Liquid Interfaces Using

J. Phys. Chem. B 2010, 114, 8067–8075

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Assembling Fibrinogen at Air/Water and Solid/Liquid Interfaces Using Langmuir and Langmuir-Blodgett Films Kamatchi Sankaranarayanan and A. Dhathathreyan* Chemical Laboratory, CLRI (CSIR), Adyar, Chennai 600020, India

Reinhard Miller Max-Planck-Institut fu¨r Kolloid- und Grenzfla¨chenforschung, Am Mu¨hlenberg 1, 14424 Potsdam, FRG ReceiVed: January 30, 2010; ReVised Manuscript ReceiVed: May 17, 2010

Langmuir films of pure fibrinogen (Fg) and Fg spread at the air/buffer interface and subphase containing electrolytes, NaCl, KCl, CaCl2, and ZnCl2, have been analyzed to understand the role of the surface/interface in mediating the organization of the protein eventually to fibrils. These films have been characterized by the surface pressure and surface potential-molecular area ((π-A) and (∆V-A)) isotherms and Brewster angle microscopy (BAM). The Langmuir-Blodgett (LB) films of the protein transferred to the solid substrates have been characterized by scanning electron microscopy (SEM) and circular dichroism (CD). Our results suggest that fibrils are formed during organization at air/solution interface and also in LB films. The rate of formation of the fibril is the maximum for Fg with ZnCl2. Adsorption of Fg to surfaces coated with a neutral lipid, dimyristoylphosphatidylcholine (DMPC), and a cationic lipid, dioctadecyldimethylammonium bromide (DOMA), from a range of solution concentrations has been studied using a quartz crystal microbalance (QCM). The work of adhesion of the protein on the solid surface shows fibril formation and positive adhesion for Fg in the presence of electrolytes. SEM results show that the adherent protein exhibits the widely reported nodulelike structure in the presence of CaCl2 and ZnCl2. These results provide definite evidence that specifically designed surfaces can promote adhesion of Fg and also activate fibril formation even in the absence of thrombin. I. Introduction Adsorption of proteins to solid substrates is an important event and has implications in biotechnology, immunoassays, biosensors, and biomaterials. In almost all these applications, plasma fibrinogen (Fg) is the most relevant protein in the body that adsorbs to the foreign material surfaces.1 Fg takes part in blood coagulation and facilitates adhesion as well as aggregation of platelets, which are very important properties in the processes of both hemeostasis and thrombosis.2 Human plasma fibrinogen is a 340 kDa dimeric protein and its adsorption is a complex process involving noncovalent interactions, electrostatic forces, hydrogen bonding, and van der Waals forces.3 The end product of the blood clotting process is the proteolytic enzyme thrombin,4 which selectively cleaves short arginyl peptides from the precursor molecules to form fibrin.5 These fibrin monomers have the property of self-assembly at physiological pH to form fibrils that form the elastic network of blood clots. The biological response to biomaterials is thus strongly correlated to the structure and conformation of Fg and its role in fibril formation. Recently, the mechanical properties of electrospun fibers of Fg have been studied and results show that the properties of the macroscopic structures depend on the conformation of individual molecules.6 Adsorption of fibrinogen onto nanorough platinum surfaces has been investigated using radiolabeling and a quartz crystal microbalance with dissipation (QCM-D), and factors of nanoroughness, surface morphology, and bound water on the adsorption have been analyzed.7 Orientation and conformation of fibrinogen molecules at the * Author for correspondence. Tel: +91-44-24411630. Fax: +91-4424911589. E-mail: [email protected].

