Role of viscogens on the macromolecular assemblies of fibrinogen at liquid/air and solid/air interfaces Kamatchi Sankaranarayanan
Citation: Biointerphases 10, 021009 (2015); doi: 10.1116/1.4922291 View online: http://dx.doi.org/10.1116/1.4922291 View Table of Contents: http://avs.scitation.org/toc/bip/10/2 Published by the American Vacuum Society
Role of viscogens on the macromolecular assemblies of fibrinogen at liquid/air and solid/air interfaces Kamatchi Sankaranarayanana) DST-INSPIRE Faculty, Department of Energy and Environment, National Institute of Technology (NIT), Tiruchirapalli 620015, India
(Received 4 April 2015; accepted 28 May 2015; published 10 June 2015) In this study, an attempt has been made to understand the organization and association of fibrinogen (Fg) in solvent environment induced by viscogens such as 1-ethyl 3-methyl imidazolium ethyl sulfate (IL-emes), Ficoll, and Trehalose. The author observed that Fg in IL-emes adsorbed on solid surface shows higher b-sheet conformation. Shear viscosity measured using quartz crystal microbalance, for Fg in IL-emes was highest with a corresponding higher adsorbed mass 3.26 lg/cm2. Associated assemblies of the protein at the liquid/air interface were monitored with changes in surface tension and were used to calculate work of adhesion. Changes in work of adhesion were used as a tool to measure the adsorption of Fg to solid surfaces in presence of viscogens and highest adsorption was observed for hydrophilic surfaces. Scanning electron microscopy images show Fg in trehalose forms elongated bead like structures implying organization of the protein at the interface. Crowding in the solvent environment induced by viscogens can slow down organization of Fg, C 2015 American Vacuum Society. leading to macromolecular assemblies near the interface. V [http://dx.doi.org/10.1116/1.4922291]
I. INTRODUCTION Adsorption of soluble proteins at solid–liquid interfaces plays a fundamental role in biomedical devices. Protein adsorption is the first interaction with the implanted biomaterials.1,2 It is crucial for tissue compatibility with the implants, cellular interfacing with semiconductor materials, molecular electronics, biomaterials. Especially, adsorption of serum proteins, such as fibrinogen (Fg), plays a major role in designing suitable bioimplantable materials.3,4 The interactions between protein and surface depend on various factors such as surface charge, protein size, protein dipole moment, ionic strength, and solvent viscosity. In spite of extensive study on protein adsorption, basic questions concerning the structural details of a protein in its adsorbed state is yet unanswered. Especially, effect of viscogens on adsorption of proteins to surface remains elusive. Experimental strategies for controlling protein adsorption in a real-life situation mimicking crowding are crucial to create a biocompatible surface. Biochemical process in living systems occur in highly crowded environments. Macromolecular self-association, formation of aggregates in protein, and amyloid inclusion bodies in some diseases such as Parkinson’s disease, Alzheimer’s disease, etc., are specific reactions that trigger crowding.5 The use of high solute concentrations to mimic crowded environments results in an excluded volume effect, an increased viscosity, and also a secondary solvation effect. Out of this, increase in viscosity is attributed to crowding density as well as the size of cosolute. Experiments mimicking crowding generally use high-molecular weight cosolutes, such as Dextran and Ficoll 70, to mimic all three effects, or
a)
Electronic addresses: [email protected]; [email protected]
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small molecules such as sucrose and trimethylamine N-oxide, a bacterial osmolyte, to primarily act upon viscosity and/or solvation of the protein backbone. Several studies have been carried out to understand the possible effects of crowding on folding and association of proteins.6–10 Effect of crowding from experimental and theoretical point of view on protein folding,11–17 stability,18,19 protein association,20–22 and protein aggregation23 have been studied by a number of groups. Plasma fibrinogen is an important component of the coagulation cascade, as well as a major determinant of blood viscosity and blood flow. In many diseases including ischemic heart disease, stroke have been reported due to elevated plasma fibrinogen levels and also associated with an increased risk of cardiovascular disorders.24–26 Even though a number of research papers have appeared in the literature on the role of Fg and its importance in many fields, a fundamental understanding of its organization at different interfaces, adsorption kinetics, and the corresponding conformational change is still lacking, which shows the complexity of this problem. Quartz crystal microbalance (QCM) with dissipation monitoring has been used by Weber et al. for the real time study of Fg adsorption to model biomaterial surfaces.27 Fg as lyophilisome to design nanoclusters of nickel oxide and nickel hydroxides has been reported.28 While fibril formation during adsorption of fibrinogen to surfaces is known to be catalyzed by thrombin, there still exists a debate about whether the enzyme is solely responsible for the organized structures or is it a response to the substrate or the adsorbing conformation of Fg. Recently, the role of surface roughness on adsorption of Fg to silica surfaces has been studied by Lord et al. using stochastic nanolithography.29 In this work, the influence of viscogens on the organization, association, and subsequent adsorption of plasma
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021009-2 Kamatchi Sankaranarayanan: Role of viscogens on the macromolecular assemblies R
protein Fg has been carried out. FicollV 400 (FL), ionic liquid: 1-ethyl 3-methyl imidazolium ethyl sulfate (IL-emes), and trehalose (Treh) have been used as viscogens. Ficoll is a highly branched polymer formed by the copolymerization of sucrose and epichlorohydrin. It is completely nonionic and because of the abundance of hydroxyl groups, it is very hydrophilic and extremely water-soluble. Ficoll is recognized as one of the crowding agents.30 IL-emes increases the viscosity of the solvent environment and also involves in hydrogen bonding interactions with water.31 Trehalose interacts very efficiently with water, i.e., the interaction between trehalose/water is much stronger than water/water interaction and can inhibit protein folding during aggregation.32,33 Quartz crystal microbalance has been used to analyze the adsorbed protein to the solid/liquid interface and also measuring the shear viscosity. Shear viscosity reported in this study is a 2D rheological measurement carried out at isothermal conditions. Surface tension measurements at water/air interface and contact angle analysis at the solid/liquid interfaces have been carried out to analyze the role of interfaces on Fg adsorption. Together with conformation of the adsorbed protein, the role of viscosity in Fg adsorption has been discussed. II. EXPERIMENTAL SECTION Fg from bovine plasma, IL-emes, FL (Product No. F4375), and Treh were purchased from Sigma Aldrich chemicals, USA, and was more than 99% pure. Lipids dioctadecyldimethylammonium bromide (DOMA) and 1,2dipalmitoyl-sn-glycero-3-phospho-(10 -rac-glycerol) (sodium salt) (DPPG) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were purchased from Larodon, Sweden (99.9% pure), and used as such for the preparation of Langmuir–Blodgett (LB) films. Chloroform and methanol High Performance Liquid Chromatography grade were purchased from Merck. All aqueous solutions were prepared with distilled water further purified with a four-stage Milli-Q water system (Millipore, resistivity greater than 18.2 MX). Average molecular weight of Ficoll 400 is 450 000 and radius of Ficoll is approximately 10 nm. The solutions of Fg were prepared at pH 7.5 (10 mM phosphate buffer) with final concentration of 0.1 lM under mild agitation. The resulting solution was clear and was used for all the experiments. The additives concentration was fixed at 10 lM for IL-emes and Trehalose, whereas 2% (w/v) for FL which is about 50 lM. All the concentrations were fixed based on the previous study by the authors.34 The pH of the protein solution with the additives remained the same. All measurements reported here were done a minimum of three times to ensure reproducibility. A. UV2VIS and fluorescence spectroscopy
The samples were analyzed using UV-1800-Shimadzu spectrophotometer with quartz cuvettes of 1 cm path length. The corresponding hydrated FL, IL, and Treh were used as reference for the measurements. The steady state fluorescence Biointerphases, Vol. 10, No. 2, June 2015
spectroscopy was performed using Spectrometer with the kex ¼ 280 nm.
