- Email: [email protected]
Interaction of Myoglobin colloids with BSA in solution: Insights into complex formation and elastic compliance
G Model
ARTICLE IN PRESS
BIOMAC-7965; No. of Pages 10
International Journal of Biological Macromolecules xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Interaction of Myoglobin colloids with BSA in solution: Insights into complex formation and elastic compliance D. Madhumitha, Aruna Dhathathreyan ∗ Advanced Materials Lab., CSIR-CLRI, Adyar, Chennai 600020, India
a r t i c l e
i n f o
Article history: Received 20 June 2017 Received in revised form 19 July 2017 Accepted 26 July 2017 Available online xxx Keywords: Myoglobin Colloids Bovine serum albumin
a b s t r a c t This work focusses on the supramolecular complex formed between Myoglobin (Mb) and Bovine Serum Albumin (BSA) at colloids/solution interface at pH 4.0 and pH 7.5. Electrostatic interactions between Mb as colloids and BSA solution (pH = 7.5 and 4.0) have been confirmed by Zeta potential that suggest that while Mb has a narrow interaction range, BSA has a wider interaction space. The organization of Mb colloids in BSA characterized using dilational rheological parameters show that the Mb colloids are elastic and the strong adsorbed water layers on the surface restrict the deformation, regulated by the viscoelastic surface layer. Stability of the complexes analyzed using UV–vis, Fluorescence and Circular dichroic spectroscopy indicate that there is a 1:1 interaction between Mb and BSA with a binding constant of about 105 M−1 . Quartz Crystal microbalance with dissipation has been used to evaluate the elastic compliance of the complexes of Mb colloids dispersed in very dilute BSA solution. The higher elastic compliance at pH = 4.0 (than at pH = 7.5) and the complex sizes correlate with changes in zeta potential suggesting that the mechanical properties of the protein in colloids are dependent on both the electrostatic interaction as well as the degree of hydration of the colloids. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Particles with directional interactions that serve as models for biological structures can be used to build new functional bio materials [1–3]. Among such particles, anisotropic colloids with attractive patches have a tendency to self-assemble into open lattices and colloidal equivalents of molecules and micelles [4–6]. Till recently, there has been no detailed studies on the design and properties of these model systems that combine mutual attraction, anisotropy, and deformability, particularly in the context of protein based materials. Analysis of biological functions indicates the crucial role played by protein-protein interactions [7–9]. A detailed characterization of these interactions can provide information about the function of protein complexes which would be helpful in drug research [10–12]. Understanding the relationship between physico-chemical properties of the hydrated or solvated proteins and their ability to form complexes with other polyelectrolytes through non-covalent bonds is essential for food and pharmaceutical industries.
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (A. Dhathathreyan).
Protein-protein interactions at interfaces have been analysed using a number of biophysical techniques [13]. The study of the intermolecular or non-covalent interactions that occur between the proteins has long been key to our understanding of complex molecular systems, particularly when considering the assembly of molecules into supramolecular systems. For a charged macromolecule like a protein at infinite dilution, the interactions involve only one macroion and the co-ions present in the bulk solution. In the majority of the theoretical approaches to modelling protein solutions, the model usually ignores the atomistic representation of molecule and the solvent by replacing the charged groups or monomers of polymeric chain with a necklace of spheres carrying assigned charges, either positive (polycation) or negative (polyanion), respectively. The presence of solvent is replaced by a continuum usually by using a fixed dielectric constant. A total potential energy of the polyion is a sum of potentials of covalent and non-covalent interactions, where chemical bonding is given by harmonic potential of a defined monomer bond length. For nonchemical bonding the energies defining the interactions are the screened Coulomb potential between charged monomers, and the Lennard-Jones potential for the short-range interactions. Such interactions in colloid science focus on anisotropy and asymmetry in design of particles and deviations from spherical
http://dx.doi.org/10.1016/j.ijbiomac.2017.07.157 0141-8130/© 2017 Elsevier B.V. All rights reserved.
Please cite this article in press as: D. http://dx.doi.org/10.1016/j.ijbiomac.2017.07.157
Madhumitha,
A.
Dhathathreyan,
Int.
J.
Biol.
Macromol.
(2017),
G Model BIOMAC-7965; No. of Pages 10 2
ARTICLE IN PRESS D. Madhumitha, A. Dhathathreyan / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
symmetry has led to new methodologies for self-assembling nano structures [14,15]. In this regard, patchy colloids whose surfaces are coated with different materials of varied properties are of particular interest. Study and analysis of the role of heterogeneous charge patches is important not only for exploiting these new material design routes, but also for studying proteins. Proteins often carry heterogeneous surface charge, which primarily drives aggregation behavior under definite conditions [16,17]. The anisotropy of protein interactions is essential for understanding aggregation behavior, and general, high-fidelity coarsegrain models of proteins need to account for charged as well as hydrophobic patches [18,19]. Controlling protein aggregation is important in the food and pharmaceutical industries and even for understanding neurodegenerative diseases such as Alzheimer’s [20–22]. In the present work, we focus on the mechanism of complex formation between Myoglobin designed as capsule and Bovine serum albumin (BSA) in solution for application in therapeutics, protein stabilization and extending the shelf-life with a view to propose a methodology for storing blood proteins. Mb has been designed in the colloidal form and its interactions with the globular protein BSA in solution form have been studied at the colloid/solution interface by balancing both electrostatic and steric stabilization. 2 different pH: 4.0 and 7.5 have been chosen for both Mb and BSA. The positively charged hydrated colloid of Mb (at pH = 4.0) interacting with negatively charged BSA (pH = 7.5) and the reverse case of Mb colloid (pH = 7.5) interacting with BSA (pH = 4.0) have been studied. Myoglobin (Mb) is a cytosolic heme protein and is a member of the globin family together with hemoglobin (Hb), neuroglobin (Ngb) and cytoglobin (Cygb). The most important condition for functioning of all living aerobic organisms is continuous supply of sufficient oxygen. The access of oxygen to metabolic processes in cells is mediated by the respiratory proteins, tetrameric blood hemoglobin (Hb) and monomeric muscle myoglobin (Mb). Myoglobin is expressed in red muscles in response to mitochondrial demand for oxygen, transporting it from sarcolemma to mitochondria [23–25]. O2 release from MbO2 at physiological po2 values proceeds only upon direct contact of the protein with mitochondria [26,27]. Monitoring myoglobin levels in blood and urine has been used as means to detect skeletal or cardiac muscle cell injury. Myoglobin is rapidly released after muscle damage, and thus can be a useful biomarker in the early phases of injury. Under specific conditions of muscle injuries the Mb interacts with the albumin and other proteins in order to bind to the mitochondrial membrane. Applications in pharmacotherapy and the development of an artificial cornea, require an understanding of limits of human corneal diffusion and its clinical relevance, particularly in the context of BSA interacting with myoglobin [28,29]. Albumin is the major plasma protein circulating in blood. Albumin potently decreases capillary permeability, although the mechanisms are not understood completely. Important factors in this study are; the electrostatic interactions between charged groups and charged patches on the two protein surfaces-one in the capsule form and the other in solution, dependence on the polyion properties such as charge density, and mechanical stiffness in terms of elastic compliance. These features of local and global structure of the protein-protein complexes are still poorly understood. The protein-protein complex formation is mainly driven by various non-covalent interactions, like electrostatic, H-bonding, hydrophobic, and steric interactions [30–34]. Protein carries +ve or −ve zeta potential based on the pH of the medium (+ve at pH lower than pI and vice-versa).
Please cite this article in press as: D. http://dx.doi.org/10.1016/j.ijbiomac.2017.07.157
2. Experimental 2.1. Materials and methods Bovine Serum Albumin (BSA) of 99.9% purity has been purchased from Sigma Aldrich, USA and used as such. Its isoelectric point is 4.8.Horse heart myoglobin was purchased from Biozyme (Reinheitszahl 1/4 4.8) (San Diego, CA) and was used as such. For every experiment, the protein dissolved in 20 mM 4-morpholinepropanesulfonic acid (Boehringer Mannheim, Ingelheim am Rhein, Germany) at pH 7.0 was filtered with Millipore (Billerica, MA) Millex SV filters before use. Before preparation of Mb colloids, a few crystals of sodium dithionite (final concentration 0.2 mg/mL) were added to the Mb, in the respective buffer to remove the oxygen and stored in a gastight sample container. The excess of dithionite was eliminated by gel filtration chromatography on a column (20 cm 1.5 cm) filled with Sephadex G-25 (Pharmacia, Sweden). Mb colloids have been prepared at two different pH using buffers pH 4.0 and pH 7.5, below and above the isoelectric point of Mb (pI = 6.9). These colloids have been prepared by the freeze-thaw technique of Kuppevelt et al. [35]. The respective buffer solutions are introduced in liquid nitrogen for about two minutes and then the protein solution is added dropwise. The frozen droplets of protein solution have been allowed to warm up to −35 ◦ C for 3 h and then stored at 8 ◦ C overnight before using them for measurements. The purified Mb colloids formed by the above procedure are hereafter referred as colloids. Mb colloids of either positive or negative charge (pH 4.0 or 7.5) are mixed with BSA solution (pH 7.5/4.0) in increasing concentrations. The pH variations, as a function of concentration allows for systematic changes in the electrostatic interactions of the components in the system and have been examined using zeta potential, UV−Vis spectroscopy, CD, fluorescence spectroscopy, DLS. The adsorption and interfacial organization of BSA/Mb mixtures on to solid surface have been analysed using Quartz Crystal Microbalance with dissipation (QCM-D) technique and the elastic compliance of the complexes formed have been evaluated. Stability of the complexes have been analysed using the interfacial elasticity and viscosity at colloid/solution interface. 2.2. UV–vis and steady state fluorescence measurements UV visible measurements have been carried out using Shimadzu UV1800 spectrophotometer (Japan), with quartz cells of 1 cm path length in the region 200–600 nm at 0.2 nm interval. Steady state fluorescence spectra have been recorded using VARIAN CARY Eclipse spectrophotometer and measured in the range 300–500 nm with the excitation wavelength at 280 nm. The spectra have been checked at intervals of 5 mins. for every sample of Mb complexed with BSA. 2.3. CD spectroscopy The far UV CD measurements for the colloids have been carried out in Jasco J 815 polarimeter at room temperature (295 K) using a 1 mm cell in the normal range 190–240 nm. The secondary structure distributions are fitted using Dichroweb online database [36]. The stability of the colloids and the surface charge interaction of the BSA/Mb have been monitored by particle size analyser and zeta potential measurements using Malvern Nano-ZS zeta sizer 2.4. Dilational rheology of Mb colloids in BSA solution Freshly prepared solutions of the Mb capsules and BSA solution have been used for each measurement. The surface tension of
Madhumitha,
A.