polystyrene (PS)/protein solution interface have been studied using sum frequency generation (SFG) vibrational spectroscopy, supplemented by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR).8 This study showed that the fibrinogen adopts a bent structure during adsorption. Studies on Fg adsorption, platelet adhesion, and activation on mixed hydroxyl-/methyl-terminated self-assembled monolayers have shown that, more important than the amount of adsorbed Fg, its conformation is primarily responsible for platelet adhesion and activation.9 In this regard, substrate properties such as surface energy, surface roughness, and surface chemistry have been identified as key factors influencing the structure of adsorbed Fg.10 An understanding of the influence of the biomaterial’s surface chemistry on the conformation of adsorbed proteins is crucial for improvement of the biocompatibility of foreign-body materials.11,12 A number of research papers have reported on the interaction of globular proteins like ovalbumin with lipids, interaction of alcohol dehydrogenase with electrolytes at air/ water interface and effect of pH on surface organized assemblies of hemoglobin.13-15 Adsorption and adhesion of Fg to different surfaces like heptanized silica surface, TiO2 and its behavior on glass have been addressed by a number of researchers.16-23 Fibrinogen and its adsorption and adhesion to various surfaces have been studied using different scanning probe microscopy techniques. Variations in the dimensions of molecular organizates of Fg at interface and changes with bulk concentration, temperature, and pH have been addressed.24 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

10.1021/jp100896b  2010 American Chemical Society Published on Web 06/02/2010

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interfaces, adsorption kinetics and the corresponding conformational change is still lacking, which testifies to the complexity of this problem. In a recent study, Weber and co-workers used a quartz crystal microbalance with dissipation monitoring (QCM-D) for the real time study of Fg adsorption to model biomaterial surfaces.25 Fg as lyophilisome to design nanoclusters of nickel oxide and nickel hydroxides has been reported.26 While fibril formation during adsorption of fibrinogen (Fg) to surfaces is known to be catalyzed by thrombin, there still exists a controversy 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. In the present work, surface activity of pure Fg at the air/water interface and in the presence of electrolytes like NaCl, KCl, ZnCl2, and CaCl2 and the viscoelastic response have been studied. Further, the role of metal ions in the organization and in the assembly of fibrils has been analyzed. The electrolytes chosen here belong to the Hofmeister family. Hofmeister ions are generally known to exert their effects indirectly by changing the hydrogen-bonding properties of water. The question of how Hofmeister ions affect the structure and hydrogen-bonding properties of water has been reviewed by Collins and Washabaugh27 and the problem is being actively investigated today by many research groups.28-32 Surface viscoelasticity is also one of the main factors controlling the mechanical behavior of the protein solution as a whole under shear. Although there has been ongoing research on the relation between surface rheology and bulk rheology,33,34 the rheological behavior of protein films is still an area that is not fully understood from either the experimental or modeling points of view. Using the oscillating drop method, conformational preferences of β-casein on adsorption to air/solution interface and the dilatational viscosity in protein-surfactant mixtures have been analyzed.35-37 In this work the role of air/water interface and solid surfaces in organizing Fg molecules and fibril formation have been studied using Langmuir films and Langmuir-Blodgett films (LB films) of Fg. Changes in the organization of the protein in the films in the presence of electrolytes have been analyzed using NaCl, KCl, CaCl2, and ZnCl2 in the subphase. The dilational elasticity and viscosity of the protein and protein + electrolytes have been studied using the oscillating drop technique. The films of Fg spread at the air/water interface adsorbed onto a quartz surface coated with Langmuir-Blodgett films (LB films) of a neutral phospholipid, dimyristoylphosphatidylcholine (DMPC), and a cationic lipid, dimethyldioctadecylammonium bromide (DOMA), have been analyzed using a quartz crystal microbalance. 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.38-42 This fact together with the essential irreversibility of Fg adsorption to most surfaces has resulted in a rigorous analysis of binding data according to the conventional Langmuir adsorption isotherm.43,44 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.