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Cary
Eclipse
B. CD spectroscopy
The Circular Dichroic (CD) spectra of pure Fg at pH 7.5 and with IL-emes, FL, and Treh were carried out using a JASCO J-715 spectropolarimeter (JASCO Corp., Tokyo). The far-UV (240–190 nm) spectra of the protein in different levels of hydrated IL obtained using 0.1 cm path length quartz cells were analyzed using the Dichroweb fitting to three structural parameters: a-helix, b-sheet, and aperiodic.35 C. Picosecond-time-resolved fluorescence spectroscopy
Fluorescence lifetime measurements of samples have been carried out in a picosecond-time-correlated single-photon-counting spectrometer. The excitation source is the tunable Ti-sapphire laser (Spectra Physics, USA). The laser pulse (pulse width of 2 ps and repetition rate of 4 MHz) has been derived from the frequency-doubled output (532 nm) of mode-locked laser (Spectra Physics, USA). The picosecond standard tuning range is 720–850 nm. The sample was excited by the laser pulse at 280 nm, and emission was monitored at right angles to the excitation path. The first photon was detected by the microchannel plate photomultiplier tube (Hamamatsu-C 4878). When the first excitation pulse occurred, a synchronization pulse triggered the charging of the capacitor in the time to amplitude convertor through the discriminator. The voltage on the capacitor increased linearly until a stop timing pulse was detected on repeating the start–stop cycle, and a histogram representative of the fluorescence decay was obtained, which has been further analyzed using the IBH (Glasgow, United Kingdom) software.36 D. Quartz crystal microbalance measurements
Plasma cleaned (exposed to UV/ozone for 10 min) gold-coated quartz substrates were used for all QCM measurements. Measurements were performed with 50 ll of temperature-stabilized and degassed sample liquid, which was delivered to the chamber containing the sensor crystal to ensure a complete exchange of the liquid. This ensures that processes of adsorption and surface adlayer changes can be followed in situ while subsequently exposing different solutions to the surface. All measurements were performed at a temperature of 2425 C. QCM sensors quartz crystal microbalance, crystal holders, and polished gold AT cut 5 MHz gold crystals of 25 mm diameter crystals from Maxtek were used for the study. The oscillation frequency was measured using a Maxtek RQCM with phase lock oscillator with independent crystal measurement channel. Data acquisition was performed using the Maxtek RQCM Data Logging software (v. 1.6.0) on a personal computer connected through an RS 232 serial interface. A sampling rate of 1/60 Hz was employed for all experiments. Any baseline drift was regulated using the coarse and fine capacitance adjustments. Upon interaction of protein with the surface of
021009-3 Kamatchi Sankaranarayanan: Role of viscogens on the macromolecular assemblies
a sensor crystal, changes in the resonance frequency, Df, are related to attached mass governed by the Sauerbray’s equation Dm ¼ ðlqÞ1=2 ðDfÞ=ð2f 0 Þ2 :
(1)
f0 is the resonance frequency of the crystal (5 MHz at 25 C), l is the shear modulus of the quartz crystal (2.947 1011 g s2 cm1 at room temperature), and q is the density of quartz (2.648 g cm3). Shear viscosity is then given by the equation g ¼ ð19:627 ðDfÞ2 107 Þ=q:
(2)
E. Langmuir–Blodgett films
LB films of cationic lipid DOMA, neutral DMPC, and anionic DPPG monolayers were transferred to quartz slides (ERMA, FRG) for forming hydrophobic surfaces with a transfer ratio of 0.85. The solid surfaces used for the transfer of the films have been cleaned using freshly prepared chromic acid and further in a Plasma Cleaner (Harrick Plasma, USA) to ensure complete hydrophilicity. F. Contact angle measurements
The contact angles of the protein solution during adsorption to bare quartz and quartz coated with one layer LB of the lipid have been measured by the sessile drop method using a video Holmarc contact angle system (Model HO-IAD-CAM-01), India. Accuracy of the angle measured was 60.1 . A drop of the protein was placed on the quartz substrate and simultaneously viewed from the front using a CCD camera attached to a microscope. The drop images have been digitized and stored in a computer. The contact angle is measured by placing markers around the circumference of the drop. Contact angle as a function of time is recorded and using Eq. (3), initial and final work of adhesion was calculated DW ¼ clv ð1 þ cos hÞ:
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analyzed for its ground state absorbance and the presence of Ficoll slightly shifts the Trp maximum very slightly by 4 nm to the blue region (Fig. 1—supplementary material37). Blue shifts in Trp peak position in UV absorption spectra are generally indicative of increased solvent exposure of these residues. Steady state fluorescence emission spectra did not show significant changes in emission maximum as well as emission wavelength (Fig. 2—supplementary material).37 Protein solution with different additives can have reversible processes consisting of different adsorption/desorption rate constants, which cannot be differentiated by analyzing ground state and fluorescence steady state spectroscopy.38 Picosecond fluorescence lifetime measurements were carried out and the different timescales were fitted using a triexponential function and the decay times for Fg with viscogens are presented in supplementary material (Table I of Ref. 37). Since the protein is large with about 78 tryptophans,39 s1and s2 is attributed to the average of different orientations of the tryptophans and s3 to the tryptophan-solvent environment. On an average the time scales s1 and s2 remain constant for different additives showing that the protein in the solution is not altered much. But in presence of Ficoll, longer timescale with a higher population for s3 is observed. This is due to the long chain polysaccharide Ficoll having a shielding effect on the protein with a high population of the longer decay time species of the protein resulting in aggregation. CD spectra of the solutions were carried out and presented in Fig. 1 and corresponding secondary structures (Fig. 2) remain almost same with changes in the microviscosity. For very low concentrations of protein (107 M) used in the study, it is expected that no aggregation or intermolecular interactions interfere with the interfacial measurements. Thus, dilute solutions of Fg used in this study should lead to an optimal assembly process at the air/solution interface. Adsorption of Fg from the subphase of pure buffer and buffer with Ficoll, trehalose, and IL-emes at the air/water interface was monitored through changes in surface tension as a function of time (Table II—supplementary material).37
(3)
G. Surface tension measurements and scanning electron microscopy
Surface tension (clv) has been measured using a Wilhelmy plate technique in a NIMA Tensiometer (UK) at T ¼ 25 C. On reaching equilibrium, the films have been transferred onto cleaned solid substrates using the LangmuirSchafer film transfer technique and characterized using scanning electron microscopy (SEM). A thin layer of ˚ ) was sputtered on these samples, and SEM studgold (200 A ies were undertaken using a Hitachi S4800 model. III. RESULTS AND DISCUSSION Before studying the role of viscogens on adsorption of Fg to surfaces, the effect of solvent environment on Fg was studied. Fg with viscogens IL-emes, Ficoll, Trehalose was Biointerphases, Vol. 10, No. 2, June 2015
FIG. 1. CD spectra of Fg in the presence of viscogens (Inset: Control Fg).