Dhathathreyan,
Int.
J.
Biol.
Macromol.
(2017),
G Model
ARTICLE IN PRESS
BIOMAC-7965; No. of Pages 10
D. Madhumitha, A. Dhathathreyan / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
3
Fig. 1. UV Visible spectra of Mb colloids at (a) pH = 4.0 and (b) pH = 7.5 in BSA soln (pH = 7.5 and 4.0 respectively).
these samples are measured with the profile analysis tensiometer PAT-1 (SINTERFACE Technologies, Germany) with an accuracy of ±0.1 mN/m and thermostated at a temperature of 25 ◦ C. Drops of the solution as well as the solution containing capsules are formed at the tip of a PTFE capillary immersed into a cuvette filled with a buffer-saturated atmosphere. After having reached the adsorption equilibrium after about 1hr., the drop is subjected to harmonic oscillations with frequencies (f) 0.01–0.2 Hz in order to study the dilatational elasticity. For each frequency, the dilation/expansion was carried out for atleast 30mins. Accuracy in elasticity and viscosity values are 1 mN/m and 1 mN/ms. Each measurement is the average of three measurements and are presented in this study. 2.5. Quartz Crystal microbalance with dissipation (QCM-D) QCM-D sensors with a silica coating used in the study have been cleaned using a 2% sodium dodecyl sulfate solution (for 30 min), rinsing with ultrapure water, blow-drying with nitrogen. Cleaned substrates have been stored in air and again exposed to UV/ozone (30 min) prior to use. Adsorption and interfacial processes have been monitored simultaneously using a home-built, impedancebased QCM consisting of an impedance (network) analyzer for determining resonance frequencies and bandwidths of a quartz crystal [37] and a fluid cell, also home-built, where the crystals have been mounted, to allow adsorption, fluid exchange, and temperature control. The network analyzer was purchased from Ivan Makarov (makarov.ca, Canada). The resonance frequencies and bandwidths have been collected for overtones, n, between 3 and 11, corresponding to frequencies between 15 and 55 MHz. Following the conventional methodology, we quantify dissipative processes using the dissipation factor D, defined as the ratio of full bandwidth of the resonance to the resonance frequency (the inverse Q-factor). The frequency shift values are scaled by the overtone order, that is, f/n. This is because f/n is the same for all overtones if the frequency shift is caused by gravimetric effects. Dissipation changes are not scaled by the overtone order because such a scaling by n is implicitly contained in the definition of D itself. 2.6. Scanning electron micrographs Scanning electron micrographs have been obtained using a field emission scanning electron microscope (FE-SEM, Hitachi S-4200). The films transferred onto the substrates have been dried in a desPlease cite this article in press as: D. http://dx.doi.org/10.1016/j.ijbiomac.2017.07.157
iccator for at least 1 h and then coated with gold prior to SEM observation.
2.7. Results and discussion The complex formed from Mb colloids of pH 4.0 and pH 7.5. mixed with BSA solutions of pH 7.5 and pH 4.0 respectively in increasing concentrations have been characterized using UV–vis and fluorescence spectra. The characteristic absorbance spectra for the mixture are given in Fig. 1(a) and (b). In both the cases, addition of Mb colloids to BSA solution shows an increase in absorbance intensity as observed at Soret band at 409 nm without much difference shift in its maximum wavelength. The Soret band is very sensitive to the heme environment and is commonly used to monitor heme pocket disruption in presence of external perturbation. Circular dichroism (CD) and steady state fluorescence experiments have been performed to monitor the interactions between Mb colloids and BSA solution. The fluorescence spectra of the two mixtures are shown in Fig. 2(a) and (b). The emission spectra of Trp of BSA have been recorded in the range 300–500 nm with the excitation wavelength at 280 nm. BSA solution at pH 4.0 shows emission at 347 nm with an intensity of 33.91a.u. As the concentrations of Mb colloids are increased in BSA soln., the fluorescence intensity starts to quench but no dramatic change shift in the wavelength. The fluorescence quenching of BSA is due to the proximity of Mb colloids which reveal nearly a 1:1 interaction with a binding constant of about 105 M−1 . The heme group of myoglobin’s is buried into the protein globule rather than exposed on its surface, so the quenching of donor emission by myoglobin cannot occur by a dynamic mechanism. Energy transfer to the heme only occurs in a complex whose lifetime exceeds that of donor fluorescence (static quenching). The stability of the proteins at the colloid/solution interface have been monitored using Circular dichroism measurements are shown in Fig. 3. Here the spectra shown are that of the Mb colloids measured by using BSA soln. as reference. The corresponding secondary structure distributions are fitted using CD PRO shown in Table 1. From the table it is observed that, the Mb proteins at capsule/solution interface are fairly stable. At a concentration of 2.5 × 10−7 M of BSA solution in the mixture, Mb in the colloids (pH 4.0), has a maximum of 68.3% of helix. For the reverse case, Mb colloids (pH 7.5) for a conc. of 1 × 10−7 in BSA solution show 80% of
Madhumitha,
A.
Dhathathreyan,
Int.
J.
Biol.
Macromol.
(2017),
G Model
ARTICLE IN PRESS
BIOMAC-7965; No. of Pages 10
D. Madhumitha, A. Dhathathreyan / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
4
Fig. 2. Fluorescence Spectra of BSA Soln. with (a) Mb colloids (pH 7.5) (b) Mb colloids (pH 4.0).
Fig. 3. Circular dichroic spectra of Mb colloids at (a) pH = 4.0 and (b) pH = 7.5 in BSA soln (pH = 7.5 and 4.0 respectively).
Table 1% Secondary Structural Population of Mb in complex of Mb colloids with BSA solution. Conc. of Mb colloids in BSA soln. (x10−7 M)
0.5 1 1.5 2 2.5 3 5 6 8
Mb colloids (pH 4.0)
Mb colloids (pH 7.5)
␣ helix
 sheet
 turn
Unrd
␣ helix
 sheet
 turn
Unrd
27.6 28 27.8 27.2 68.3 64.9 46.8 48.1 41.2
20.3 20.2 20.6 21.3 4.1 4.9 12.5 11 14
21.7 21.4 22 21.4 10.9 13.2 15.9 15 19.4
30.4 30.3 29.5 30.1 16.7 17 24.9 26 25.7
69.6 80.6 74.8 71.6 70.6 71.3 56.7 57 49.3
5.8 3.2 2.7 4.3 4.7 5.1 7.7 9.1 13.1
7.3 7.1 9.3 8.8 5.7 6.5 11.4 11 17.3
17.3 9.2 13.2 15.3 19.1 17.3 24.2 22.9 20.3
helicity. This shows that the secondary structure of Mb gets more helicity at pH 7.5 suggesting the levels of hydration are higher. The stability of the colloids at colloid/solution interface has been monitored using zeta potential and particle size measurements. Fig. 4 shows the zeta potential plot for BSA soln. (pH 4.0) with Mb colloids (pH 7.5) and vice versa. The size of Mb colloids at pH 4.0 in BSA soln. at pH 7.5 varies from 200 to 600 nm. In the reverse case, the average size varies from 200 nm to 400 nm showing that
Please cite this article in press as: D. http://dx.doi.org/10.1016/j.ijbiomac.2017.07.157
changes in overall charge density on the Mb leads to altered level of hydration which in turn can make the colloids compacter. Fig. 5(a) and (b) shows the SEM micrographs of the Mb colloids mixed with the maximum BSA concentration for both pH = 4.0 and 7.5 respectively. From the Figure it is seen clearly that the colloids are nearly spherical and the fused colloids are a result of the drying process.
Madhumitha,
A.
Dhathathreyan,
Int.
J.
Biol.
Macromol.
(2017),
G Model
ARTICLE IN PRESS
BIOMAC-7965; No. of Pages 10
D. Madhumitha, A. Dhathathreyan / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
5
Fig. 4. Zeta potential plots for Mb colloids at pH = 4.0 and pH = 7.5 in BSA soln. (pH = 7.5 and 4.0 respectively).
Fig. 5. Scanning electron micrographs of Mb colloids (a) pH = 4.0 (b) pH = 7.5 in BSA solution (pH = 7.5 and pH = 4.0 respectively) (Scale bar = 200 nm).
2.8. Size of the colloids and the complex The sizes of the colloids are shown in Fig. 6.The largest size for Mb colloids (270 nm) at the lowest BSA concentration is driven by partially folded protein, whereas at higher pH the size is smaller (∼200 nm). This could be due to repulsive interactions between negatively charged myoglobin monomers, while at higher pH the Mb molecules still maintain their native form.