Sankaranarayanan et al. II. Materials and Methods Sample Preparation. Bovine plasma Fg, type IV, was purchased from Sigma (catalog no. F-4753) and was 95% clottable. Gel electrophoresis has shown no detectable impurities and consistent results in the current study have been obtained for the same Fg product from different lots. The buffer solutions employed salts from Fisher Scientific. The phosphate buffer was composed of 0.008 M Na2HPO4 and 0.002 M KH2PO4 (pH 7.5). All aqueous solutions were prepared with distilled water further purified with a four-stage Milli-Q water system (Millipore, resistivity greater than 18.2 MΩ). Protein was dissolved under mild agitation (1 mg/mL or 3 µM). Solution pH was adjusted to pH 6.5 using a phosphate buffer. pH was measured both before and after the experiments and exhibited little variation. Stock solution of 1 mM solutions of all electrolytes (Fisher Scientific, Pittsburgh, PA, Certified ACS S271-500) was prepared from the salts roasted at about 400 °C for 24 h to drive off organic contaminants. The final solution concentrations were determined by UV spectroscopy at 280 nm, for the protein. Freshly prepared solutions of the protein and the electrolytes were used for all measurements. Langmuir and LB Films of Fg. Langmuir films of Fg in buffer and in the presence of electrolytes were prepared in a NIMA 601 S thermostated (T ) 22 °C) single barrier trough (32 × 20 × 0.5 cm3) with a Wilhelmy balance for the surface pressure measurement (accuracy in surface pressure ) (0.01 mN/m). The protein film from the stock was spread by pouring a small volume of protein solution along a glass rod implanted across the water/air interface45,46 on a subphase whose pH was adjusted to 7.5 (pI of Fg ) 5.5). After a waiting period of about 5 min, the film was compressed at a speed of 5 mm/min. The films formed were compressed and expanded repeatedly to check for any instability in the film. No appreciable hysteresis was seen in the films. The surface potential was measured using a Kelvin vibrating plate method.47The accuracy in surface potential values was (0.1 V. The surface potential ∆V was measured as a difference between potential of a pure water surface and that of water surface covered with a monolayer. From the experimental ∆V values the dipole moment component µ⊥ (normal component of dipole moment value) are estimated from the Helmholtz equation

µ⊥ ) ε0A∆V Here ∆V and A are the surface potential and average area of a close-packed monolayer and ε0 is the permittivity of free space; µ⊥ is normal component of dipoles perpendicular to the monolayer plane. The films were transferred onto solid substrates like freshly cleaned quartz or coverslips (ERMA, FRG) using the LB film technique and a constant surface pressure of π ) 20 mN/m was maintained for all the films. The transfer ratio was nearly 0.85 for all the films. Langmuir-Blodgett films (LB films) of cationic lipid dimethyldioctadecylammonium bromide (DOMA) and neutral phospholipid dimyristoyloleoylphosphatidylcholine (DMPC) monolayers served as model hydrophobic surfaces. Close packed LB films of these lipids were formed on microscope slides, resulting in water contact angles ranging from 69° to 71°. CD Spectroscopy. The CD spectra of the LB films of pure Fg and Fg with electrolytes were carried out using a JASCO J-715 spectropolarimeter (JASCO Corp., Tokyo). The near-UV (340-250 nm) and far-UV (260-180 nm) spectra of Fg in 0.1

Fibrinogen at Air/Water and Solid/Liquid Interfaces

J. Phys. Chem. B, Vol. 114, No. 24, 2010 8069

mm path length quartz cells obtained were analyzed using the Selcon method48 fitting to three structural parameters: alpha helix, β-sheet, and aperiodic. They were compared with the solution spectra, and it was found that secondary structures of fresh films on solid surfaces agreed with the solution structures quite well. But on aging (about 24 h) the films showed denaturation. Brewster Angle Microscopy (BAM). Brewster angle microscopy (BAM) of the Langmuir films at the air/water interface were studied using a Mini BAM model from NFT with a setup similar to the one used by Honig and Mo¨bius.49 The Brewster angle microscopy technique in particular allows phase transitions in molecular films at the liquid/air interface to be studied. In this technique, a parallel light beam polarized in the plane of incidence is reflected at the Brewster angle on the air/water interface. The reflected light is detected by a microscope connected to a video camera. The reflected light is sensitive to local density or thickness differences that appear in the molecular film during compression. The trough has a working area of 30 cm × 20 cm, which is large enough to mount the BAM directly on it. BAM images were performed with Mini BAM equipped with a 30 mW laser emitting p-polarized light with a wavelength of 532 nm that was reflected off the air/water interface at approximately 53.1° (incident Brewster angle). Under such conditions, the reflectivity of the beam was almost zero on the clean water surface. The reflected beam passes through a focal lens into an analyzer at a known angle of incident polarization, and finally to a CCD camera. Quartz Crystal Microbalance (QCM). The films of cationic DOMA and nonionic DMPC amphiphiles were formed on the clean gold coated quartz substrates by the Langmuir-Blodgett film (LB film) technique using a 1 mM chloroform solution (transferred at p ) 25 mN/m, three layers, Y-type transfer with transfer ratio ) 0.9) and were left to dry overnight in a vacuum desiccator. The static contact angles of water in contact with bare surface and surface with the LB films were first measured before the start of each experiment. Known volumes of the Fg and Fg with electrolytes were then injected into the QCM cell for each experiment for measuring the contact angle. The protein solution adsorbed to a gold coated quartz surface was studied using a quartz crystal microbalance (QCM). The adsorbed mass in QCM is calculated from the change in resonance frequency, ∆f, upon adsorption of the film using the Sauerbrey equation,