021009-4 Kamatchi Sankaranarayanan: Role of viscogens on the macromolecular assemblies
FIG. 2. % secondary structures of Fg with additives in solution and adsorbed state.
CD spectroscopy for the protein films at the air/water interface (skimmed from the interface after attaining constant surface tension) and it was seen that the protein did not show any change in conformation compared to the liquid state. Thus, air/liquid interface has not led the protein to any denaturation. Table III and Fig. 3 in supplementary material37 shows the % secondary structure and CD spectra of Fg in viscogens before and after surface tension measurements. In a separate experiment, protein films at the interface were transferred to solid quartz surfaces, analyzed for changes in secondary structures, and morphology was studied by SEM. Circular dichroic spectroscopy for Fg in adsorbed state was carried out and the signals were fitted using Dichroweb software. Figure 2 compares % secondary structures of protein with viscogens, both in the solution phase as well as on the solid quartz substrate. There is an increase in the overall b-sheet content when Fg adsorbs to solid surfaces compared to its conformation in solvent. Other secondary structural features such as b-turn and unordered structures remain constant for the protein with additives, in solution and adsorbed state. It can be seen that protein in IL-emes shows higher b sheet content. The CD spectra of Fg in IL both in solution and adsorbed state are presented as Fig. 3. A minimum at 215 nm clearly indicates the conformational transition to b sheet on adsorption. This is due to the charges on ionic liquid, which contributes to hydrophilic–hydrophobic interaction altering the H-bond interactions in the protein, in addition to viscosity, and leading to changes in the conformation. Such changes in H-bond interactions due to ionic liquids are reported.31,40,41 Thus, IL-emes plays a major role in altering the conformation of Fg in the adsorbed state. Protein adsorption by itself is an intricate process, which is driven by different protein–surface forces, including van der Waals, hydrophobic, electrostatic forces. In addition, adsorption of proteins along with viscogens is a quite complex process. Viscosity, though a macroscopic quantity, originates from these intramolecular Biointerphases, Vol. 10, No. 2, June 2015
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interactions at the nanoscale. This study thus focuses on role of viscosity in protein adsorption at different interfaces. Shear viscosities arising due to adsorption at the solid/liquid interface of Fg were calculated from quartz crystal microbalance measurements. Table I presents the shear viscosities and adsorbed mass of Fg in the presence of viscogens and the corresponding control samples and it is maximum for IL-emes. This adsorption is characterized by change in adsorbed mass (Dm) of the protein. It suggests that Fg adsorbs more with a higher adsorbed mass in IL-emes environment. Measurements of Fg adsorption on different surfaces has shown a maximum coverage of Fg with the lowest and highest value equal to 0.14 and 2.7 lg/cm2 on Si-nanofibers and this divergence has been attributed to various adsorption mechanisms of Fg depending on the ionic strength, pH, and bulk concentration.42–44 Our studies show a higher adsorbed mass of 3.26 lg/cm2 with IL-emes indicating multilayer formation due to adsorption. Together with CD studies, it can be seen that Fg with IL-emes on adsorption leads to more b-sheet corresponding to the higher viscosity and adsorbed mass. Assuming an interfacial tension value c at the interface, a force balance along the solid surface at the three-phase contact line gives the contact angle. In the present study, the lipid component, DOMA, DPPG, or DMPC film, is immobilized in a 2D plane while the other (Fg) is distributed in solution. It is well established that adsorption of Fg to surfaces differs quite dramatically at high protein flux compared to conditions of low protein flux, suggesting a dependence on the final surface coverage. This fact together with the essential irreversibility of Fg adsorption to most surfaces has resulted in a careful analysis of binding data.45–48 The rate of change in work of adhesion during adsorption of the protein onto these surfaces has been evaluated using the contact angles of Fg solutions at the quartz surface. By fitting the appropriate rate equation to these plots, we have arrived at the conditions for optimal adsorption and thus adhesion. The results suggest that the rate of change of work of adhesion can be used to assess the quality of adhesion. In biomedical applications, the rate of adsorption is limited by mass transport to the surface, where binding of Fg to
FIG. 3. CD spectra of Fg/IL in solution and adsorbed state.
021009-5 Kamatchi Sankaranarayanan: Role of viscogens on the macromolecular assemblies TABLE I. Shear viscosity, adsorbed mass for Fg in viscogens and corresponding control samples.
Fg/
Shear viscosity (cP)
Dm (lg/cm2)
Shear viscosity of control (cP)
Dm of control (lg/cm2)
0.21 0.30 0.25 0.32
2.55 3.04 2.95 3.26
0.13 0.22 0.22 0.19
0.72 1.52 1.11 1.46
Buffer Ficoll 400 Trehalose IL-emes
various surfaces takes place. Two aspects are dealt with in this study: (1) nature of the interface and its influence on rate of adsorption and (2) effect of viscogens. Thus, adsorption of Fg with viscogens to model lipid coated hydrophobic surfaces were compared with bare surface. Contact angle measurements of Fg to different surfaces were carried out by allowing a 5 ll drop of protein solution to spread on bare/ hydrophobic surfaces, and contact angle was obtained with respect to time. Work of adhesion, a parameter that connects adsorption at liquid/air interface and solid/liquid interface was calculated as a function of time for these samples to different lipid coated surfaces from the contact angle titrations. The work of adhesion is given by Wads ¼ cð1 þ cos hÞ;
(4)
where c is the surface tension of Fg at liquid/vapor interface From Eq. (4), DW can be rewritten as DW ¼ cFg ð1 þ cos ht Þ cFg ð1 þ cos h1 Þ;
(5)
DW ¼ cFg cos ht cos h1 :
(6)
cFg is the surface tension of Fg and Fg with viscogens at the liquid/vapor interface and is given in Table II—supplementary material.37 Hence, for h values between 0 and 180 , based on Eq. (6) DW > 0; h1 > ht
;
hindered adsorption;
(7)
DW < 0; h1 < ht
;
promotion of adsorption;
(8)
TABLE II. Initial and final work of adhesion for pure Fg and Fg with additives adsorbed to bare substrate and DOMA, DPPG, and DMPC coated substrate. Substrate Bare
DOMA coated
DPPG coated
DMPC coated
(mJ/m2)
Fg
Fg/Ficoll
Fg/IL
Fg/Treh
Wi Wf DW Wi Wf DW Wi Wf DW Wi Wf DW
211.30 114.24 325.54 40.28 116.82 76.54 444.51 118.57 563.08 173.45 18.62 192.07
53.76 136.99 83.22 465.95 130.03 595.98 102.88 108.68 5.8 81.632 109.09 27.46
101.74 138.34 36.6 461.30 140 601.3 318.77 113.62 432.39 89.971 115.32 25.35
109.32 113.27 3.95 36.21 133.59 169.8 125.45 110.9 236.35 74.076 113.22 39.14
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DW ¼ 0; h1 ¼ ht
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;
no adsorption:
(9)
There may exist a condition between that of Eqs. (7) and (8) where difference between h1 and ht may tend toward zero. This corresponds to poor adsorption due to poor spreading of the solution on solid surface. Such a condition, therefore, can lead to localized aggregation of proteins on the surface or poor surface coverage. Therefore, most negative value for DW must correspond to best adsorption condition. DW calculated using Eq. (3) has been plotted as a function of time (supplementary material—Fig. 3)37 and fitted using sigmoidal Boltzmann function of the form y ¼ ðA1 A2 Þ=ð1 þ exp ðx x0 Þ=dxÞ þ A2 ;
(10)
which can be rewritten in our case as DW ¼ ðA1 A2 Þ=ð1 þ exp ðt t0 Þ=dtÞ þ A2 :
(11)