D/f vs Time plots for the different overtones are shown in Fig. 7(a) and (b). Based on the work of Du and Johannsmann, for simple thin viscoelastic film in a Newtonian liquid, one can show that the dissipation induced by the film is proportional to the elastic compliance [39]. Based on the plots of D/f versus overtone order, the elastic compliance may be evaluated from the slope using the following equation D/-f = 2nf 0 J
2.9. QCM-D The adsorption and interfacial organization of BSA/Mb mixtures on to quartz surface have been analysed using Quartz Crystal Microbalance with dissipation (QCM-D) technique. When the Mb colloids dispersed in BSA solution contacts the Quartz surface, adsorption is initiated. As the resonant frequency gets damped due to the adsorbing material, mass is increasing. However, since hydrodynamically trapped solvent contributes to the mass, mass cannot be determined independently. The focus here is on the intrinsic properties of the film and for this one considers half band half width to the negative frequency shift [38]. Both these parameters are proportional to the film mass at the thin film limit. The Please cite this article in press as: D. http://dx.doi.org/10.1016/j.ijbiomac.2017.07.157
J is the elastic compliance, f0 the fundamental frequency (5 MHz), the viscosity of the solution. Fig. 8(a) and (b) shows the estimated elastic compliance as a function of the BSA in solution on addition to the Mb colloids. The elastic compliance has been estimated for both pH 4.0 and pH 7.5 colloids of Mb for titration with BSA in solution.Tthe elastic compliance for the positively charged Mb colloids are lower than that of Mb colloids at pH = 7.5. The derived elastic compliance is in the same range for all harmonics. Proteins organized biomolecules, with characteristic secondary structures, such as ␣helices, -strands, turns and loops, are folded and assembled to assume a characteristic three-dimensional structure that defines
Madhumitha,
A.
Dhathathreyan,
Int.
J.
Biol.
Macromol.
(2017),
G Model BIOMAC-7965; No. of Pages 10 6
ARTICLE IN PRESS D. Madhumitha, A. Dhathathreyan / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
Fig. 6. Sizes of complex formed between Mb colloids (pH = 4.0 and 7.5 respectively) with BSA in soln. (at pH = 7.5 and 4.0 respectively).
the to the protein’s function, dynamics, and mechanical properties [40,41]. An analysis of the secondary structure population of Mb, the size changes, zeta potential and the elastic compliance gives a direct correlation between the structure, intrinsic rigidity and stability of the complex. The results show clearly at pH 7.5, Mb colloids are larger in size, have higher helicity (∼80%) and higher elastic compliance. These results are similar to that of Perticaroli et al. who analyzed the nanomechanical properties of few proteins with different secondary structures: ␣-helices (myoglobin and bovine serum albumin), -barrels (green fluorescent protein), and ␣ +  + loop structures (lysozyme) [42]. In their work they demonstrated that ␣-proteins have lower elastic moduli than those dominated by  motifs. From the plot in this work, it is seen that the change in elastic compliance follows the change in zeta potential suggesting the role of hydration and the charge-charge interactions at the capsule/solution interface. It is known that water engages in two important types of interactions near proteins: it forms ordered cages around exposed hydrophobic regions, and it participates in hydrogen bonds with surface polar groups. A special role in the inter-protein interactions may be played by intermolecular salt bridges between negatively charged carboxylate groups of the Asp residues and the positively charged side groups of the lysine and arginine residues. These salt bridges can best form at near-neutral pH values and can contribute to the intermolecular connectivity. At low pH, the carboxylate groups are neutralized to carboxylic acid groups, which imply that the salt bridges would be disrupted, which would lead to a decrease of the strength of the inter-protein interactions. Where liquid water is absent, salt bridges could be strong and could fulfill an important role in maintaining a strong water-repellent film. In aqueous solutions containing positively charged Mb colloids dispersed in negatively charged BSA solution, a complex is formed through the non-covalent interaction between oppositely charged groups. This process is affected by the charge density of each macro ion. Within this we distinguish two major categories of noncovalent binding depending on strength of attractive interactions between Mb colloids and BSA. At pH = 4 for the Mb colloids, First is a weak interaction where the BSA from solution binds to small, multiple positive patches on the Mb colloid surface without affect-
Please cite this article in press as: D. http://dx.doi.org/10.1016/j.ijbiomac.2017.07.157
ing the native conformation of Mb. The second type of attractive interactions is described as a strong binding, where the BSA binds to a single, large positive patch on the protein surface. The overall scheme for formation of the complex is given in Fig. 9. In the case of Mb colloids of a particular charge at either pH = 4 or 7.5 present in the BSA solution, it is surrounded by an ionic cloud containing an excess of counterions. Under these conditions electrostatic interactions between the Mb charged groups themselves are screened. Consequently, the long-range repulsive interactions between the charged colloids are weakened exponentially with the increasing distance between them. Increasing BSA concentrations leads to increased screening which can cause structural changes in the Mb colloids which in turn gets reflected in the zeta potential size and the elastic compliance. Thus decreasing sizes for Mb colloids at pH = 4 with increasing BSA could indicate an effect like the ‘salting out’ thus supporting screening of the long-range attractive electrostatic interactions. The corresponding elastic compliance is smaller than that of the Mb colloids at near neutral pH value. Stoichiometric ([+]/[−] = 1) complexes are formed from BSA +Mb colloids in buffer. In general, the complex between BSA and Mb colloids is recognized to be electrostatically driven. For example, Porfiri et al. reported that shielding by increasing strength of the solution will lead to a reduction in complex formation [43,44]. Boeris et al. studied the aqueous solution behavior of cationic polyethyleneimine in the presence of anions with two or more electrical charges like citrate, phosphate, sulphate, malate, malonate and succinate using turbidimetry and light scattering. Their results suggest that the non-soluble polyethyleneimine–anion complexes have the property to precipitate macromolecules charged with an opposite electrical charge at specific concentrations [45]. Milioni et al. have used the D/f to derive information on the conformation of surface attached biomolecules. The basis for this measurement is that the (D/F) is a measure of the hydrodynamic volume of the attached entity, expressed by its intrinsic viscosity [] [46]. The method proposed in this work thus can be used to evaluate the stability of hydrodynamic volume of charged biomolecules interacting with small molecules as well as other biomolecules as well as quantify it. The dilational elasticity and viscosity of pure Mb colloids at pH = 4 and 7.5 and complexing with the BSA soln. at the point where zeta potential shows near neutralization have been studied.
Madhumitha,
A.
Dhathathreyan,
Int.
J.
Biol.
Macromol.
(2017),
G Model BIOMAC-7965; No. of Pages 10
ARTICLE IN PRESS D. Madhumitha, A. Dhathathreyan / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
7
Fig. 7. (a). D vs. f plots for 3rd, 5th, 7th, 9th and 11th overtones for (a) Mb colloids (pH = 4.0) for different effective concentrations of BSA solution, (pH = 7.5) (b) Mb colloids (pH = 7.5) for different effective concentrations of BSA solution, (pH = 4.0).
Please cite this article in press as: D. http://dx.doi.org/10.1016/j.ijbiomac.2017.07.157
Madhumitha,
A.
Dhathathreyan,
Int.
J.
Biol.
Macromol.
(2017),
G Model BIOMAC-7965; No. of Pages 10 8
ARTICLE IN PRESS D. Madhumitha, A. Dhathathreyan / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
Fig. 8. Elastic compliance of (a) Mb colloids (pH = 4.0) for different effective concentrations of BSA solution, pH = 7.5 (b) Mb colloids (pH = 7.5) for different effective concentrations of BSA solution, pH = 4.0.
Fig. 9. Scheme of complex formation of Mb colloids with BSA in solution and changes in the elastic compliance.
Fig. 10(a) and (b) shows the values of the elasticity and viscosity. It is seen that as frequency of dilation increases elasticity increases (3–7 mN/m) and viscosity decreases (50-15 mN/m.S) with a sharp change as frequency for the pure Mb colloids at pH = 4.0. However on complex formation with BSA solution there is an overall decrease in elasticity values (3–5 mN/m) while the viscosity a slightly higher value initially (80-7 mN/m S) The pure Mb colloids show lower elasticity as well as viscosity values at pH = 7.5 compared with that at pH = 4.0. However, there is an increase in both elasticity and viscosity values as complex is formed between Mb colloids and BSA in solution. These are in agreement with the changes in zeta potential and the sizes of the
Please cite this article in press as: D. http://dx.doi.org/10.1016/j.ijbiomac.2017.07.157
complex formed at the interface due to the charge-charge interaction and the degree of hydration associated with it. Here water acts truly as a plasticizer and such a property of water giving rise to anomalous dilational elasticity and viscosity in the presence of different counterions to different extent have been reported by Lakshmanan et al. [47,48]. In the present work, the Mb colloids interacting with BSA undergo direct adsorption to the gold coated quartz surface. We find that the extent of dissipation caused by the film and the evolution of dissipation as a function of surface coverage is dependent on the internal properties of these particles and their attachment to the surface. A limited rotation of the colloids in the buffer
Madhumitha,
A.
Dhathathreyan,
Int.
J.
Biol.
Macromol.