∆f ) -2f02∆m(Fqνq) ) -f0∆m(Fqtq) ) -C∆m

(1)

f0 is the resonance frequency of the solvent (before any solute is present), ∆m is mass of the adsorbed film (from the solution), Fq and νq are the specific density and shear wave velocity in quartz, and tq is the thickness of the quartz. The mass measured includes a solvent contribution that is hydrodynamically coupled to the solute used. The contact angle of the Fg solution has been estimated using the method of Lin and Ward.50 The contact angles were measured as a function of time simultaneously with the measurements of delta frequency and thickness changes. Here the assumption is that the refractive indices of the vesicles and the buffer are close to each other at the time of spreading and the frequency change associated with an area of liquid droplet in contact.

∆f )

∫ ∫ S(r,θ) m(r,θ)r dr dθ

for limits 0 to 2π and 0 to re

(2) S is the differential sensitivity (∂f∂m). For a Newtonian fluid m(r,θ) is described as the effective mass contained within decay length δ

δ ) (ηl /πf0Fl)1/2

(3)

Using Sauerbrey’s equation (1) and eqs 2 and 3

∆f ) C[(3Vd /π)2/3(1 - cos2 θ)]/[(2 - 3 cos θ + cos3 θ)2/3]

(4)

The contact angle θ is evaluated. From this value, the work of adhesion for the protein interacting with the gold surface is given by

Wads ) γ(1 + cos θ)

(5)

Here γ was obtained by using the conventional Wilhelmy balance technique and the solutions containing the vesicles were used to measure the surface tension values. Assuming a constant interfacial tension value γ for the different interfaces, a force balance along the solid surface at the three phase contact line gives the contact angle. Dilational Viscosity and Elasticity Measurements. Freshly prepared solutions of the protein and the electrolytes were used for each measurement. The surface tension of pure protein solution and with the various electrolytes were measured with the profile analysis tensiometer. PAT-1 (SINTERFACE Technologies) had an accuracy of (0.01 mN/m and thermostated at a temperature of 22 °C. The solution drops were formed at the tip of a PTFE capillary immersed into a cuvette filled with a water-saturated atmosphere. After having reached the adsorption equilibrium, the solution drop was subjected to harmonic oscillations with frequencies (f) 0.01-0.2 Hz to study the dilatational elasticity.51 Accuracy in elasticity and viscosity values are 0.01 and 0.01 mN/ms. Percent error in elasticity and viscosity lie around 0.02%. Each measurement was repeated at least three times, and the averages of these three measurements are presented in this study. In this study, it is assumed that for small deformations of the liquid drop containing the protein, the mechanical response of the surface layer will be linear with the applied strain. The experiments presented here are in this linear response regime. There are two fundamental modes of mechanical deformation for a surface: shear (at constant area) and compression (at constant shape). Scanning Electron Microscopy (SEM). The LB films of protein samples were coated on cover slips and thereafter airdried. The SEM images of the coated substrates have been viewed using an FET Quanta 200 scanning electron microscope at 10-20 kV. The samples were mounted on aluminum stubs using double side adhesive tape and coated with gold (around 20-25 nm thick) in an Edward E-306 sputter coater. The film thickness was measured with a quartz crystal film thickness monitor (Balzers QSG 201D, Balzers).

