adsorb more efficiently than 2(C12Cys) GS alone. The negative value of both ΔGmic° and ΔGads indicates that both processes are spontaneous in the medium. The intermolecular or intramolecular hydrophobic interactions of 2(C12Cys) GS and HSA are significantly favored, possibly owing to the protein unfolding and complex formation of surfactant–protein, therefore resulting in the adsorption at the interface before micelle formation in solution.

2.2. Steady-State Fluorescence Measurements

Steady-state fluorescence spectroscopy is a useful method to investigate the microenvironment differences in the area of the fluorophore and to examine the protein tertiary structure. Figure 2a reveals the HSA fluorescence spectra with and without 2(C12Cys) GS concentration (0.1–1 μM), which exhibits quenching fluorescence intensity of HSA. This behavior may be attributed to HSA–2(C12Cys) GS complex formation and burial residues of aromatic amino acids, namely, Trp (tryptophan), Tyr (tyrosine), and Phe (phenylalanine) to a nonpolar environment. The deviations in the fluorescence spectra acquired by protein excitation at 295 nm have been ascribed to the existence of Trp residues while the variations that result from protein excitation at 280 nm are connected with Trp and Tyr residues.45 As the intensity of fluorescence and λmax (emission maximum) changes, protein conformation occurs. Such parameters become essential tools in examining protein folding or unfolding methods.46−48 The quantum yield of tryptophan residues was much higher than tyrosine, and in proteins where both Tyr and Trp residues are present; Tyr is generally quenched by Trp residues. The deviations that were shown at 280 and 295 nm were found to be similar and were not revealed.

Figure 2.

Figure 2

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Fluorescence quenching results of the HSA/2(C12Cys) GS system: (a) quenching profiles with varying concentrations of the surfactant, (b) Stern–Volmer plot, and (c) modified Stern–Volmer plot.

Fluorescence quenching mechanisms are explained by the two ways; static and dynamic. The static mechanism involves the arrangement of ground state complex formation among the fluorophore and quencher, whereas dynamic quenching proceeds through the excited state complex formation. The improved fluorescence quenching mechanisms were obtained from the analysis of quantitative results. The quenching process was calculated via the Stern–Volmer equation.49

2.2. 6

where F0 and F stand for the fluorescence intensities without and with the quencher, and [Q] and KSV specify the quencher concentration and the Stern–Volmer quenching constant, respectively.

The value of KSV (4.41 × 105 L mol–1) found from the slope of Figure 2b, gives substantial quenching of the HSA emission spectra by 2(C12Cys) GS. The value of KSV was then used to calculate the Kq (quenching rate constant) from the following eq 7

2.2. 7

where, τ0 is the average fluorophore life time and is 10–8 s for biomolecules.

From Table 2, it is fairly evident that the quenching rate constant values were in the order of 1013 L mol–1 s–1, whereas scatter collision quenching constants with biopolymers were in the order of 1010 L mol–1 s–1. The greater magnitude of the quenching rate constant value reveals that quenching occurs through a static mechanism rather than the dynamic procedure and consists of the ground state complex formation among 2(C12Cys) GS and HSA.50−52

Table 2. Stern–Volmer Quenching Constant (KSV), Bimolecular Quenching Constant (kq), Binding Constant (Ka), and Binding Sites (n) for the Binding of 2(C12Cys) GS with HSA in Buffer Solution (pH 7.4) at 25 °C.

sample Ksv (105 L mol–1) kq (1013L mol –1 s–1) R2 Ka (104 mol L–1) n ΔGb° (kJ/mol−1)
2(C12Cys) + HSA 4.41 4.41 0.989 3.11 1.19 –25.63
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The modified Stern–Volmer eq 8 was used to calculate the Kb (binding constant) and n (binding sites number). Log Kb (antilog of the intercept) and n (slope) of Figure 2c were used to estimate the Kb and the n, respectively.

2.2. 8

where Kb represents the apparent binding constant of 2(C12Cys) GS and n is the number of binding sites. The intercept and slope shown in Figure 2c were utilized to determine Kb and n, respectively. In Table 2 the n value exhibits only one binding class for 2(C12Cys) GS. The binding Gibbs free energy upon surfactant combination was determined by the equation53

2.2. 9

The negative value of Gibbs free energy indicates that the combination of 2(C12Cys) GS and HSA was spontaneous and thermodynamically favored.

2.3. Synchronous Fluorescence Measurements

The fluctuation of the microenvironment near fluorophores (Tyr/Trp) was also examined via the synchronous fluorescence measurement. As compared to conventional fluorescence, the measurement of synchronous fluorescence was appropriate owing to its specificity toward aromatic residues. It also diminishes spectral overlaps via weakening spectral bands and simplifies the spectra by using a suitable wavelength.54 Observance of the Δλ among the excitation and emission wavelengths at 15 and 60 nm, respectively, and the deviations of the microenvironment around tyrosine and tryptophan residues were probed.55,56 The related synchronous fluorescence spectra (Figure 3a,b) depicts that addition of 2(C12Cys) GS creates quenching around the fluorophores (Tyr or Trp), showing the interaction and binding of 2(C12Cys) GS with HSA. This quenching in fluorescence is due to an exposure of an aromatic residue to hydrophobic tail of 2(C12Cys) GS. The interactions are more successfully apparent in Figure 3a than in Figure 3b; the cause was due to the greater affinity of 2(C12Cys) GS molecules toward tryptophan (Trp) than that of the tyrosine (Tyr) residue. This statement is in coherence with the results found in steady-state fluorescence.