(2017),
G Model BIOMAC-7965; No. of Pages 10
ARTICLE IN PRESS D. Madhumitha, A. Dhathathreyan / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
9
Fig. 10. Dilational elasticity and viscosity of pure Mb colloids at (a) pH = 4.0 and with BSA solution at pH = 7.5 (b) pH = 7.5 and with BSA solution at pH = 4.0.
containing the BSA (rocking) and a small sliding contribute to the dissipation. For every change in concentration of BSA in the solution, charge-charge interaction, followed by some% of water displacement takes place which results in change in the frequency. As adsorption progresses, at higher coverage, lateral hydrodynamic interactions between neighboring colloids compete with the modes of dissipation, which results in a maximum in dissipation at some intermediate adsorption times. In study of adsorption of biomolecules, determining an adsorbate’s amount, shape, orientation, and internal structure is a challenge. One requires to combine many different techniques based on different sensing principles to get a more realistic representation of the adsorbate’s organization on the surface. The work has attempted to focus on the nature of the binding, the structure and properties of the complexes that result due to Mb in the colloidal state interacting with BSA in the solution. The work demonstrates the correlation between charge-charge interaction, ‘salting-in/out’ effects, degree of hydration and the elastic compliance of the Mb colloids. Results show clearly that as the Mb colloids get compacter at low pH, the levels of hydration decrease resulting in low elastic compliance. By evaluating the D/f ratio one can measure elastic compliance in liquid medium for very thin films and may have significance in sensing of the biological absorbates. The results suggest that the this ratio is sensitive to the charge-charge
Please cite this article in press as: D. http://dx.doi.org/10.1016/j.ijbiomac.2017.07.157
interaction between Mb colloids and BSA and their lateral organization of molecules on the surface. QCM-D does not have an intrinsic lateral resolution on the nanometer-scale but the time-resolved correlation of both D and F can provide quantitative insight into the organization of proteins on the molecular scale. Understanding the intrinsic rigidity of ␣-helices or -sheets, is crucial to unravelling the relationships between structure, mechanical properties, dynamics, and function in biological systems. The analytical approach to evaluate elastic compliance as a function of charge densities on the Mb colloids, should be useful for the investigation of molecular processes at model membranes, the organization of nanosized particles, including viruses or drug delivery vehicles, at surfaces. Acknowledgements The authors would like to thank DST-SERB, Govt. of India for a project grant under which part of the work was carried out. D.M. would like to thank DST-SERB for the Senior Research fellowship. References
Madhumitha,
[1] Zhang, S.C. GlotzerZhan, Nano Lett. 4 (2004) 1407–1413. [2] S.C. Glotzer, M.J. Solomon, Nat. Mater. 6 (2007) 557–562. [3] Y. Gi-Ra, J.P. David, S. Stefano, J. Phys.: Condens. Matter 25 (2013) 193101.
A.
Dhathathreyan,
Int.
J.
Biol.
Macromol.
(2017),
G Model BIOMAC-7965; No. of Pages 10 10
ARTICLE IN PRESS D. Madhumitha, A. Dhathathreyan / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
[4] P. Akcora, H. Liu, S.K. Kumar, J. Moll, Y. Li, B.C. Benicewicz, L.S. Schadler, D. Acehan, A.Z. Panagiotopoulos, V. Pryamitsyn, V. Ganesan, J. Ilavsky, P. Thiyagarajan, R.H. Colby, J.F. Douglas, Nat. Mater. 8 (2009) 354–359. [5] M.S. Nikolic, C. Olsson, A. Salcher, A. Kornowski, A. Rank, R. Schubert, A. Frömsdorf, H. Weller, S. Förster, Angew. Chem. Int. Ed. 48 (2009) 2752–2754. [6] J.A. Luiken, P.G. Bolhuis, Phys. Rev. E 88 (2013) 012303. [7] C. Janin, A.-C. Taylor, Introduction, Presses Universitaires de France, 2009. [8] E.D. Levy, J.B. Pereira-Leal, Curr. Opin. Struct. Biol. 18 (2008) 349–357. [9] D. Reichmann, O. Rahat, M. Cohen, H. Neuvirth, G. Schreiber, Curr. Opin. Struct. Biol. 17 (2007) 67–76. [10] M. Vidal, M.E. Cusick, A.L. Barabasi, Cell 144 (2011) 986–998. [11] F.P. Davis, D.T. Barkan, N. Eswar, J.H. McKerrow, A. Sali, Protein Sci. 16 (2007) 2585–2596. [12] Y. Loewenstein, D. Raimondo, O.C. Redfern, J. Watson, D. Frishman, M. Linial, C. Orengo, J. Thornton, A. Tramontano, Genome Biol. 10 (2009) 207. [13] S.H. Khan, F. Ahmad, N. Ahmad, D.C. Flynn, R. Kumar, J. Biomol. Struct. Dyn. 28 (2011) 929–938. [14] J. Zhang, E. Luijten, S. Granick, Annu. Rev. Phys. Chem. 66 (2015) 581–600. [15] J.M. Dempster, M. Olvera de la Cruz, ACS Nano 10 (2016) 5909–5915. [16] J.M. Park, B.B. Muhoberac, P.L. Dubin, J. Xia, Macromolecules 25 (1992) 290–295. [17] P.R. Majhi, R.R. Ganta, R.P. Vanam, E. Seyrek, K. Giger, P.L. Dubin, Langmuir 22 (2006) 9150–9159. [18] H. Liu, S.K. Kumar, F. Sciortino,J. Chem. Phys. 127 (2007), 084902. [19] L.J. Quang, S.I. Sandler, A.M. Lenhoff, J. Chem. Theory Comput. 10 (2014) 835–845. [20] E. Bianchi, R. Blaak, C.N. Likos, Phys. Chem. Chem. Phys. 13 (2011) 6397–6410. [21] M.E.M. Cromwell, E. Hilario, F. Jacobson, AAPS J. 8 (2006) E572–E579. [22] C.A. Ross, M.A. Poirier, Nat. Med. 10 (Suppl) (2004) S10–S17. [23] R. Hill, Nature 132 (1933) 897–898. [24] G.A. Millikan, Physiol. Rev. 19 (1939) 503–523. [25] R. Starodub, V. Korobov, V. Nazarenko, Naukova Dumka, Kiev (1992) 1. [26] G.B. Postnikova, S.V. Tselikova, Biofizika 50 (2005) 297–306. [27] G.B. Postnikova, S.V. Tselikova, E.A. Shekhovtsova, Biochemistry 74 (2009) 1211–1218. [28] R.A. Charalel, K. Engberg, J. Noolandi, J.R. Cochran, C. Frank, C.N. Ta, Ophthalmic Res. 48 (2012) 50–55.
Please cite this article in press as: D. http://dx.doi.org/10.1016/j.ijbiomac.2017.07.157
[29] G.B.P. Ekaterina, A. Shekhovtsova, J. Phys. Chem. Biophys. 03 (2013). [30] C.G. de Kruif, R. Tuinier, Food Hydrocolloids 15 (2001) 555–563. [31] S.K. Chaturvedi, M.K. Siddiqi, P. Alam, M. Zaman, R.H. Khan, RSC Adv. 6 (2016) 85860–85869. [32] P. Alam, S.K. Chaturvedi, T. Anwar, M.K. Siddiqi, M.R. Ajmal, G. Badr, M.H. Mahmoud, R. Hasan Khan, J. Lumin. 164 (2015) 123–130. [33] M.K. Siddiqi, P. Alam, S.K. Chaturvedi, R.H. Khan, Int. J. Biol. Macromol. 92 (2016) 1220–1228. [34] M.K. Siddiqi, P. Alam, S.K. Chaturvedi, S. Nusrat, M.R. Ajmal, A.S. Abdelhameed, R.H. Khan, Int. J. Biol. Macromol. (2017), http://dx.doi.org/10. 1016/j.ijbiomac.2017.07.036. [35] W.F. Daamen, P.J. Geutjes, H.T.B. van Moerkerk, S.T.M. Nillesen, R.G. Wismans, T. Hafmans, L.P.W.J. Van den Heuvel, A.M.A. Pistorius, J.H. Veerkamp, J.C.M. vanHest, T.H. van Kuppevelt, Adv. Mater. 19 (2007) 673–677. [36] L. Whitmore, B.A. Wallace, Biopolymers 89 (2008) 392–400. [37] R. Beck, U. Pittermann, K.G. Weil, Berichte der Bunsengesellschaft für Physikalische Chemie 92 (1988) 1363–1368. [38] M.A. Plunkett, Z. Wang, M.W. Rutland, D. Johannsmann, Langmuir 19 (2003) 6837–6844. [39] B. Du, D. Johannsmann, Langmuir 20 (2004) 2809–2812. [40] M.J. Buehler, Proc. Natl. Acad. Sci. 103 (2006) 12285–12290. [41] T. Ackbarow, X. Chen, S. Keten, M.J. Buehler, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 16410–16415. [42] S. Perticaroli, J.D. Nickels, G. Ehlers, H. O’Neill, Q. Zhang, A.P. Sokolov, Soft Matter 9 (2013) 9548–9556. [43] M.C. Porfiri, M. Braia, B. Farruggia, G. Picó, D. Romanini, Process Biochem. 44 (2009) 1046–1049. [44] M.C. Porfiri, G. Picó, B. Farruggia, D. Romanini, Process Biochem. 45 (2010) 1753–1756. [45] V. Boeris, C. Cassane, J. Wagner, G. Picó, Colloids Surf. B: Biointerfaces 82 (2011) 354–358. [46] D. Milioni, A. Tsortos, M. Velez, E. Gizeli, Anal. Chem. 89 (2017) 4198–4203. [47] M. Lakshmanan, R. Parthasarathi, A. Dhathathreyan, Biochim. Biophys. Acta 1764 (2006) 1767–1774. [48] M. Lakshmanan, A. Dhathathreyan, R. Miller, Coll. Surf. A 324 (2008) 194–201.
Madhumitha,
A.