Thus, A1 correlates to initial work of adhesion (Wi) at t ¼ 0, A2 final work of adhesion (Wf) at steady state, t0 point of inflexion in the plot, and dt the time constant. Initial and final work of adhesion Wi and Wf were calculated using Boltzmann function given in Eq. (7). All samples had a good fit to Boltzmann function with an R2 of 0.97. Work of adhesion of Fg and Fg with additives on different surfaces was calculated and tabulated (Table II). It is well accepted that adsorption of proteins from aqueous solutions onto any solid should be in three steps: (1) diffusion of protein molecules from bulk to interface, (2) attachment of protein molecules to active sites on the surface, and (3) restructuring of the protein after adsorption. Step 3 decides the subsequent adsorption kinetics and also determines final surface properties of implants in biomedical devices. Here, it is seen that Fg with viscogens adsorbs preferentially on bare hydrophilic surface as indicated by low work of adhesion compared to hydrophobic surfaces prepared using cationic DOMA, anionic DPPG and neutral DMPC. In case of neutral DMPC, Fg with viscogens show lowest work of adhesion because the water–viscogen interaction overrides the solvated protein–lipid interaction. The adsorption data and surface tensions measurements confirm this picture drawn from the shear rheology. Increase in viscosity (as in IL-emes and Ficoll) is connected with unfolding of the protein as studied from CD spectroscopy, which on the other hand leads to a increase in adsorption near the interfaces.49 This is seen if the protein is considered a surface-active molecule; its adsorption can result in disruption of hydration layers at the interface affecting hydrophobicity of the surface. The physical interpretation of W is the amount of work needed to remove water from the surface. One can consider W to be a simple measure of surface hydrophobicity, and it is known that hydrophobic surfaces (e.g., Teflon) have lower W values than hydrophilic surfaces (e.g., glass). In general, protein adsorption decreases with surface energy and increases with hydrophobicity.50,51 The real model of protein adsorption is complex and involves many processes such as protein–interface interactions, protein orientations on the surface,
021009-6 Kamatchi Sankaranarayanan: Role of viscogens on the macromolecular assemblies
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FIG. 4. SEM images of (a) Fg, (b) Fg/Treh, (c) Fg/Ficoll, and (d) Fg/IL (Scale bar: 1 lm).
conformational changes accompanied with protein unfolding, lateral protein–protein interactions, and desorption that lead to multiple states of adsorbed proteins on the surface.52 Results from this study suggest that the adsorption of Fg preceded by local association and organization is dependent not only on the nature of interactions with bare or hydrophobic surface coated with lipids, but also on its diffusion arising due to the changes in local densities. Such nanostructural organization and its role in determining the macroscopic interfacial rheology of b-lactoglobulin fibrils at liquid interfaces has been reported recently.53 Since the adsorption profiles on bare hydrophilic surface showed a favorable DW, these samples were examined using SEM. Initially, the protein was allowed to assemble itself at the water/air interface, and after attaining a constant surface tension, the films were transferred horizontally to clean quartz surfaces and observed under SEM (Fig. 4). Fg in the presence of trehalose showed a bead like morphology and with other additives no characteristic morphology was seen. Such bead like morphology in presence of sugars is reported.54 Sankaranarayanan et al. have shown that Fg near interfaces and with electrolytes showed clusters
of about 50–80 nm in size55 whereas in the current study clusters are about 2 lm. This reinforces the point that slowing down of protein molecules due to changes in viscosity leads to larger sized clusters. Local viscosity causes changes in the hydrophilic/hydrophobic balance at the surface which in turn triggers the organization of Fg. To verify the role of local aggregation, Fg and Fg in trehalose were transferred to carbon coated grids and HRTEM was carried out (Fig. 5). It was seen that Fg showed a typical trinodular shape whereas Fg/Treh showed a bead shaped structure, confirming the morphology obtained using SEM. IV. CONCLUSIONS This work is an attempt to link the microenvironmental changes of the protein near interfaces to its macroscopic properties by combining rheology and tensiometry. In conclusion, assemblies of Fg organizing at interfaces, both at air/water interface and near solid surfaces seem to lead to local aggregation of the protein. IL-emes mediates local viscosity by organizing Fg near the surfaces with more b-sheet content. Trehalose induces bead like morphology near the interface and Ficoll leads to a shielding effect on the protein. Adsorption of Fg to solid surfaces suggests that there seem to exist a synergy between hydrophobic interaction and diffusion that leads to better adsorption. This work demonstrates organization of Fg molecules at the interface or near a surface ultimately leading to organized assemblies even in the absence of thrombin. Thus, surfaces initiate macromolecular assemblies of Fg with viscogens suggesting that the interface plays a vital role. ACKNOWLEDGMENT
FIG. 5. HRTEM images of (a) Fg and (b) Fg/Trehalose. Biointerphases, Vol. 10, No. 2, June 2015
K.S. thanks Department of Science and Technology, Government of India, for the Inspire Faculty Award (Dy. No. 108 Dt. 8.1.2014).
021009-7 Kamatchi Sankaranarayanan: Role of viscogens on the macromolecular assemblies 1
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M. S. Lord, J. M. Whitelock, A. Simmons, R. L. Williams, and B. K. Milthorpe, Biointerphases 9, 041002 (2014). 30 A. P. Minton, J. Cell Sci. 119, 2863 (2006). 31 Q. G. Zhang, N. N. Wang, and Z. W. Yu, J. Phys. Chem. B 114, 4747 (2010). 32 N. K. Jain and I. Roy, Eur. J. Pharm. Biopharm. 69, 824 (2008). 33 C. Branca, S. Maccarrone, S. Magaz u, G. Maisano, S. M. Bennington, and J. Taylor, J. Chem. Phys. 122, 174513 (2005). 34 K. Sankaranarayanan, B. Sreedhar, B. U. Nair, and A. Dhathathreyan, J. Phys. Chem. B. 117, 1234 (2013). 35 L. Whitemore and B. Wallace, Nucleic Acids Res. 32, W668 (2004). 36 V. Karunakaran, P. Ramamurthy, T. Josephrajan, and V. T. Ramakrishnan, Spectrochim. Acta, Part A 58, 1443 (2002). 37 See supplementary material at http://dx.doi.org/10.1116/1.4922291 for details on UV-Vis spectra, Fluorescence spectra, fluorescence lifetime, surface tension, %secondary structures before and after surface tension and work of adhesion for Fg in viscogens. 38 P. A. Cuypers, G. M. Willems, H. C. Hemker, and W. T. Hermens, Ann. N. Y. Acad. Sci. 516, 244 (1987). 39 Y. Ishida, H. Takiuchi, A. Matsushima, and Y. Inada, Biochim. Biophys. Acta 536, 70 (1978). 40 K. Dong, Y. Song, X. Liu, W. Cheng, X. Yao, and S. Zhang, J. Phys. Chem. B 116, 1007 (2012). 41 A. Wulf, K. Fumino, and R. Ludwig, Angew. Chem. 49, 449 (2010). 42 A. Bratek-Skicki, P. Zeliszewska, and Z. Adamczyk, Colloids Surf., B 103, 482 (2013). 43 N. Hassan, V. Verdinelli, J. M. Ruso, and P. V. Messina, Soft Matter 8, 6582 (2012). 44 Z. Bai, M. J. Filiaggi, and J. R. Dahn, Surf. Sci. 603, 839 (2009). 45 R. I. Thacker and G. S. Retzinger, Exp. Mol. Pathol. 84, 122 (2008). 46 T. M. Massa, M. L. Yang, J. Y. C. Ho, J. L. Brash, and J. P. Santerre, Biomaterials 26, 7367 (2005). 47 T. Ishizaki, N. Saito, Y. Sato, and O. Takai, Surf. Sci. 601, 3861 (2007). 48 J. Hu, D. Yang, Q. Kang, and D. Shen, Sens. Actuators, B 96, 390 (2003). 49 Progress in Colloid and Interface Science: Interfacial Rheology, edited by R. Miller and L. Liggieri (Brill, Boston, 2009), Vol. 1. 50 D. R. Absolom, W. Zingg, and A. W. Neumann, J. Biomed. Mater. Res. 21, 161 (1987). 51 D. L. Kirchman, D. L. Henry, and S. C. Dexter, Mar. Chem. 27, 201 (1989). 52 P. Becher, Proteins at Interfaces II, Fundamentals and Applications, ACS Symposium Series Vol. 602, edited by T. A. Horbett and J. L. Brash (American Chemical Society, Washington, DC, 1995). 53 S. Jordens, P. A. R€ uhs, C. Sieber, L. Isa, P. Fischer, and R. Mezzenga, Langmuir 30, 10090 (2014). 54 P. Roach, D. Farrar, and C. C. Perry, J. Am. Chem. Soc. 127, 8168 (2005). 55 K. Sankaranarayanan, A. Dhathathreyan, and R. Miller, J. Phys. Chem. B. 114, 8067 (2010).