Figure 3.

Figure 3

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Synchronous spectra of HSA in the presence of varying concentrations of 2(C12Cys) GS at 298 K (a) Δλ = 15 nm (represents contributions due to tyrosine), and (b) Δλ = 60 nm (represents contributions due to tryptophan).

2.4. UV–Visible Absorption Spectra

UV–visible absorption spectroscopy is a valuable tool to examine the complex formation of ligand–protein.57,58 The transitions from highest occupied molecular orbital to lowest unoccupied molecular orbital include σ → σ*, π → π*, n → σ*, and n → π*, which are allowed transitions. In proteins, the essential amino acids and binding cofactors usually have conjugated π systems which can absorb the light. The HSA absorption spectra without and with the increasing concentrations of 2(C12Cys) GS are shown in Figure 4.

Figure 4.

Figure 4

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UV–vis absorption spectra of HSA (4 μM) in the absence and presence of various concentrations of 2(C12Cys) GS.

The typical absorption maximum at 280 nm can be ascribed to the occurrence of chromophores in the protein. A hyperchromic effect is detected in the absorption spectra of HSA with rising concentration of 2(C12Cys) GS from Figure 4. However, there was no significant change in the absorption maxima. Such variations in the intensity of absorption show that there was no effect on the chromophore microenvironment as a result of complex formation (i.e., static quenching) in the ground state among chromophores of HSA and 2(C12Cys) GS.

2.5. Far-UV CD Analysis

The results of far-UV circular dichroism (CD) experiments can provide the evidence regarding the secondary configuration of proteins.59 The CD spectra for HSA without and with the 2(C12Cys) GS are given in Figure 5. The two-negative double humped peaks at 208 nm (arise due to π → π* transition) and 222 nm (arise due to n → π* transition) in the ultraviolet region can be seen in CD spectra of HSA which is a distinguishing feature of the α-helical content of protein. At small concentrations of 2(C12Cys) GS (0.1 mm), there was small variation in the helicity, signifying that the secondary construction of HSA was probably balanced via 2(C12Cys) GS. In accordance with the literature60 the secondary structure of HSA was stabilized using a cross-linking purpose at small concentration of 2(C12Cys) GS. Because 2(C12Cys) GS has binary tails, it may form a link between specific nonpolar and negatively charged residues situated on dissimilar loops of HSA. The liberated micelles start to form after the saturation binding of HSA with 2(C12Cys) GS at high concentration, which indicates that HSA has a prolonged structure with exposed hydrophobic residues. The significant decrease in α-helix content for HSA in the presence of 2(C12Cys) GS clearly revealed the more hydrophobic nature. On the basis of the above results, it has been concluded that the binding of 2(C12Cys) GS with HSA leads to its unfolding. The results also pronounced more unfolding of HSA in the presence of 2(C12Cys) GS because of the fact that the electrostatic and hydrophobic forces that control this phenomenon are more significant in the case of 2(C12Cys) GS.

Figure 5.

Figure 5

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Far UV-CD spectra of HSA (4 μM) in the absence and presence of various concentrations of 2(C12Cys) GS.

2.6. Molecular Docking

The molecular docking studies are useful in exploring the details related to interaction and the binding sites of a ligand on protein and binding energies of small molecules with proteins.61 Therefore, the results of HEX 8.0 calculations illustrated hydrophobic interactions between drug site I and II of HSA and the 2(C12Cys) GS (Figure 6). Figure 6a,b displays binding sites of HSA and the binding patches of 2(C12Cys) GS fitted into the hydrophobic pockets of HSA, respectively.62 Further Figure 6c shows the detailed hydrogen bonded interactions with the HSA residues viz., Asn 429, Asn 458, Val 455, Val 424, Ser 454, Glu 425, Tyr 411, and Lys 205 which play a critical role for GS interaction to HSA. The binding free energy of the 2(C12Cys) GS with the HSA was found to be −9.23 kcal/mol. From the docking results, it was found that the 2(C12Cys) GS binds to HSA via hydrophobic interactions into the binding pockets of HSA.

Figure 6.

Figure 6

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(a) binding sites of HSA, (b) docked pose of the compound into the binding sites of HSA, (compound is shown in colored spheres), (c) zoomed-in view showing the interactions of the compound with the residues of HSA (compound is shown in spheres).