Dhathathreyan,
Int.
J.
Biol.
Macromol.
(2017),
ARTICLE IN PRESS
BIOMAC-7965; No. of Pages 10
International Journal of Biological Macromolecules xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Interaction of Myoglobin colloids with BSA in solution: Insights into complex formation and elastic compliance D. Madhumitha, Aruna Dhathathreyan ∗ Advanced Materials Lab., CSIR-CLRI, Adyar, Chennai 600020, India
a r t i c l e
i n f o
Article history: Received 20 June 2017 Received in revised form 19 July 2017 Accepted 26 July 2017 Available online xxx Keywords: Myoglobin Colloids Bovine serum albumin
a b s t r a c t This work focusses on the supramolecular complex formed between Myoglobin (Mb) and Bovine Serum Albumin (BSA) at colloids/solution interface at pH 4.0 and pH 7.5. Electrostatic interactions between Mb as colloids and BSA solution (pH = 7.5 and 4.0) have been confirmed by Zeta potential that suggest that while Mb has a narrow interaction range, BSA has a wider interaction space. The organization of Mb colloids in BSA characterized using dilational rheological parameters show that the Mb colloids are elastic and the strong adsorbed water layers on the surface restrict the deformation, regulated by the viscoelastic surface layer. Stability of the complexes analyzed using UV–vis, Fluorescence and Circular dichroic spectroscopy indicate that there is a 1:1 interaction between Mb and BSA with a binding constant of about 105 M−1 . Quartz Crystal microbalance with dissipation has been used to evaluate the elastic compliance of the complexes of Mb colloids dispersed in very dilute BSA solution. The higher elastic compliance at pH = 4.0 (than at pH = 7.5) and the complex sizes correlate with changes in zeta potential suggesting that the mechanical properties of the protein in colloids are dependent on both the electrostatic interaction as well as the degree of hydration of the colloids. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Particles with directional interactions that serve as models for biological structures can be used to build new functional bio materials [1–3]. Among such particles, anisotropic colloids with attractive patches have a tendency to self-assemble into open lattices and colloidal equivalents of molecules and micelles [4–6]. Till recently, there has been no detailed studies on the design and properties of these model systems that combine mutual attraction, anisotropy, and deformability, particularly in the context of protein based materials. Analysis of biological functions indicates the crucial role played by protein-protein interactions [7–9]. A detailed characterization of these interactions can provide information about the function of protein complexes which would be helpful in drug research [10–12]. Understanding the relationship between physico-chemical properties of the hydrated or solvated proteins and their ability to form complexes with other polyelectrolytes through non-covalent bonds is essential for food and pharmaceutical industries.
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (A. Dhathathreyan).
Protein-protein interactions at interfaces have been analysed using a number of biophysical techniques [13]. The study of the intermolecular or non-covalent interactions that occur between the proteins has long been key to our understanding of complex molecular systems, particularly when considering the assembly of molecules into supramolecular systems. For a charged macromolecule like a protein at infinite dilution, the interactions involve only one macroion and the co-ions present in the bulk solution. In the majority of the theoretical approaches to modelling protein solutions, the model usually ignores the atomistic representation of molecule and the solvent by replacing the charged groups or monomers of polymeric chain with a necklace of spheres carrying assigned charges, either positive (polycation) or negative (polyanion), respectively. The presence of solvent is replaced by a continuum usually by using a fixed dielectric constant. A total potential energy of the polyion is a sum of potentials of covalent and non-covalent interactions, where chemical bonding is given by harmonic potential of a defined monomer bond length. For nonchemical bonding the energies defining the interactions are the screened Coulomb potential between charged monomers, and the Lennard-Jones potential for the short-range interactions. Such interactions in colloid science focus on anisotropy and asymmetry in design of particles and deviations from spherical
http://dx.doi.org/10.1016/j.ijbiomac.2017.07.157 0141-8130/© 2017 Elsevier B.V. All rights reserved.
Please cite this article in press as: D. http://dx.doi.org/10.1016/j.ijbiomac.2017.07.157
Madhumitha,
A.
Dhathathreyan,
Int.
J.
Biol.
Macromol.
(2017),
G Model BIOMAC-7965; No. of Pages 10 2
ARTICLE IN PRESS D. Madhumitha, A. Dhathathreyan / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
symmetry has led to new methodologies for self-assembling nano structures [14,15]. In this regard, patchy colloids whose surfaces are coated with different materials of varied properties are of particular interest. Study and analysis of the role of heterogeneous charge patches is important not only for exploiting these new material design routes, but also for studying proteins. Proteins often carry heterogeneous surface charge, which primarily drives aggregation behavior under definite conditions [16,17]. The anisotropy of protein interactions is essential for understanding aggregation behavior, and general, high-fidelity coarsegrain models of proteins need to account for charged as well as hydrophobic patches [18,19]. Controlling protein aggregation is important in the food and pharmaceutical industries and even for understanding neurodegenerative diseases such as Alzheimer’s [20–22]. In the present work, we focus on the mechanism of complex formation between Myoglobin designed as capsule and Bovine serum albumin (BSA) in solution for application in therapeutics, protein stabilization and extending the shelf-life with a view to propose a methodology for storing blood proteins. Mb has been designed in the colloidal form and its interactions with the globular protein BSA in solution form have been studied at the colloid/solution interface by balancing both electrostatic and steric stabilization. 2 different pH: 4.0 and 7.5 have been chosen for both Mb and BSA. The positively charged hydrated colloid of Mb (at pH = 4.0) interacting with negatively charged BSA (pH = 7.5) and the reverse case of Mb colloid (pH = 7.5) interacting with BSA (pH = 4.0) have been studied. Myoglobin (Mb) is a cytosolic heme protein and is a member of the globin family together with hemoglobin (Hb), neuroglobin (Ngb) and cytoglobin (Cygb). The most important condition for functioning of all living aerobic organisms is continuous supply of sufficient oxygen. The access of oxygen to metabolic processes in cells is mediated by the respiratory proteins, tetrameric blood hemoglobin (Hb) and monomeric muscle myoglobin (Mb). Myoglobin is expressed in red muscles in response to mitochondrial demand for oxygen, transporting it from sarcolemma to mitochondria [23–25]. O2 release from MbO2 at physiological po2 values proceeds only upon direct contact of the protein with mitochondria [26,27]. Monitoring myoglobin levels in blood and urine has been used as means to detect skeletal or cardiac muscle cell injury. Myoglobin is rapidly released after muscle damage, and thus can be a useful biomarker in the early phases of injury. Under specific conditions of muscle injuries the Mb interacts with the albumin and other proteins in order to bind to the mitochondrial membrane. Applications in pharmacotherapy and the development of an artificial cornea, require an understanding of limits of human corneal diffusion and its clinical relevance, particularly in the context of BSA interacting with myoglobin [28,29]. Albumin is the major plasma protein circulating in blood. Albumin potently decreases capillary permeability, although the mechanisms are not understood completely. Important factors in this study are; the electrostatic interactions between charged groups and charged patches on the two protein surfaces-one in the capsule form and the other in solution, dependence on the polyion properties such as charge density, and mechanical stiffness in terms of elastic compliance. These features of local and global structure of the protein-protein complexes are still poorly understood. The protein-protein complex formation is mainly driven by various non-covalent interactions, like electrostatic, H-bonding, hydrophobic, and steric interactions [30–34]. Protein carries +ve or −ve zeta potential based on the pH of the medium (+ve at pH lower than pI and vice-versa).
Please cite this article in press as: D. http://dx.doi.org/10.1016/j.ijbiomac.2017.07.157
2. Experimental 2.1. Materials and methods Bovine Serum Albumin (BSA) of 99.9% purity has been purchased from Sigma Aldrich, USA and used as such. Its isoelectric point is 4.8.Horse heart myoglobin was purchased from Biozyme (Reinheitszahl 1/4 4.8) (San Diego, CA) and was used as such. For every experiment, the protein dissolved in 20 mM 4-morpholinepropanesulfonic acid (Boehringer Mannheim, Ingelheim am Rhein, Germany) at pH 7.0 was filtered with Millipore (Billerica, MA) Millex SV filters before use. Before preparation of Mb colloids, a few crystals of sodium dithionite (final concentration 0.2 mg/mL) were added to the Mb, in the respective buffer to remove the oxygen and stored in a gastight sample container. The excess of dithionite was eliminated by gel filtration chromatography on a column (20 cm 1.5 cm) filled with Sephadex G-25 (Pharmacia, Sweden). Mb colloids have been prepared at two different pH using buffers pH 4.0 and pH 7.5, below and above the isoelectric point of Mb (pI = 6.9). These colloids have been prepared by the freeze-thaw technique of Kuppevelt et al. [35]. The respective buffer solutions are introduced in liquid nitrogen for about two minutes and then the protein solution is added dropwise. The frozen droplets of protein solution have been allowed to warm up to −35 ◦ C for 3 h and then stored at 8 ◦ C overnight before using them for measurements. The purified Mb colloids formed by the above procedure are hereafter referred as colloids. Mb colloids of either positive or negative charge (pH 4.0 or 7.5) are mixed with BSA solution (pH 7.5/4.0) in increasing concentrations. The pH variations, as a function of concentration allows for systematic changes in the electrostatic interactions of the components in the system and have been examined using zeta potential, UV−Vis spectroscopy, CD, fluorescence spectroscopy, DLS. The adsorption and interfacial organization of BSA/Mb mixtures on to solid surface have been analysed using Quartz Crystal Microbalance with dissipation (QCM-D) technique and the elastic compliance of the complexes formed have been evaluated. Stability of the complexes have been analysed using the interfacial elasticity and viscosity at colloid/solution interface. 2.2. UV–vis and steady state fluorescence measurements UV visible measurements have been carried out using Shimadzu UV1800 spectrophotometer (Japan), with quartz cells of 1 cm path length in the region 200–600 nm at 0.2 nm interval. Steady state fluorescence spectra have been recorded using VARIAN CARY Eclipse spectrophotometer and measured in the range 300–500 nm with the excitation wavelength at 280 nm. The spectra have been checked at intervals of 5 mins. for every sample of Mb complexed with BSA. 2.3. CD spectroscopy The far UV CD measurements for the colloids have been carried out in Jasco J 815 polarimeter at room temperature (295 K) using a 1 mm cell in the normal range 190–240 nm. The secondary structure distributions are fitted using Dichroweb online database [36]. The stability of the colloids and the surface charge interaction of the BSA/Mb have been monitored by particle size analyser and zeta potential measurements using Malvern Nano-ZS zeta sizer 2.4. Dilational rheology of Mb colloids in BSA solution Freshly prepared solutions of the Mb capsules and BSA solution have been used for each measurement. The surface tension of
Madhumitha,
A.