Citation: Biointerphases 10, 021009 (2015); doi: 10.1116/1.4922291 View online: http://dx.doi.org/10.1116/1.4922291 View Table of Contents: http://avs.scitation.org/toc/bip/10/2 Published by the American Vacuum Society
Role of viscogens on the macromolecular assemblies of fibrinogen at liquid/air and solid/air interfaces Kamatchi Sankaranarayanana) DST-INSPIRE Faculty, Department of Energy and Environment, National Institute of Technology (NIT), Tiruchirapalli 620015, India
(Received 4 April 2015; accepted 28 May 2015; published 10 June 2015) In this study, an attempt has been made to understand the organization and association of fibrinogen (Fg) in solvent environment induced by viscogens such as 1-ethyl 3-methyl imidazolium ethyl sulfate (IL-emes), Ficoll, and Trehalose. The author observed that Fg in IL-emes adsorbed on solid surface shows higher b-sheet conformation. Shear viscosity measured using quartz crystal microbalance, for Fg in IL-emes was highest with a corresponding higher adsorbed mass 3.26 lg/cm2. Associated assemblies of the protein at the liquid/air interface were monitored with changes in surface tension and were used to calculate work of adhesion. Changes in work of adhesion were used as a tool to measure the adsorption of Fg to solid surfaces in presence of viscogens and highest adsorption was observed for hydrophilic surfaces. Scanning electron microscopy images show Fg in trehalose forms elongated bead like structures implying organization of the protein at the interface. Crowding in the solvent environment induced by viscogens can slow down organization of Fg, C 2015 American Vacuum Society. leading to macromolecular assemblies near the interface. V [http://dx.doi.org/10.1116/1.4922291]
I. INTRODUCTION Adsorption of soluble proteins at solid–liquid interfaces plays a fundamental role in biomedical devices. Protein adsorption is the first interaction with the implanted biomaterials.1,2 It is crucial for tissue compatibility with the implants, cellular interfacing with semiconductor materials, molecular electronics, biomaterials. Especially, adsorption of serum proteins, such as fibrinogen (Fg), plays a major role in designing suitable bioimplantable materials.3,4 The interactions between protein and surface depend on various factors such as surface charge, protein size, protein dipole moment, ionic strength, and solvent viscosity. In spite of extensive study on protein adsorption, basic questions concerning the structural details of a protein in its adsorbed state is yet unanswered. Especially, effect of viscogens on adsorption of proteins to surface remains elusive. Experimental strategies for controlling protein adsorption in a real-life situation mimicking crowding are crucial to create a biocompatible surface. Biochemical process in living systems occur in highly crowded environments. Macromolecular self-association, formation of aggregates in protein, and amyloid inclusion bodies in some diseases such as Parkinson’s disease, Alzheimer’s disease, etc., are specific reactions that trigger crowding.5 The use of high solute concentrations to mimic crowded environments results in an excluded volume effect, an increased viscosity, and also a secondary solvation effect. Out of this, increase in viscosity is attributed to crowding density as well as the size of cosolute. Experiments mimicking crowding generally use high-molecular weight cosolutes, such as Dextran and Ficoll 70, to mimic all three effects, or
a)
Electronic addresses: [email protected]; [email protected]
021009-1 Biointerphases 10(2), June 2015
small molecules such as sucrose and trimethylamine N-oxide, a bacterial osmolyte, to primarily act upon viscosity and/or solvation of the protein backbone. Several studies have been carried out to understand the possible effects of crowding on folding and association of proteins.6–10 Effect of crowding from experimental and theoretical point of view on protein folding,11–17 stability,18,19 protein association,20–22 and protein aggregation23 have been studied by a number of groups. Plasma fibrinogen is an important component of the coagulation cascade, as well as a major determinant of blood viscosity and blood flow. In many diseases including ischemic heart disease, stroke have been reported due to elevated plasma fibrinogen levels and also associated with an increased risk of cardiovascular disorders.24–26 Even though a number of research papers have appeared in the literature on the role of Fg and its importance in many fields, a fundamental understanding of its organization at different interfaces, adsorption kinetics, and the corresponding conformational change is still lacking, which shows the complexity of this problem. Quartz crystal microbalance (QCM) with dissipation monitoring has been used by Weber et al. for the real time study of Fg adsorption to model biomaterial surfaces.27 Fg as lyophilisome to design nanoclusters of nickel oxide and nickel hydroxides has been reported.28 While fibril formation during adsorption of fibrinogen to surfaces is known to be catalyzed by thrombin, there still exists a debate about whether the enzyme is solely responsible for the organized structures or is it a response to the substrate or the adsorbing conformation of Fg. Recently, the role of surface roughness on adsorption of Fg to silica surfaces has been studied by Lord et al. using stochastic nanolithography.29 In this work, the influence of viscogens on the organization, association, and subsequent adsorption of plasma
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C 2015 American Vacuum Society V
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021009-2 Kamatchi Sankaranarayanan: Role of viscogens on the macromolecular assemblies R
protein Fg has been carried out. FicollV 400 (FL), ionic liquid: 1-ethyl 3-methyl imidazolium ethyl sulfate (IL-emes), and trehalose (Treh) have been used as viscogens. Ficoll is a highly branched polymer formed by the copolymerization of sucrose and epichlorohydrin. It is completely nonionic and because of the abundance of hydroxyl groups, it is very hydrophilic and extremely water-soluble. Ficoll is recognized as one of the crowding agents.30 IL-emes increases the viscosity of the solvent environment and also involves in hydrogen bonding interactions with water.31 Trehalose interacts very efficiently with water, i.e., the interaction between trehalose/water is much stronger than water/water interaction and can inhibit protein folding during aggregation.32,33 Quartz crystal microbalance has been used to analyze the adsorbed protein to the solid/liquid interface and also measuring the shear viscosity. Shear viscosity reported in this study is a 2D rheological measurement carried out at isothermal conditions. Surface tension measurements at water/air interface and contact angle analysis at the solid/liquid interfaces have been carried out to analyze the role of interfaces on Fg adsorption. Together with conformation of the adsorbed protein, the role of viscosity in Fg adsorption has been discussed. II. EXPERIMENTAL SECTION Fg from bovine plasma, IL-emes, FL (Product No. F4375), and Treh were purchased from Sigma Aldrich chemicals, USA, and was more than 99% pure. Lipids dioctadecyldimethylammonium bromide (DOMA) and 1,2dipalmitoyl-sn-glycero-3-phospho-(10 -rac-glycerol) (sodium salt) (DPPG) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were purchased from Larodon, Sweden (99.9% pure), and used as such for the preparation of Langmuir–Blodgett (LB) films. Chloroform and methanol High Performance Liquid Chromatography grade were purchased from Merck. All aqueous solutions were prepared with distilled water further purified with a four-stage Milli-Q water system (Millipore, resistivity greater than 18.2 MX). Average molecular weight of Ficoll 400 is 450 000 and radius of Ficoll is approximately 10 nm. The solutions of Fg were prepared at pH 7.5 (10 mM phosphate buffer) with final concentration of 0.1 lM under mild agitation. The resulting solution was clear and was used for all the experiments. The additives concentration was fixed at 10 lM for IL-emes and Trehalose, whereas 2% (w/v) for FL which is about 50 lM. All the concentrations were fixed based on the previous study by the authors.34 The pH of the protein solution with the additives remained the same. All measurements reported here were done a minimum of three times to ensure reproducibility. A. UV2VIS and fluorescence spectroscopy