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Sankaranarayanan et al. TABLE 1: Average Area/Molecule, Collapse Pressure, and Surface Potential Values for Fg and Fg with Different Electrolytes Fg on subphase buffer (pH7.5) NaCl KCl ZnCl2 CaCl2

Figure 1. Surface pressure and surface potential-molecular area (π-A) and (∆V-A) isotherms of fibrinogen on buffer (pH 7.5) and NaCl, KCl, ZnCl2, and CaCl2.

collapse pressure surface area/molecule (mN/m) potential (V) (nm2) at π ) 20 mN/m 97.71 87.45 90.69 77.24 75.54

32.32 32.00 33.89 24.19 26.36

0.110 0.135 0.113 0.050 0.089

surface pressure values. The isotherms with the protein spread on different electrolytes in the subphase show that all the films show the typical “LE” to “LC” phase. However, the area/ molecule reduces from 97.71 nm2 to about 75.54 nm2, showing that the electrolytes lead most likely to faster aggregation of the molecules at the interface. Also, the corresponding surface potential values that are indicative of the orientation of the molecules in the monolayer plane show that the molecules are tilted with respect to the monolayer plane. From the area values of the protein on NaCl and KCl it is seen that the size of the ions influences the area for Fg at the air/solution interface. Small ions of high valency are highly hydrated and increase the organization of the water molecules by compacting the molecules around themselves while large ions of low valency are weakly hydrated and have the opposite effect. Thus the protein with the changing solvation shell around it shows the changes in its interfacial interaction. The isotherms of Fg with CaCl2 and ZnCl2 show contraction in area, suggesting compact packing of Fg in the presence of divalent metals like Ca2+ and Zn2+. The LB films of these transferred onto quartz substrates were characterized by absorbance spectra. Figure 2 shows the absorbance values of the LB films of Fg for different concentrations spread on buffer and on buffer with different electrolytes. Figure 3 is a scheme of organization of the protein molecules at the air/water interface on pure buffer and in the presence of electrolytes. One form of Fg is trinodular with a length of about 60 nm and, when spread on any surface, is expected to form networks with the trinodular structure reorganizing into polymeric strands with an approximate width of about 12 nm.52 Thus on different subphases the rate of formation of the network

III. Results and Discussion For very low concentrations of protein (10-8 M) it is expected that no aggregation or intermolecular interactions interfere with the measurements of dilational moduli. Thus, dilute solutions of Fg used in this study should lead to an optimal assembly process at the air/solution interface. In both these solutions after about 16 000 s, the adsorption equilibrium was achieved and harmonic oscillations of the drop area with a magnitude of change in drop surface area ∆Ω (7-8%) and frequencies in the range between 0.01 and 0.4 Hz were generated. Panels a and b of Figure 1 show the surface pressure and surface potential-molecular area (π-A) and (∆V-A) isotherms of Fg on pure buffer and on the different electrolytes. The average area/molecule, the maximum collapse pressure, and the corresponding surface potential are given in Table 1. The (π-A) isotherm of pure Fg shows a “liquid expanded” (LE) to “liquid condensed” while the corresponding (∆V-A) isotherm shows a sudden rise in surface potential value (almost resembling a “2D condensed sold phase”), suggesting that surface potential is more sensitive to changes in surface density due to close packing of the protein molecules compared to the

Figure 2. Absorbance values of LB films of pure fibrinogen for different concentrations spread on buffer and on buffer with different electrolytes.



Figure 3. Scheme of organization of the protein molecules at the air/ water interface on pure buffer and in the presence of electrolytes.

TABLE 2: Secondary Structural Features of Pure Fg and Fg with Additives sample Fg Fg Fg Fg Fg