Dhathathreyan,
Int.
J.
Biol.
Macromol.
(2017),
G Model
ARTICLE IN PRESS
BIOMAC-7965; No. of Pages 10
D. Madhumitha, A. Dhathathreyan / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
3
Fig. 1. UV Visible spectra of Mb colloids at (a) pH = 4.0 and (b) pH = 7.5 in BSA soln (pH = 7.5 and 4.0 respectively).
these samples are measured with the profile analysis tensiometer PAT-1 (SINTERFACE Technologies, Germany) with an accuracy of ±0.1 mN/m and thermostated at a temperature of 25 ◦ C. Drops of the solution as well as the solution containing capsules are formed at the tip of a PTFE capillary immersed into a cuvette filled with a buffer-saturated atmosphere. After having reached the adsorption equilibrium after about 1hr., the drop is subjected to harmonic oscillations with frequencies (f) 0.01–0.2 Hz in order to study the dilatational elasticity. For each frequency, the dilation/expansion was carried out for atleast 30mins. Accuracy in elasticity and viscosity values are 1 mN/m and 1 mN/ms. Each measurement is the average of three measurements and are presented in this study. 2.5. Quartz Crystal microbalance with dissipation (QCM-D) QCM-D sensors with a silica coating used in the study have been cleaned using a 2% sodium dodecyl sulfate solution (for 30 min), rinsing with ultrapure water, blow-drying with nitrogen. Cleaned substrates have been stored in air and again exposed to UV/ozone (30 min) prior to use. Adsorption and interfacial processes have been monitored simultaneously using a home-built, impedancebased QCM consisting of an impedance (network) analyzer for determining resonance frequencies and bandwidths of a quartz crystal [37] and a fluid cell, also home-built, where the crystals have been mounted, to allow adsorption, fluid exchange, and temperature control. The network analyzer was purchased from Ivan Makarov (makarov.ca, Canada). The resonance frequencies and bandwidths have been collected for overtones, n, between 3 and 11, corresponding to frequencies between 15 and 55 MHz. Following the conventional methodology, we quantify dissipative processes using the dissipation factor D, defined as the ratio of full bandwidth of the resonance to the resonance frequency (the inverse Q-factor). The frequency shift values are scaled by the overtone order, that is, f/n. This is because f/n is the same for all overtones if the frequency shift is caused by gravimetric effects. Dissipation changes are not scaled by the overtone order because such a scaling by n is implicitly contained in the definition of D itself. 2.6. Scanning electron micrographs Scanning electron micrographs have been obtained using a field emission scanning electron microscope (FE-SEM, Hitachi S-4200). The films transferred onto the substrates have been dried in a desPlease cite this article in press as: D. http://dx.doi.org/10.1016/j.ijbiomac.2017.07.157
iccator for at least 1 h and then coated with gold prior to SEM observation.
2.7. Results and discussion The complex formed from Mb colloids of pH 4.0 and pH 7.5. mixed with BSA solutions of pH 7.5 and pH 4.0 respectively in increasing concentrations have been characterized using UV–vis and fluorescence spectra. The characteristic absorbance spectra for the mixture are given in Fig. 1(a) and (b). In both the cases, addition of Mb colloids to BSA solution shows an increase in absorbance intensity as observed at Soret band at 409 nm without much difference shift in its maximum wavelength. The Soret band is very sensitive to the heme environment and is commonly used to monitor heme pocket disruption in presence of external perturbation. Circular dichroism (CD) and steady state fluorescence experiments have been performed to monitor the interactions between Mb colloids and BSA solution. The fluorescence spectra of the two mixtures are shown in Fig. 2(a) and (b). The emission spectra of Trp of BSA have been recorded in the range 300–500 nm with the excitation wavelength at 280 nm. BSA solution at pH 4.0 shows emission at 347 nm with an intensity of 33.91a.u. As the concentrations of Mb colloids are increased in BSA soln., the fluorescence intensity starts to quench but no dramatic change shift in the wavelength. The fluorescence quenching of BSA is due to the proximity of Mb colloids which reveal nearly a 1:1 interaction with a binding constant of about 105 M−1 . The heme group of myoglobin’s is buried into the protein globule rather than exposed on its surface, so the quenching of donor emission by myoglobin cannot occur by a dynamic mechanism. Energy transfer to the heme only occurs in a complex whose lifetime exceeds that of donor fluorescence (static quenching). The stability of the proteins at the colloid/solution interface have been monitored using Circular dichroism measurements are shown in Fig. 3. Here the spectra shown are that of the Mb colloids measured by using BSA soln. as reference. The corresponding secondary structure distributions are fitted using CD PRO shown in Table 1. From the table it is observed that, the Mb proteins at capsule/solution interface are fairly stable. At a concentration of 2.5 × 10−7 M of BSA solution in the mixture, Mb in the colloids (pH 4.0), has a maximum of 68.3% of helix. For the reverse case, Mb colloids (pH 7.5) for a conc. of 1 × 10−7 in BSA solution show 80% of
Madhumitha,
A.
Dhathathreyan,
Int.
J.
Biol.
Macromol.
(2017),
G Model
ARTICLE IN PRESS
BIOMAC-7965; No. of Pages 10
D. Madhumitha, A. Dhathathreyan / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
4
Fig. 2. Fluorescence Spectra of BSA Soln. with (a) Mb colloids (pH 7.5) (b) Mb colloids (pH 4.0).
Fig. 3. Circular dichroic spectra of Mb colloids at (a) pH = 4.0 and (b) pH = 7.5 in BSA soln (pH = 7.5 and 4.0 respectively).
Table 1% Secondary Structural Population of Mb in complex of Mb colloids with BSA solution. Conc. of Mb colloids in BSA soln. (x10−7 M)
0.5 1 1.5 2 2.5 3 5 6 8
Mb colloids (pH 4.0)
Mb colloids (pH 7.5)
␣ helix
 sheet
 turn
Unrd
␣ helix
 sheet
 turn
Unrd
27.6 28 27.8 27.2 68.3 64.9 46.8 48.1 41.2
20.3 20.2 20.6 21.3 4.1 4.9 12.5 11 14
21.7 21.4 22 21.4 10.9 13.2 15.9 15 19.4
30.4 30.3 29.5 30.1 16.7 17 24.9 26 25.7
69.6 80.6 74.8 71.6 70.6 71.3 56.7 57 49.3
5.8 3.2 2.7 4.3 4.7 5.1 7.7 9.1 13.1
7.3 7.1 9.3 8.8 5.7 6.5 11.4 11 17.3
17.3 9.2 13.2 15.3 19.1 17.3 24.2 22.9 20.3
helicity. This shows that the secondary structure of Mb gets more helicity at pH 7.5 suggesting the levels of hydration are higher. The stability of the colloids at colloid/solution interface has been monitored using zeta potential and particle size measurements. Fig. 4 shows the zeta potential plot for BSA soln. (pH 4.0) with Mb colloids (pH 7.5) and vice versa. The size of Mb colloids at pH 4.0 in BSA soln. at pH 7.5 varies from 200 to 600 nm. In the reverse case, the average size varies from 200 nm to 400 nm showing that
Please cite this article in press as: D. http://dx.doi.org/10.1016/j.ijbiomac.2017.07.157
changes in overall charge density on the Mb leads to altered level of hydration which in turn can make the colloids compacter. Fig. 5(a) and (b) shows the SEM micrographs of the Mb colloids mixed with the maximum BSA concentration for both pH = 4.0 and 7.5 respectively. From the Figure it is seen clearly that the colloids are nearly spherical and the fused colloids are a result of the drying process.
Madhumitha,
A.
Dhathathreyan,
Int.
J.
Biol.
Macromol.
(2017),
G Model
ARTICLE IN PRESS
BIOMAC-7965; No. of Pages 10
D. Madhumitha, A. Dhathathreyan / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
5
Fig. 4. Zeta potential plots for Mb colloids at pH = 4.0 and pH = 7.5 in BSA soln. (pH = 7.5 and 4.0 respectively).
Fig. 5. Scanning electron micrographs of Mb colloids (a) pH = 4.0 (b) pH = 7.5 in BSA solution (pH = 7.5 and pH = 4.0 respectively) (Scale bar = 200 nm).
2.8. Size of the colloids and the complex The sizes of the colloids are shown in Fig. 6.The largest size for Mb colloids (270 nm) at the lowest BSA concentration is driven by partially folded protein, whereas at higher pH the size is smaller (∼200 nm). This could be due to repulsive interactions between negatively charged myoglobin monomers, while at higher pH the Mb molecules still maintain their native form.