The samples were analyzed using UV-1800-Shimadzu spectrophotometer with quartz cuvettes of 1 cm path length. The corresponding hydrated FL, IL, and Treh were used as reference for the measurements. The steady state fluorescence Biointerphases, Vol. 10, No. 2, June 2015
spectroscopy was performed using Spectrometer with the kex ¼ 280 nm.
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Cary
Eclipse
B. CD spectroscopy
The Circular Dichroic (CD) spectra of pure Fg at pH 7.5 and with IL-emes, FL, and Treh were carried out using a JASCO J-715 spectropolarimeter (JASCO Corp., Tokyo). The far-UV (240–190 nm) spectra of the protein in different levels of hydrated IL obtained using 0.1 cm path length quartz cells were analyzed using the Dichroweb fitting to three structural parameters: a-helix, b-sheet, and aperiodic.35 C. Picosecond-time-resolved fluorescence spectroscopy
Fluorescence lifetime measurements of samples have been carried out in a picosecond-time-correlated single-photon-counting spectrometer. The excitation source is the tunable Ti-sapphire laser (Spectra Physics, USA). The laser pulse (pulse width of 2 ps and repetition rate of 4 MHz) has been derived from the frequency-doubled output (532 nm) of mode-locked laser (Spectra Physics, USA). The picosecond standard tuning range is 720–850 nm. The sample was excited by the laser pulse at 280 nm, and emission was monitored at right angles to the excitation path. The first photon was detected by the microchannel plate photomultiplier tube (Hamamatsu-C 4878). When the first excitation pulse occurred, a synchronization pulse triggered the charging of the capacitor in the time to amplitude convertor through the discriminator. The voltage on the capacitor increased linearly until a stop timing pulse was detected on repeating the start–stop cycle, and a histogram representative of the fluorescence decay was obtained, which has been further analyzed using the IBH (Glasgow, United Kingdom) software.36 D. Quartz crystal microbalance measurements
Plasma cleaned (exposed to UV/ozone for 10 min) gold-coated quartz substrates were used for all QCM measurements. Measurements were performed with 50 ll of temperature-stabilized and degassed sample liquid, which was delivered to the chamber containing the sensor crystal to ensure a complete exchange of the liquid. This ensures that processes of adsorption and surface adlayer changes can be followed in situ while subsequently exposing different solutions to the surface. All measurements were performed at a temperature of 2425 C. QCM sensors quartz crystal microbalance, crystal holders, and polished gold AT cut 5 MHz gold crystals of 25 mm diameter crystals from Maxtek were used for the study. The oscillation frequency was measured using a Maxtek RQCM with phase lock oscillator with independent crystal measurement channel. Data acquisition was performed using the Maxtek RQCM Data Logging software (v. 1.6.0) on a personal computer connected through an RS 232 serial interface. A sampling rate of 1/60 Hz was employed for all experiments. Any baseline drift was regulated using the coarse and fine capacitance adjustments. Upon interaction of protein with the surface of
021009-3 Kamatchi Sankaranarayanan: Role of viscogens on the macromolecular assemblies
a sensor crystal, changes in the resonance frequency, Df, are related to attached mass governed by the Sauerbray’s equation Dm ¼ ðlqÞ1=2 ðDfÞ=ð2f 0 Þ2 :
(1)
f0 is the resonance frequency of the crystal (5 MHz at 25 C), l is the shear modulus of the quartz crystal (2.947 1011 g s2 cm1 at room temperature), and q is the density of quartz (2.648 g cm3). Shear viscosity is then given by the equation g ¼ ð19:627 ðDfÞ2 107 Þ=q:
(2)
E. Langmuir–Blodgett films
LB films of cationic lipid DOMA, neutral DMPC, and anionic DPPG monolayers were transferred to quartz slides (ERMA, FRG) for forming hydrophobic surfaces with a transfer ratio of 0.85. The solid surfaces used for the transfer of the films have been cleaned using freshly prepared chromic acid and further in a Plasma Cleaner (Harrick Plasma, USA) to ensure complete hydrophilicity. F. Contact angle measurements
The contact angles of the protein solution during adsorption to bare quartz and quartz coated with one layer LB of the lipid have been measured by the sessile drop method using a video Holmarc contact angle system (Model HO-IAD-CAM-01), India. Accuracy of the angle measured was 60.1 . A drop of the protein was placed on the quartz substrate and simultaneously viewed from the front using a CCD camera attached to a microscope. The drop images have been digitized and stored in a computer. The contact angle is measured by placing markers around the circumference of the drop. Contact angle as a function of time is recorded and using Eq. (3), initial and final work of adhesion was calculated DW ¼ clv ð1 þ cos hÞ:
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analyzed for its ground state absorbance and the presence of Ficoll slightly shifts the Trp maximum very slightly by 4 nm to the blue region (Fig. 1—supplementary material37). Blue shifts in Trp peak position in UV absorption spectra are generally indicative of increased solvent exposure of these residues. Steady state fluorescence emission spectra did not show significant changes in emission maximum as well as emission wavelength (Fig. 2—supplementary material).37 Protein solution with different additives can have reversible processes consisting of different adsorption/desorption rate constants, which cannot be differentiated by analyzing ground state and fluorescence steady state spectroscopy.38 Picosecond fluorescence lifetime measurements were carried out and the different timescales were fitted using a triexponential function and the decay times for Fg with viscogens are presented in supplementary material (Table I of Ref. 37). Since the protein is large with about 78 tryptophans,39 s1and s2 is attributed to the average of different orientations of the tryptophans and s3 to the tryptophan-solvent environment. On an average the time scales s1 and s2 remain constant for different additives showing that the protein in the solution is not altered much. But in presence of Ficoll, longer timescale with a higher population for s3 is observed. This is due to the long chain polysaccharide Ficoll having a shielding effect on the protein with a high population of the longer decay time species of the protein resulting in aggregation. CD spectra of the solutions were carried out and presented in Fig. 1 and corresponding secondary structures (Fig. 2) remain almost same with changes in the microviscosity. For very low concentrations of protein (107 M) used in the study, it is expected that no aggregation or intermolecular interactions interfere with the interfacial measurements. Thus, dilute solutions of Fg used in this study should lead to an optimal assembly process at the air/solution interface. Adsorption of Fg from the subphase of pure buffer and buffer with Ficoll, trehalose, and IL-emes at the air/water interface was monitored through changes in surface tension as a function of time (Table II—supplementary material).37
(3)
G. Surface tension measurements and scanning electron microscopy
Surface tension (clv) has been measured using a Wilhelmy plate technique in a NIMA Tensiometer (UK) at T ¼ 25 C. On reaching equilibrium, the films have been transferred onto cleaned solid substrates using the LangmuirSchafer film transfer technique and characterized using scanning electron microscopy (SEM). A thin layer of ˚ ) was sputtered on these samples, and SEM studgold (200 A ies were undertaken using a Hitachi S4800 model. III. RESULTS AND DISCUSSION Before studying the role of viscogens on adsorption of Fg to surfaces, the effect of solvent environment on Fg was studied. Fg with viscogens IL-emes, Ficoll, Trehalose was Biointerphases, Vol. 10, No. 2, June 2015
FIG. 1. CD spectra of Fg in the presence of viscogens (Inset: Control Fg).
