Article Cite This: Langmuir XXXX, XXX, XXX−XXX
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Conformational and Organizational Insights into Serum Proteins during Competitive Adsorption on Self-Assembled Monolayers Abshar Hasan, Gyan Waibhaw, and Lalit M. Pandey* Bio-Interface & Environmental Engineering Lab, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam 781039, India
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ABSTRACT: Physicochemical interactions of proteins with surfaces mediate the interactions between the implant and the biological system. Surface chemistry of the implant is crucial as it regulates the events at the interface. The objective of this study was to explore the performance of modified surfaces for such interactions relevant to various biomedical applications. Because of a wide range of surface wettability, we aimed to study protein behavior (i.e., conformational changes and their packing) during competitive protein adsorption. Three serum proteins (bovine serum albumin, BSA; fibrinogen, FB; and immunoglobulin G, IgG) were tested for their conformational changes and orientation upon adsorption on hydrophilic (COOH and amine), moderately hydrophobic (mixed and hybrid), and hydrophobic (octyl) surfaces generated via silanization. Modified surfaces were characterized using Fourier-transform infrared spectroscopy, contact angle, and atomic force microscopy (AFM) techniques. Adsorbed masses of proteins from single and binary protein solutions on different surfaces were quantified along with their secondary structure analyses. Maximum adsorbed protein masses were found to be on negatively charged and hydrophobic (octyl) surfaces because of ionic and hydrophobic interactions between protein molecules and surfaces, respectively. Side-on and end-on orientations of adsorbed protein molecules were analyzed using theoretical and AFM analyses. We observed compact and elongated forms of BSA molecules on hydrophilic and hydrophobic surfaces, respectively. We further found a linear increase in the α-helix content of BSA and β-sheet contents of FB and IgG proteins with the increasing side-on (%)-oriented protein molecules on the surfaces. This indicates that side-on orientations of adsorbed FB and IgG lead to the formation of β-sheets. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was employed to quantify the protein types and their ratio in competitively adsorbed proteins on different surfaces. A theoretical analysis was also used to determine the % secondary structures of competitively adsorbed proteins from BSA/FB and BSA/IgG solutions, which very well agreed with experimental results. The competitive protein adsorption from both BSA/FB and BSA/IgG solutions was found to be entropy-driven, as revealed by thermodynamic studies performed using isothermal titration calorimetry.
1. INTRODUCTION Protein adsorption on surfaces is one of the most common events but is complicated and is of paramount importance for various applications in biomedical fields, drug delivery, and food packaging industries.1−4 Although researchers have been trying to unfold its highly complicated mechanism, there exists a scope of research due to disparity within the scientific community arising from various proposed mechanisms and theories.4 The adsorption process is complicated because it is governed not only by the type of protein, solution pH, temperature, etc. but also by the physiochemical and nano-/ microscopic properties of surfaces.4−6 The protein−surface interaction is the first event that occurs when any foreign biomaterial comes in contact with the biological fluid, which is later followed by a cascade of other biological processes. The interaction between protein molecules and the surface determines the reversible or irreversible nature of the adsorption process. Irreversible adsorption on the surface is © XXXX American Chemical Society
unfavorable as it causes an increase in free energy, driving proteins to maximize their footprint via conformational rearrangement, leading to a change in the native conformation of the protein.7,8 Such conformational changes may cause loss of biological activity (in case of enzymes) and biofouling, ultimately resulting in the implant failure.9 Generally, the hydrophobic surfaces thermodynamically favor irreversible adsorption of proteins, whereas the hydrophilic surfaces exhibit insignificant protein adsorption.10 Various zwitterionic materials have been utilized for surface coating and hydrogel preparations to prevent fouling.11,12 Zwitterionic polymers containing carboxybetaine and sulfobetaine offered advantage of long-term stability in comparison to that of poly(ethylene glycol) (degrade eventually).13,14 Received: April 5, 2018 Revised: May 30, 2018 Published: June 25, 2018 A
DOI: 10.1021/acs.langmuir.8b01110 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir Scheme 1. Schematic Representation of Three Serum Proteins (BSA, IgG, and FB) with Their Dimensionsa
a
(A) Heart-shaped BSA molecule, (B, C) side-on- and end-on-oriented IgG molecules, respectively, and (D) FB molecule.