D/f vs Time plots for the different overtones are shown in Fig. 7(a) and (b). Based on the work of Du and Johannsmann, for simple thin viscoelastic film in a Newtonian liquid, one can show that the dissipation induced by the film is proportional to the elastic compliance [39]. Based on the plots of D/f versus overtone order, the elastic compliance may be evaluated from the slope using the following equation D/-f = 2nf 0 J
2.9. QCM-D The adsorption and interfacial organization of BSA/Mb mixtures on to quartz surface have been analysed using Quartz Crystal Microbalance with dissipation (QCM-D) technique. When the Mb colloids dispersed in BSA solution contacts the Quartz surface, adsorption is initiated. As the resonant frequency gets damped due to the adsorbing material, mass is increasing. However, since hydrodynamically trapped solvent contributes to the mass, mass cannot be determined independently. The focus here is on the intrinsic properties of the film and for this one considers half band half width to the negative frequency shift [38]. Both these parameters are proportional to the film mass at the thin film limit. The Please cite this article in press as: D. http://dx.doi.org/10.1016/j.ijbiomac.2017.07.157
J is the elastic compliance, f0 the fundamental frequency (5 MHz), the viscosity of the solution. Fig. 8(a) and (b) shows the estimated elastic compliance as a function of the BSA in solution on addition to the Mb colloids. The elastic compliance has been estimated for both pH 4.0 and pH 7.5 colloids of Mb for titration with BSA in solution.Tthe elastic compliance for the positively charged Mb colloids are lower than that of Mb colloids at pH = 7.5. The derived elastic compliance is in the same range for all harmonics. Proteins organized biomolecules, with characteristic secondary structures, such as ␣helices, -strands, turns and loops, are folded and assembled to assume a characteristic three-dimensional structure that defines
Madhumitha,
A.
Dhathathreyan,
Int.
J.
Biol.
Macromol.
(2017),
G Model BIOMAC-7965; No. of Pages 10 6
ARTICLE IN PRESS D. Madhumitha, A. Dhathathreyan / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
Fig. 6. Sizes of complex formed between Mb colloids (pH = 4.0 and 7.5 respectively) with BSA in soln. (at pH = 7.5 and 4.0 respectively).
the to the protein’s function, dynamics, and mechanical properties [40,41]. An analysis of the secondary structure population of Mb, the size changes, zeta potential and the elastic compliance gives a direct correlation between the structure, intrinsic rigidity and stability of the complex. The results show clearly at pH 7.5, Mb colloids are larger in size, have higher helicity (∼80%) and higher elastic compliance. These results are similar to that of Perticaroli et al. who analyzed the nanomechanical properties of few proteins with different secondary structures: ␣-helices (myoglobin and bovine serum albumin), -barrels (green fluorescent protein), and ␣ +  + loop structures (lysozyme) [42]. In their work they demonstrated that ␣-proteins have lower elastic moduli than those dominated by  motifs. From the plot in this work, it is seen that the change in elastic compliance follows the change in zeta potential suggesting the role of hydration and the charge-charge interactions at the capsule/solution interface. It is known that water engages in two important types of interactions near proteins: it forms ordered cages around exposed hydrophobic regions, and it participates in hydrogen bonds with surface polar groups. A special role in the inter-protein interactions may be played by intermolecular salt bridges between negatively charged carboxylate groups of the Asp residues and the positively charged side groups of the lysine and arginine residues. These salt bridges can best form at near-neutral pH values and can contribute to the intermolecular connectivity. At low pH, the carboxylate groups are neutralized to carboxylic acid groups, which imply that the salt bridges would be disrupted, which would lead to a decrease of the strength of the inter-protein interactions. Where liquid water is absent, salt bridges could be strong and could fulfill an important role in maintaining a strong water-repellent film. In aqueous solutions containing positively charged Mb colloids dispersed in negatively charged BSA solution, a complex is formed through the non-covalent interaction between oppositely charged groups. This process is affected by the charge density of each macro ion. Within this we distinguish two major categories of noncovalent binding depending on strength of attractive interactions between Mb colloids and BSA. At pH = 4 for the Mb colloids, First is a weak interaction where the BSA from solution binds to small, multiple positive patches on the Mb colloid surface without affect-
Please cite this article in press as: D. http://dx.doi.org/10.1016/j.ijbiomac.2017.07.157
ing the native conformation of Mb. The second type of attractive interactions is described as a strong binding, where the BSA binds to a single, large positive patch on the protein surface. The overall scheme for formation of the complex is given in Fig. 9. In the case of Mb colloids of a particular charge at either pH = 4 or 7.5 present in the BSA solution, it is surrounded by an ionic cloud containing an excess of counterions. Under these conditions electrostatic interactions between the Mb charged groups themselves are screened. Consequently, the long-range repulsive interactions between the charged colloids are weakened exponentially with the increasing distance between them. Increasing BSA concentrations leads to increased screening which can cause structural changes in the Mb colloids which in turn gets reflected in the zeta potential size and the elastic compliance. Thus decreasing sizes for Mb colloids at pH = 4 with increasing BSA could indicate an effect like the ‘salting out’ thus supporting screening of the long-range attractive electrostatic interactions. The corresponding elastic compliance is smaller than that of the Mb colloids at near neutral pH value. Stoichiometric ([+]/[−] = 1) complexes are formed from BSA +Mb colloids in buffer. In general, the complex between BSA and Mb colloids is recognized to be electrostatically driven. For example, Porfiri et al. reported that shielding by increasing strength of the solution will lead to a reduction in complex formation [43,44]. Boeris et al. studied the aqueous solution behavior of cationic polyethyleneimine in the presence of anions with two or more electrical charges like citrate, phosphate, sulphate, malate, malonate and succinate using turbidimetry and light scattering. Their results suggest that the non-soluble polyethyleneimine–anion complexes have the property to precipitate macromolecules charged with an opposite electrical charge at specific concentrations [45]. Milioni et al. have used the D/f to derive information on the conformation of surface attached biomolecules. The basis for this measurement is that the (D/F) is a measure of the hydrodynamic volume of the attached entity, expressed by its intrinsic viscosity [] [46]. The method proposed in this work thus can be used to evaluate the stability of hydrodynamic volume of charged biomolecules interacting with small molecules as well as other biomolecules as well as quantify it. The dilational elasticity and viscosity of pure Mb colloids at pH = 4 and 7.5 and complexing with the BSA soln. at the point where zeta potential shows near neutralization have been studied.
Madhumitha,
A.
Dhathathreyan,
Int.
J.
Biol.
Macromol.
(2017),
G Model BIOMAC-7965; No. of Pages 10
ARTICLE IN PRESS D. Madhumitha, A. Dhathathreyan / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
7
Fig. 7. (a). D vs. f plots for 3rd, 5th, 7th, 9th and 11th overtones for (a) Mb colloids (pH = 4.0) for different effective concentrations of BSA solution, (pH = 7.5) (b) Mb colloids (pH = 7.5) for different effective concentrations of BSA solution, (pH = 4.0).
Please cite this article in press as: D. http://dx.doi.org/10.1016/j.ijbiomac.2017.07.157
Madhumitha,
A.
Dhathathreyan,
Int.
J.
Biol.
Macromol.
(2017),
G Model BIOMAC-7965; No. of Pages 10 8
ARTICLE IN PRESS D. Madhumitha, A. Dhathathreyan / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
Fig. 8. Elastic compliance of (a) Mb colloids (pH = 4.0) for different effective concentrations of BSA solution, pH = 7.5 (b) Mb colloids (pH = 7.5) for different effective concentrations of BSA solution, pH = 4.0.
Fig. 9. Scheme of complex formation of Mb colloids with BSA in solution and changes in the elastic compliance.
Fig. 10(a) and (b) shows the values of the elasticity and viscosity. It is seen that as frequency of dilation increases elasticity increases (3–7 mN/m) and viscosity decreases (50-15 mN/m.S) with a sharp change as frequency for the pure Mb colloids at pH = 4.0. However on complex formation with BSA solution there is an overall decrease in elasticity values (3–5 mN/m) while the viscosity a slightly higher value initially (80-7 mN/m S) The pure Mb colloids show lower elasticity as well as viscosity values at pH = 7.5 compared with that at pH = 4.0. However, there is an increase in both elasticity and viscosity values as complex is formed between Mb colloids and BSA in solution. These are in agreement with the changes in zeta potential and the sizes of the
Please cite this article in press as: D. http://dx.doi.org/10.1016/j.ijbiomac.2017.07.157
complex formed at the interface due to the charge-charge interaction and the degree of hydration associated with it. Here water acts truly as a plasticizer and such a property of water giving rise to anomalous dilational elasticity and viscosity in the presence of different counterions to different extent have been reported by Lakshmanan et al. [47,48]. In the present work, the Mb colloids interacting with BSA undergo direct adsorption to the gold coated quartz surface. We find that the extent of dissipation caused by the film and the evolution of dissipation as a function of surface coverage is dependent on the internal properties of these particles and their attachment to the surface. A limited rotation of the colloids in the buffer
Madhumitha,
A.
Dhathathreyan,
Int.
J.
Biol.
Macromol.