021009-4 Kamatchi Sankaranarayanan: Role of viscogens on the macromolecular assemblies
FIG. 2. % secondary structures of Fg with additives in solution and adsorbed state.
CD spectroscopy for the protein films at the air/water interface (skimmed from the interface after attaining constant surface tension) and it was seen that the protein did not show any change in conformation compared to the liquid state. Thus, air/liquid interface has not led the protein to any denaturation. Table III and Fig. 3 in supplementary material37 shows the % secondary structure and CD spectra of Fg in viscogens before and after surface tension measurements. In a separate experiment, protein films at the interface were transferred to solid quartz surfaces, analyzed for changes in secondary structures, and morphology was studied by SEM. Circular dichroic spectroscopy for Fg in adsorbed state was carried out and the signals were fitted using Dichroweb software. Figure 2 compares % secondary structures of protein with viscogens, both in the solution phase as well as on the solid quartz substrate. There is an increase in the overall b-sheet content when Fg adsorbs to solid surfaces compared to its conformation in solvent. Other secondary structural features such as b-turn and unordered structures remain constant for the protein with additives, in solution and adsorbed state. It can be seen that protein in IL-emes shows higher b sheet content. The CD spectra of Fg in IL both in solution and adsorbed state are presented as Fig. 3. A minimum at 215 nm clearly indicates the conformational transition to b sheet on adsorption. This is due to the charges on ionic liquid, which contributes to hydrophilic–hydrophobic interaction altering the H-bond interactions in the protein, in addition to viscosity, and leading to changes in the conformation. Such changes in H-bond interactions due to ionic liquids are reported.31,40,41 Thus, IL-emes plays a major role in altering the conformation of Fg in the adsorbed state. Protein adsorption by itself is an intricate process, which is driven by different protein–surface forces, including van der Waals, hydrophobic, electrostatic forces. In addition, adsorption of proteins along with viscogens is a quite complex process. Viscosity, though a macroscopic quantity, originates from these intramolecular Biointerphases, Vol. 10, No. 2, June 2015
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interactions at the nanoscale. This study thus focuses on role of viscosity in protein adsorption at different interfaces. Shear viscosities arising due to adsorption at the solid/liquid interface of Fg were calculated from quartz crystal microbalance measurements. Table I presents the shear viscosities and adsorbed mass of Fg in the presence of viscogens and the corresponding control samples and it is maximum for IL-emes. This adsorption is characterized by change in adsorbed mass (Dm) of the protein. It suggests that Fg adsorbs more with a higher adsorbed mass in IL-emes environment. Measurements of Fg adsorption on different surfaces has shown a maximum coverage of Fg with the lowest and highest value equal to 0.14 and 2.7 lg/cm2 on Si-nanofibers and this divergence has been attributed to various adsorption mechanisms of Fg depending on the ionic strength, pH, and bulk concentration.42–44 Our studies show a higher adsorbed mass of 3.26 lg/cm2 with IL-emes indicating multilayer formation due to adsorption. Together with CD studies, it can be seen that Fg with IL-emes on adsorption leads to more b-sheet corresponding to the higher viscosity and adsorbed mass. Assuming an interfacial tension value c at the interface, a force balance along the solid surface at the three-phase contact line gives the contact angle. In the present study, the lipid component, DOMA, DPPG, or DMPC film, is immobilized in a 2D plane while the other (Fg) is distributed in solution. It is well established that adsorption of Fg to surfaces differs quite dramatically at high protein flux compared to conditions of low protein flux, suggesting a dependence on the final surface coverage. This fact together with the essential irreversibility of Fg adsorption to most surfaces has resulted in a careful analysis of binding data.45–48 The rate of change in work of adhesion during adsorption of the protein onto these surfaces has been evaluated using the contact angles of Fg solutions at the quartz surface. By fitting the appropriate rate equation to these plots, we have arrived at the conditions for optimal adsorption and thus adhesion. The results suggest that the rate of change of work of adhesion can be used to assess the quality of adhesion. In biomedical applications, the rate of adsorption is limited by mass transport to the surface, where binding of Fg to
FIG. 3. CD spectra of Fg/IL in solution and adsorbed state.
021009-5 Kamatchi Sankaranarayanan: Role of viscogens on the macromolecular assemblies TABLE I. Shear viscosity, adsorbed mass for Fg in viscogens and corresponding control samples.