+ + + +

KCl NaCl CaCl2 ZnCl2

% helix

% sheet

% β Turn

% unordered

38.6 24.2 28.2 20.5 24.7

24.7 37.2 33.8 46.7 43.3

16 16.4 15.0 14.2 19.2

20.7 22.2 23.0 18.6 12.8

preceded by fibril formation may differ and thus lead to changes in average area measured from the isotherms. The LB films of the protein spread on buffer and on buffer with electrolytes have been transferred onto solid substrates and CD spectra were recorded. Circular dichroism (CD) spectra of LB films of Fg on buffer and with the different electrolytes were measured in UV-VUV region. Since these are thin films transferred onto solid substrates, there can exist some linear anisotropy. For such films with linear anisotropy the apparent CD signal is known to be perturbed. To obtain the true CD spectra, the thin films of Fg were first examined as a function of sample rotation angle and the linear dichroism spectra were measured. Since the linear anisotropy was very small (about 0.3), the CD spectra measured as a function of sample rotation angle did not show any dramatic changes, suggesting that the contribution of linear anisotropy to CD is small. The accurate thickness of the films was measured and was smaller than 100 nm. The corresponding secondary structural features are presented in Table 2. The estimated secondary structure contents indicate that the native molecule contains about 38% R-helix, 25% β-sheet, 16% β-turn, and 21% random coil, which is in good agreement with the published CD data.53 However, on the subphase containing electrolytes, there seems to be an overall decrease of helical content with films on CaCl2 showing the least helical percent. Azpiazu and Chapman54 studied the secondary structure of human Fg and its plasmin fragments by FTIR and reported that fragment E and the coiledcoil portion of fragment D are particularly rich in R-helical segments. The results suggest that the electrolytes bind to the fragment E and the coiled-coil portion of fragment D. This in turn may actually accelerate the formation of fibrils. This is in accordance with the area contraction seen in the isotherms. Panels a-e of Figure 4 show the Brewster angle micrographs of the Langmuir films of Fg on pure buffer and on buffer with electrolytes at a surface pressure π ) 5 and 20 mN/m, respectively. The micrographs show that on buffer and with KCl small structures are seen and with NaCl the shapes appear slightly elongated. However, in the presence of CaCl2 and ZnCl2, the threadlike structures appear and these seem to cluster

Figure 4. Brewster angle micrographs of the Langmuir films of (a) fibrinogen on pure buffer and on buffer with (b) KCl, (c) NaCl, (d) CaCl2, and (e) ZnCl2 at surface pressure 20 mN/m (scale bar ) 70 µm).

together in the presence of ZnCl2. A number of research articles have appeared in the literature on BAM studies on changes in phases due to protein adsorbing to lipid monolayers,55 due to changes in molecular conformation in annexin,56 displacement of protein films due to surfactants,57 and changes in phase of Ferritin due to varying iron content.58 The BAM micrographs of Fg presented here report for the first time morphology of the protein aggregates and possibly “fibril” formation. The results of the experiments with harmonic oscillations of the surface area were analyzed and the elasticity and viscosity calculated using the software of the instrument PAT-1 in the following way. The viscoelasticity modulus can be presented in a complex form

E ) E′ + IE′′ ) E′ + Iωηd

(6)

where E′ is the dilational elasticity (storage modulus) and ηd is the dilational viscosity obtained from the imaginary part, E′′ divided by the imposed angular frequency ω. This viscous contribution to the viscoelasticity modulus corresponds to the measurable phase difference, Φ, between the stress (dγ) and the strain (dA). This means that the dilational elasticity and viscosity can be determined as a function of frequency from measured absolute value of the modulus, |E|, and the phase angle Φ via

E′ ) |E| cos Φ

(7)

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E′′ ) |E| sin Φ

Sankaranarayanan et al.

(8)

or

TABLE 3: Dilational Elasticity and Viscosity of Fg and Fg with Electrolytes at the Lowest and Highest Frequency of Dilation frequency

ηd ) [|E|/ω] sin Φ

0.005 (Hz)

(9)

The initial experimental data were first filtered to exclude the scattering errors. Also, the constant shift of the surface tension with time was eliminated, caused by the yet small deviation of the system from equilibrium when the harmonic oscillations are started. The plots of dilational elasticity and viscosity are presented in Figure 5. The values of elasticity and viscosity for Fg and Fg with electrolytes for the lowest and highest frequency of oscillations are given in Table 3. From the values it is seen that at low frequency Fg and Fg with electrolytes show unusually high dilational viscosity, and in the presence of ZnCl2, this value is the highest. The high dilational viscosity is possibly an indicator for increased association of the protein molecules leading to the fibril formation. This result is in agreement with the initiation and onset of fibrils with divalent metal ions like Zn2+ reported earlier.59 However, in

Figure 5. Dilational elasticity and viscosity of Fg and Fg with KCl, NaCl, CaCl2, and ZnCl2.