(2017),
G Model BIOMAC-7965; No. of Pages 10
ARTICLE IN PRESS D. Madhumitha, A. Dhathathreyan / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
9
Fig. 10. Dilational elasticity and viscosity of pure Mb colloids at (a) pH = 4.0 and with BSA solution at pH = 7.5 (b) pH = 7.5 and with BSA solution at pH = 4.0.
containing the BSA (rocking) and a small sliding contribute to the dissipation. For every change in concentration of BSA in the solution, charge-charge interaction, followed by some% of water displacement takes place which results in change in the frequency. As adsorption progresses, at higher coverage, lateral hydrodynamic interactions between neighboring colloids compete with the modes of dissipation, which results in a maximum in dissipation at some intermediate adsorption times. In study of adsorption of biomolecules, determining an adsorbate’s amount, shape, orientation, and internal structure is a challenge. One requires to combine many different techniques based on different sensing principles to get a more realistic representation of the adsorbate’s organization on the surface. The work has attempted to focus on the nature of the binding, the structure and properties of the complexes that result due to Mb in the colloidal state interacting with BSA in the solution. The work demonstrates the correlation between charge-charge interaction, ‘salting-in/out’ effects, degree of hydration and the elastic compliance of the Mb colloids. Results show clearly that as the Mb colloids get compacter at low pH, the levels of hydration decrease resulting in low elastic compliance. By evaluating the D/f ratio one can measure elastic compliance in liquid medium for very thin films and may have significance in sensing of the biological absorbates. The results suggest that the this ratio is sensitive to the charge-charge
Please cite this article in press as: D. http://dx.doi.org/10.1016/j.ijbiomac.2017.07.157
interaction between Mb colloids and BSA and their lateral organization of molecules on the surface. QCM-D does not have an intrinsic lateral resolution on the nanometer-scale but the time-resolved correlation of both D and F can provide quantitative insight into the organization of proteins on the molecular scale. Understanding the intrinsic rigidity of ␣-helices or -sheets, is crucial to unravelling the relationships between structure, mechanical properties, dynamics, and function in biological systems. The analytical approach to evaluate elastic compliance as a function of charge densities on the Mb colloids, should be useful for the investigation of molecular processes at model membranes, the organization of nanosized particles, including viruses or drug delivery vehicles, at surfaces. Acknowledgements The authors would like to thank DST-SERB, Govt. of India for a project grant under which part of the work was carried out. D.M. would like to thank DST-SERB for the Senior Research fellowship. References
Madhumitha,
[1] Zhang, S.C. GlotzerZhan, Nano Lett. 4 (2004) 1407–1413. [2] S.C. Glotzer, M.J. Solomon, Nat. Mater. 6 (2007) 557–562. [3] Y. Gi-Ra, J.P. David, S. Stefano, J. Phys.: Condens. Matter 25 (2013) 193101.
A.
Dhathathreyan,
Int.
J.
Biol.
Macromol.
(2017),
G Model BIOMAC-7965; No. of Pages 10 10
ARTICLE IN PRESS D. Madhumitha, A. Dhathathreyan / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
[4] P. Akcora, H. Liu, S.K. Kumar, J. Moll, Y. Li, B.C. Benicewicz, L.S. Schadler, D. Acehan, A.Z. Panagiotopoulos, V. Pryamitsyn, V. Ganesan, J. Ilavsky, P. Thiyagarajan, R.H. Colby, J.F. Douglas, Nat. Mater. 8 (2009) 354–359. [5] M.S. Nikolic, C. Olsson, A. Salcher, A. Kornowski, A. Rank, R. Schubert, A. Frömsdorf, H. Weller, S. Förster, Angew. Chem. Int. Ed. 48 (2009) 2752–2754. [6] J.A. Luiken, P.G. Bolhuis, Phys. Rev. E 88 (2013) 012303. [7] C. Janin, A.-C. Taylor, Introduction, Presses Universitaires de France, 2009. [8] E.D. Levy, J.B. Pereira-Leal, Curr. Opin. Struct. Biol. 18 (2008) 349–357. [9] D. Reichmann, O. Rahat, M. Cohen, H. Neuvirth, G. Schreiber, Curr. Opin. Struct. Biol. 17 (2007) 67–76. [10] M. Vidal, M.E. Cusick, A.L. Barabasi, Cell 144 (2011) 986–998. [11] F.P. Davis, D.T. Barkan, N. Eswar, J.H. McKerrow, A. Sali, Protein Sci. 16 (2007) 2585–2596. [12] Y. Loewenstein, D. Raimondo, O.C. Redfern, J. Watson, D. Frishman, M. Linial, C. Orengo, J. Thornton, A. Tramontano, Genome Biol. 10 (2009) 207. [13] S.H. Khan, F. Ahmad, N. Ahmad, D.C. Flynn, R. Kumar, J. Biomol. Struct. Dyn. 28 (2011) 929–938. [14] J. Zhang, E. Luijten, S. Granick, Annu. Rev. Phys. Chem. 66 (2015) 581–600. [15] J.M. Dempster, M. Olvera de la Cruz, ACS Nano 10 (2016) 5909–5915. [16] J.M. Park, B.B. Muhoberac, P.L. Dubin, J. Xia, Macromolecules 25 (1992) 290–295. [17] P.R. Majhi, R.R. Ganta, R.P. Vanam, E. Seyrek, K. Giger, P.L. Dubin, Langmuir 22 (2006) 9150–9159. [18] H. Liu, S.K. Kumar, F. Sciortino,J. Chem. Phys. 127 (2007), 084902. [19] L.J. Quang, S.I. Sandler, A.M. Lenhoff, J. Chem. Theory Comput. 10 (2014) 835–845. [20] E. Bianchi, R. Blaak, C.N. Likos, Phys. Chem. Chem. Phys. 13 (2011) 6397–6410. [21] M.E.M. Cromwell, E. Hilario, F. Jacobson, AAPS J. 8 (2006) E572–E579. [22] C.A. Ross, M.A. Poirier, Nat. Med. 10 (Suppl) (2004) S10–S17. [23] R. Hill, Nature 132 (1933) 897–898. [24] G.A. Millikan, Physiol. Rev. 19 (1939) 503–523. [25] R. Starodub, V. Korobov, V. Nazarenko, Naukova Dumka, Kiev (1992) 1. [26] G.B. Postnikova, S.V. Tselikova, Biofizika 50 (2005) 297–306. [27] G.B. Postnikova, S.V. Tselikova, E.A. Shekhovtsova, Biochemistry 74 (2009) 1211–1218. [28] R.A. Charalel, K. Engberg, J. Noolandi, J.R. Cochran, C. Frank, C.N. Ta, Ophthalmic Res. 48 (2012) 50–55.
Please cite this article in press as: D. http://dx.doi.org/10.1016/j.ijbiomac.2017.07.157
[29] G.B.P. Ekaterina, A. Shekhovtsova, J. Phys. Chem. Biophys. 03 (2013). [30] C.G. de Kruif, R. Tuinier, Food Hydrocolloids 15 (2001) 555–563. [31] S.K. Chaturvedi, M.K. Siddiqi, P. Alam, M. Zaman, R.H. Khan, RSC Adv. 6 (2016) 85860–85869. [32] P. Alam, S.K. Chaturvedi, T. Anwar, M.K. Siddiqi, M.R. Ajmal, G. Badr, M.H. Mahmoud, R. Hasan Khan, J. Lumin. 164 (2015) 123–130. [33] M.K. Siddiqi, P. Alam, S.K. Chaturvedi, R.H. Khan, Int. J. Biol. Macromol. 92 (2016) 1220–1228. [34] M.K. Siddiqi, P. Alam, S.K. Chaturvedi, S. Nusrat, M.R. Ajmal, A.S. Abdelhameed, R.H. Khan, Int. J. Biol. Macromol. (2017), http://dx.doi.org/10. 1016/j.ijbiomac.2017.07.036. [35] W.F. Daamen, P.J. Geutjes, H.T.B. van Moerkerk, S.T.M. Nillesen, R.G. Wismans, T. Hafmans, L.P.W.J. Van den Heuvel, A.M.A. Pistorius, J.H. Veerkamp, J.C.M. vanHest, T.H. van Kuppevelt, Adv. Mater. 19 (2007) 673–677. [36] L. Whitmore, B.A. Wallace, Biopolymers 89 (2008) 392–400. [37] R. Beck, U. Pittermann, K.G. Weil, Berichte der Bunsengesellschaft für Physikalische Chemie 92 (1988) 1363–1368. [38] M.A. Plunkett, Z. Wang, M.W. Rutland, D. Johannsmann, Langmuir 19 (2003) 6837–6844. [39] B. Du, D. Johannsmann, Langmuir 20 (2004) 2809–2812. [40] M.J. Buehler, Proc. Natl. Acad. Sci. 103 (2006) 12285–12290. [41] T. Ackbarow, X. Chen, S. Keten, M.J. Buehler, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 16410–16415. [42] S. Perticaroli, J.D. Nickels, G. Ehlers, H. O’Neill, Q. Zhang, A.P. Sokolov, Soft Matter 9 (2013) 9548–9556. [43] M.C. Porfiri, M. Braia, B. Farruggia, G. Picó, D. Romanini, Process Biochem. 44 (2009) 1046–1049. [44] M.C. Porfiri, G. Picó, B. Farruggia, D. Romanini, Process Biochem. 45 (2010) 1753–1756. [45] V. Boeris, C. Cassane, J. Wagner, G. Picó, Colloids Surf. B: Biointerfaces 82 (2011) 354–358. [46] D. Milioni, A. Tsortos, M. Velez, E. Gizeli, Anal. Chem. 89 (2017) 4198–4203. [47] M. Lakshmanan, R. Parthasarathi, A. Dhathathreyan, Biochim. Biophys. Acta 1764 (2006) 1767–1774. [48] M. Lakshmanan, A. Dhathathreyan, R. Miller, Coll. Surf. A 324 (2008) 194–201.
Madhumitha,
A.
Dhathathreyan,
Int.
J.
Biol.
Macromol.
(2017),