3. Conclusions

The cmc value of the 2(C12Cys) GS was reduced in the presence of HSA, which depicted that there were significant interaction among the protein and the GS. The fluorescence results indicate that 2(C12Cys) GS binds with HSA. This result reflects that 2(C12Cys) GS is a better fit to alter the conformation of proteins. Additionally, quenching in the fluorescence technique ensures the static process. The negative ellipticity value has been reduced upon the addition of the surfactant because of unfolding HSA, which was confirmed by CD data. The shift in the UV spectral lines signifies the change in the secondary construction of the protein. Molecular docking reveals the accurate domain of residues into which 2(C12Cys) GS gets fixed and complements the spectroscopic findings. Thus, we can conclude that 2(C12Cys) GS, interacts surprisingly with HSA and is further enhanced by the ligand to modify the HSA conformation.

4. Experimental Section

4.1. Materials

Cystine (98%), n-dodecyl bromide (98%), and HSA were purchased from Sigma-Aldrich, USA. Acetone (99%, Merck, India), methanol (99%, S D Fine-Chem Limited, India), hexane (98%, Merck, India), thymolphthalein (Kemphasol 98%), and NaOH (97% Merck, India) were used as received.

The 2(C12Cys) GS was prepared and purified by following the reported literature method.38 The synthesis method of the 2(C12Cys) GS (Scheme S1) and its characterizations such as elemental analysis, 1H NMR (Figure S1), and FT-IR (Figure S2) are given in Supporting Information.

All chemicals and buffer solution used were of pure analytical grade. DDW (double distilled water) was used during the experiments. Sodium phosphate buffer (20 mM, pH 7.4) was used to prepare the stock solutions of 2(C12Cys) GS and HSA.

4.2. Methods

4.2.1. Surface Tension (γ) Measurements

The measurements of surface tension were carried out in the 2(C12Cys) GS in presence of HSA using a SD Hardson tensiometer by a ring detachment method at 25 °C. The circulating water thermostat was used to maintain the temperature. The Pt−Ir ring was rinsing perfectly before use. Prior to the experiment, the surface tension of DDW was calibrated in the range of 72.0 ± 0.3 mN/m. DDW was also used to clean the glassware.

4.2.2. Steady-State Fluorescence Measurements

Fluorescence spectra were analyzed by a RF-5301 PC fluorescence spectrophotometer (Shimadzu, Japan) equipped with a xenon flash lamp using quartz cells (1.0 cm). Before the analysis, the tool parameters were set as: excitation wavelength (295 nm), emission wavelength range (300–400 nm), and slit width (5 nm) along with a path distance of 1 cm. HSA concentration (4 μM) and increasing concentration of 2(C12Cys) GS (0.1–1 μM) were used in the experiment. The fluorescence experiment was performed at 25 °C.

4.2.3. UV–Visible Spectroscopy

UV–visible spectra were recorded on a UV-1800 Schimadzu UV spectrophotometer using a quartz cuvette of 1 cm path length at room temperature. Absorption spectra of the HSA and 2(C12Cys) GS complex were evaluated in the range of 240–320 nm.

4.2.4. CD Measurements

The spectra of CD were observed on a spectropolarimeter (Jasco J-815 model) equipped with a microcomputer. The apparatus was calibrated with (+)-10-camphorsulfonic acid and the entire CD analysis was carried out at 25 °C with a thermostatically controlled cell holder attached to a circulating water bath (Neslab RTE-110) with an accuracy of ±0.1 °C. Variation in the secondary configuration of the protein was recorded in the far-UV region (200–250 nm) via a path length (1 mm) cell. The signal from the reference sample containing the buffer and the surfactant was subtracted from the CD signal for entire measurements. The high-tension current for the spectra acquired was originated to be less than 600 V. The spectra were composed with a scan rate of 20 nm/min and a reaction time of 1 s. Every spectrum was the average of four scans. The results are indicated in conditions of mean residue ellipticity (MRE) shown below10

4.2.4. 10

where θobs = the ellipticity in millidegrees, CP = the molar concentration of the HSA, n = the number of amino acid residues of protein, and l = the path length of the cell in centimeters.

4.2.5. Molecular Docking Studies

The molecular docking studies were carried out by using HEX 8.0 software63 which is a molecular graphics program for calculating and exhibiting viable docking modes of proteins and nucleic acids. The crystal arrangement of the HSA (PDB ID: 1H9Z) was taken from the protein data bank (http://www.rcsb.org./pdb). Visualization of the docked pose was completed by using CHIMERA (www.cgl.ucsf.edu/chimera), PyMol (http://pymol.sourceforge.net/), and the Discovery Studio molecular graphics program.

Acknowledgments

J.A. is thankful to the Deanship of Scientific Research (DSR), Taibah University, Al-Madina, Saudi Arabia for providing the research grant with the project no. 60343.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.9b03315.

  • Synthesis of the N,N′-dialkyl cystine GS, 2(C12Cys); scheme of the synthesis route; and characterization figures (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao9b03315_si_001.pdf (207.9KB, pdf)

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