0.2 (Hz)

sample

elasticity (mN/m)

viscosity (mN/ms)

elasticity (mN/m)

viscosity (mN/ms)

Fg Fg/KCl Fg/NaCl Fg/CaCl2 Fg/ZnCl2

35.34 41.84 42.74 42.34 44.95

134.67 163.81 158.22 213.34 255.22

44.72 52.68 52.23 56.65 61.36

4.82 6.86 4.87 9.13 12.10

most results reported on interaction of Fg with electrolytes, fibril formation with CaCl2 is slower compared to ZnCl2. Our results show that Fg exhibits a lower viscosity with CaCl2 compared with ZnCl2. At higher frequency the viscosity drops by an order and there is only a marginal increase in elasticity. At higher frequency if the time for relaxation and organization of individual protein molecules is assumed to be of the same order as the rate of shear there seems to be no overall change in the dilational properties. Figure 6 shows the scanning electron micrographs of the films transferred to glass coverslips, and SEMs show the formation of fibrillar structures in the presence of electrolytes. Pure Fg shows dendritic patterns and either appears singly or appears in small clusters. However, nodular rodlike structures in the presence of CaCl2 and ZnCl2 were seen.

Figure 6. Scanning electron micrographs of (a) Fg and Fg with (b) KCl, (c) NaCl, (d) CaCl2, and (e) ZnCl2. Scale bar: (a) 100 µm; (b)-(e) 100 nm.



TABLE 4: Initial and Final Work of Adhesion for Pure Fg and Fg with Additives Adsorbed to Bare Substrate and DOMA and DMPC Coated Substrate substrate bare

Wi Wf Wi Wf Wi Wf

DOMA coated DMPC coated

Fg

Fg + NaCl

Fg + KCl

Fg + CaCl2

Fg + ZnCl2

37.169 -0.3516 -110.33 0.4321 -124.37 0.1783

47.254 0.0093 104.77 -7.07 -99.464 -2.0053

25.467 -0.0091 105.78 -7.057 -85.56 -0.3818

6.6311 0.1151 124.35 -1.6210 -0.1716 0.01206

491.62 0.3736 100.29 2.4776 -31.986 36.996

The SEM of Fg with KCl and NaCl shows clusters with branched structures. Assuming an interfacial tension value γ at the interface, a force balance along the solid surface at the three-phase contact line gives the contact angle. In the present method, the lipid component, DOMA or DMPC film, is immobilized in a 2D plane while the other (Fg) is distributed in solution. The rate of adsorption may be limited by the mass transport to the surface where binding of Fg to the different surfaces has to take place. Two aspects are dealt with in this study: (a) nature of the interface and its influence on the rate of the adsorption and (b) the effect of additives in solution of Fg. Table 4 gives the ∆W for Fg adsorbed to a bare surface and that coated with the lipids DOMA and DMPC. In Figure 7a-c, representative plots of the rate of change of work of adhesion for Fg on these surfaces are presented. For the sake of brevity only these plots are given here. The plots show change in work of adhesion ∆W that is the difference between the work of adhesion at t ) 0 to the work of adhesion at t ) 1 (expressed as (Wt - Weq)) as a function of time. From eq 3, ∆W can be rewritten as

∆W ) [γFg(1 + cos θt) - γFg(1 + cos θ∞)]

(10)

∆W ) γFg[cos θt - cos θ∞]

(11)

Hence, for θ values between 0° and 180°, based on eq 11,

∆W > 0; θ∞ > θt ∆W < 0; θ∞ < θt

hindered adsorption

(12)

promotion of adsorption

(13)

∆W ) 0; θ∞ ) θt

no adsorption

(14)

There may exist a condition between that of eqs 12 and 13 where the difference between θ∞ and θt may tend toward zero. This situation corresponds to poor adsorption due to poor spreading of the solution on the solid. Such a condition, therefore, can lead to localized aggregation of proteins on the surface or poor surface coverage. Therefore, the most negative value for ∆W must correspond to the best adsorption condition. From the ∆W plots, the adsorption of Fg and Fg with additives onto the three solid surfaces could be simply represented by two consecutive reactions occurring at the interface as follows