Fg/
Shear viscosity (cP)
Dm (lg/cm2)
Shear viscosity of control (cP)
Dm of control (lg/cm2)
0.21 0.30 0.25 0.32
2.55 3.04 2.95 3.26
0.13 0.22 0.22 0.19
0.72 1.52 1.11 1.46
Buffer Ficoll 400 Trehalose IL-emes
various surfaces takes place. Two aspects are dealt with in this study: (1) nature of the interface and its influence on rate of adsorption and (2) effect of viscogens. Thus, adsorption of Fg with viscogens to model lipid coated hydrophobic surfaces were compared with bare surface. Contact angle measurements of Fg to different surfaces were carried out by allowing a 5 ll drop of protein solution to spread on bare/ hydrophobic surfaces, and contact angle was obtained with respect to time. Work of adhesion, a parameter that connects adsorption at liquid/air interface and solid/liquid interface was calculated as a function of time for these samples to different lipid coated surfaces from the contact angle titrations. The work of adhesion is given by Wads ¼ cð1 þ cos hÞ;
(4)
where c is the surface tension of Fg at liquid/vapor interface From Eq. (4), DW can be rewritten as DW ¼ cFg ð1 þ cos ht Þ cFg ð1 þ cos h1 Þ;
(5)
DW ¼ cFg cos ht cos h1 :
(6)
cFg is the surface tension of Fg and Fg with viscogens at the liquid/vapor interface and is given in Table II—supplementary material.37 Hence, for h values between 0 and 180 , based on Eq. (6) DW > 0; h1 > ht
;
hindered adsorption;
(7)
DW < 0; h1 < ht
;
promotion of adsorption;
(8)
TABLE II. Initial and final work of adhesion for pure Fg and Fg with additives adsorbed to bare substrate and DOMA, DPPG, and DMPC coated substrate. Substrate Bare
DOMA coated
DPPG coated
DMPC coated
(mJ/m2)
Fg
Fg/Ficoll
Fg/IL
Fg/Treh
Wi Wf DW Wi Wf DW Wi Wf DW Wi Wf DW
211.30 114.24 325.54 40.28 116.82 76.54 444.51 118.57 563.08 173.45 18.62 192.07
53.76 136.99 83.22 465.95 130.03 595.98 102.88 108.68 5.8 81.632 109.09 27.46
101.74 138.34 36.6 461.30 140 601.3 318.77 113.62 432.39 89.971 115.32 25.35
109.32 113.27 3.95 36.21 133.59 169.8 125.45 110.9 236.35 74.076 113.22 39.14
Biointerphases, Vol. 10, No. 2, June 2015
DW ¼ 0; h1 ¼ ht
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;
no adsorption:
(9)
There may exist a condition between that of Eqs. (7) and (8) where difference between h1 and ht may tend toward zero. This corresponds to poor adsorption due to poor spreading of the solution on solid surface. Such a condition, therefore, can lead to localized aggregation of proteins on the surface or poor surface coverage. Therefore, most negative value for DW must correspond to best adsorption condition. DW calculated using Eq. (3) has been plotted as a function of time (supplementary material—Fig. 3)37 and fitted using sigmoidal Boltzmann function of the form y ¼ ðA1 A2 Þ=ð1 þ exp ðx x0 Þ=dxÞ þ A2 ;
(10)
which can be rewritten in our case as DW ¼ ðA1 A2 Þ=ð1 þ exp ðt t0 Þ=dtÞ þ A2 :
(11)
Thus, A1 correlates to initial work of adhesion (Wi) at t ¼ 0, A2 final work of adhesion (Wf) at steady state, t0 point of inflexion in the plot, and dt the time constant. Initial and final work of adhesion Wi and Wf were calculated using Boltzmann function given in Eq. (7). All samples had a good fit to Boltzmann function with an R2 of 0.97. Work of adhesion of Fg and Fg with additives on different surfaces was calculated and tabulated (Table II). It is well accepted that adsorption of proteins from aqueous solutions onto any solid should be in three steps: (1) diffusion of protein molecules from bulk to interface, (2) attachment of protein molecules to active sites on the surface, and (3) restructuring of the protein after adsorption. Step 3 decides the subsequent adsorption kinetics and also determines final surface properties of implants in biomedical devices. Here, it is seen that Fg with viscogens adsorbs preferentially on bare hydrophilic surface as indicated by low work of adhesion compared to hydrophobic surfaces prepared using cationic DOMA, anionic DPPG and neutral DMPC. In case of neutral DMPC, Fg with viscogens show lowest work of adhesion because the water–viscogen interaction overrides the solvated protein–lipid interaction. The adsorption data and surface tensions measurements confirm this picture drawn from the shear rheology. Increase in viscosity (as in IL-emes and Ficoll) is connected with unfolding of the protein as studied from CD spectroscopy, which on the other hand leads to a increase in adsorption near the interfaces.49 This is seen if the protein is considered a surface-active molecule; its adsorption can result in disruption of hydration layers at the interface affecting hydrophobicity of the surface. The physical interpretation of W is the amount of work needed to remove water from the surface. One can consider W to be a simple measure of surface hydrophobicity, and it is known that hydrophobic surfaces (e.g., Teflon) have lower W values than hydrophilic surfaces (e.g., glass). In general, protein adsorption decreases with surface energy and increases with hydrophobicity.50,51 The real model of protein adsorption is complex and involves many processes such as protein–interface interactions, protein orientations on the surface,
021009-6 Kamatchi Sankaranarayanan: Role of viscogens on the macromolecular assemblies
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FIG. 4. SEM images of (a) Fg, (b) Fg/Treh, (c) Fg/Ficoll, and (d) Fg/IL (Scale bar: 1 lm).
conformational changes accompanied with protein unfolding, lateral protein–protein interactions, and desorption that lead to multiple states of adsorbed proteins on the surface.52 Results from this study suggest that the adsorption of Fg preceded by local association and organization is dependent not only on the nature of interactions with bare or hydrophobic surface coated with lipids, but also on its diffusion arising due to the changes in local densities. Such nanostructural organization and its role in determining the macroscopic interfacial rheology of b-lactoglobulin fibrils at liquid interfaces has been reported recently.53 Since the adsorption profiles on bare hydrophilic surface showed a favorable DW, these samples were examined using SEM. Initially, the protein was allowed to assemble itself at the water/air interface, and after attaining a constant surface tension, the films were transferred horizontally to clean quartz surfaces and observed under SEM (Fig. 4). Fg in the presence of trehalose showed a bead like morphology and with other additives no characteristic morphology was seen. Such bead like morphology in presence of sugars is reported.54 Sankaranarayanan et al. have shown that Fg near interfaces and with electrolytes showed clusters
of about 50–80 nm in size55 whereas in the current study clusters are about 2 lm. This reinforces the point that slowing down of protein molecules due to changes in viscosity leads to larger sized clusters. Local viscosity causes changes in the hydrophilic/hydrophobic balance at the surface which in turn triggers the organization of Fg. To verify the role of local aggregation, Fg and Fg in trehalose were transferred to carbon coated grids and HRTEM was carried out (Fig. 5). It was seen that Fg showed a typical trinodular shape whereas Fg/Treh showed a bead shaped structure, confirming the morphology obtained using SEM. IV. CONCLUSIONS This work is an attempt to link the microenvironmental changes of the protein near interfaces to its macroscopic properties by combining rheology and tensiometry. In conclusion, assemblies of Fg organizing at interfaces, both at air/water interface and near solid surfaces seem to lead to local aggregation of the protein. IL-emes mediates local viscosity by organizing Fg near the surfaces with more b-sheet content. Trehalose induces bead like morphology near the interface and Ficoll leads to a shielding effect on the protein. Adsorption of Fg to solid surfaces suggests that there seem to exist a synergy between hydrophobic interaction and diffusion that leads to better adsorption. This work demonstrates organization of Fg molecules at the interface or near a surface ultimately leading to organized assemblies even in the absence of thrombin. Thus, surfaces initiate macromolecular assemblies of Fg with viscogens suggesting that the interface plays a vital role. ACKNOWLEDGMENT
FIG. 5. HRTEM images of (a) Fg and (b) Fg/Trehalose. Biointerphases, Vol. 10, No. 2, June 2015
K.S. thanks Department of Science and Technology, Government of India, for the Inspire Faculty Award (Dy. No. 108 Dt. 8.1.2014).
021009-7 Kamatchi Sankaranarayanan: Role of viscogens on the macromolecular assemblies 1
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