AfBfC where A represents free protein molecules, B adsorbed protein molecules, and C the rearranged adsorbed protein molecules

or the adsorbed protein molecules in the second adsorption layer. All three adsorption runs were fitted with a sigmoidal Boltzmann function of the form

y ) [(A1 - A2)/(1 + exp(x - x0)/dx] + A2

(15)

Here, A1 and A2 denote the initial work of adhesion at t ) 0, final work of adhesion (steady state), x0 the point of inflection in the plot and dx the time constant. It is well established 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 the final surface properties of the coating. Based on the conditions stated in eqs 12, 13, and 14 and from the values in Table 4, it is seen that on bare hydrophilic substrate pure Fg and Fg with the electrolytes have no dramatic enhancement in adsorption. However, in the substrate coated with DOMA (cationic lipid), except for Fg + ZnCl2 there is promotion of adsorption for all other systems, suggesting that the hydrophobic surface with some positive charges helps in the adsorption of Fg. In the case of the neutral lipid DMPC coated substrate a similar trend is seen. If ZnCl2 promotes fibril formation even before adsorption of the protein to the surface takes place, then there may be fewer multipoint interactions of the Fg with the surface. This is seen if the protein is considered a surface-active molecule; its adsorption can result in disruption of hydration layers at the interface with the effect of hydrophobizing either the surface or the solid surface. The physical interpretation of W is that it 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.60,61 The real model of protein adsorption is complex and involves many processes such as protein-interface interactions, protein orientations on the surface, conformational changes accompanied with protein unfolding, lateral protein-protein interactions, and desorption that lead to multiple states of adsorbed proteins on the surface.62 It is possible that in the case of DMPC or DOMA coated surfaces, the surface shows increased hydrophobicity and, therefore, the initial adsorption is driven by the hydrophobic residues of the protein. This initial interaction is then followed by multipoint interactions due to various degrees of unfolding and or other cooperative effects;63 a decrease in ∆W thus reflects strong adsorption forces and supports the multipoint proteinsurface interactions. While electrostatic interactions between Fg carrying a net negative charge with DOMA are easily rationalized, the promotion of adsorption in the case of a DMPC coated surface suggests that adsorption via small positively charged

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Sankaranarayanan et al. its diffusivity but also on the nature of interactions with the bare surface or the surface coated with DMPC or DOMA. In addition to any charge mediated effects, enhanced adsorption due to increased local hydrophobicity at liquid/liquid interfaces has been proposed from the studies on adsorption of ionic surfactant/protein mixtures.64 These results are in agreement with those of Jiang et al. who studied the stepwise assembly of fibrin bilayers on self-assembled monolayers of alkanethiolate.65 If such a model is extended also to the solid/liquid interface, it is likely that changes in local densities of the nonpolar regions may result in increased adsorption of Fg to lipid coated surfaces. IV. Summary In conclusion, assemblies of Fg organizing at the air/water interface and near solid surfaces seem to initiate the fibril formation. The results are in agreement with the reported fiber formation due to molecular orientation of the Fg near hydrocarbon functionalized clay surfaces.66 Adsorption of Fg to lipid coated surfaces suggests that there seem to exist a synergy between hydrophobic interaction and charge compensation that leads to better adsorption. This enhancement in adsorption is strongly dependent on the initial concentration of the protein in the bulk as well as the actual surface properties at the points of adsorption. This work demonstrates the organization of Fg molecules at the interface or near a surface ultimately leading to fibrils in the absence of thrombin. Acknowledgment. We thank the Department of Science and Technology, Government of India, for a project grant under which part of this work was carried out. References and Notes

Figure 7. ∆W vs time for Fg on (a) bare substrate, (b) DOMA coated substrate, and (c) DMPC coated substrate.

regions on the protein would mean fewer anchoring points, localization of charges, and a lower degree of denaturation. The results in this study suggest that the adsorption of Fg preceded by local association and organization is dependent not only on

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