Biological fluids comprise a mixture of low- and highmolecular-weight proteins, which compete with each other for adsorption onto the surfaces. Lower-molecular-weight proteins (e.g., albumin) found at higher concentrations undergo fast adsorption during the initial phase and later are displaced by bulky proteins (e.g., fibrinogen (FB)); this effect is called “Vroman effect” and has been confirmed using competitive and sequential adsorption processes.5,10,15−17 Since large proteins contain more surface-binding domains, have a larger surface area, and are more surface-active, they experience stronger interactions with surfaces than smaller globular proteins.18 In contrast, it was also alternatively proposed that the concentration of the protein is the major driving force that determines the composition of the adsorbed layer.19 Factors such as bulk protein concentration, 18 protein−protein interaction,20 change in protein conformation upon adsorption,21,22 and protein’s affinity toward the surface18,21 play a major role in competitive and sequential adsorptions. The size of the protein and its bulk concentration determine the protein’s affinity, transportation, and adsorption rate on the surface.23 In sequential adsorption of bovine serum albumin (BSA), fibronectin (FN), and collagen type I (Col I) on titanium alloy (Ti6Al4V) and Ti6Al4V physisorbed with poly(sodium styrenesulfonate) [poly(NaSS)], Felgueiras et al.20 used a quartz crystal microbalance. They reported a displacement of adsorbed BSA (66 kDa) with FN (250 kDa) and adsorbed FN with Col I (360 kDa). In contrast to the Vroman effect, protein adsorption from a binary mixture exhibited a different behavior. FN was predominant in the case of BSA + FN adsorption, whereas in the case of FN + Col I, both proteins were adsorbed, forming multilayers. In case of BSA + Col I, the adsorbed amount was the highest because of complex formation between BSA and Col I protein molecules. It is important to realize the effect of surface properties on regulating protein behavior during competitive and singleprotein adsorption. For example, Lassen et al. reported displacement of BSA with immunoglobulin G (IgG) and fibrinogen (FB) on hydrophilic poly acrylic acid- and poly diaminocyclohexane-coated surfaces24 but was not observed on hexamethyldisiloxane-coated and methylated surfaces.24,25
Wertz at al. observed no displacement of preadsorbed BSA with FB and vice versa on HO and C16 self-assembled monolayers (SAMs) during sequential adsorption. In contrast to the above report, the displacement of albumin with FB25 and γ-globulin17 was reported separately on polystyrene as well as on OH and CH3 SAMs, respectively. These observations clearly highlight that surface chemistry, conformation of the adsorbed proteins, and protein−protein interactions govern the competitive adsorption of proteins. Toward this direction, further studies focusing on these factors will give a better insight into the complex phenomenon of competitive protein adsorption. BSA is a small but most abundant globular serum protein (Mw ∼ 66 kDa) comprising 580 amino acid residues, which arrange themselves in α-helix (45−50%), β-sheet (20%), and (20−25%) β-turn, giving heart-shaped tertiary structures26−28 (as shown in Scheme 1A). IgG (Mw, 150 kDa) is the most abundant antibody found in all body fluids, accounting for 10− 20% of plasma proteins. It comprises two identical light and heavy chains, as shown in Scheme 1B. These chains assemble together to make three lobes, out of which two lobes contain a fragment antigen-binding (Fab) region, whereas the third lobe contains the fragment crystallizable (Fc) region. Orientations of these protein molecules upon adsorption are important for various applications such as biosensors, biomedical devices, etc. and hence require surface engineering to modulate protein behavior.29 FB is a 340 kDa dimeric protein of 47.5 nm length, consisting of two sets of three non-identical Aα, Bβ, and γ polypeptide chains.30,31 Disulfide-linked amino terminals of these chains form a central E-region, whereas disulfide-linked carboxyl terminal parts of each chain at the outer other end form the D-region. The length of the Aα chain is longer than that of its counterparts of the other chains and is termed as the αC region.30 FB adsorption on hydrophilic surfaces is promoted by the αC region, whereas the D and E regions play a major role in adsorption on hydrophobic surfaces.30 In spite of a lot of research in determining protein behavior at the solid−liquid interface, there still exists huge ambiguity in the governing mechanism of protein adsorption from single and binary solutions and the underlying adsorption kinetics. The role of interacting surfaces is also an important area of B
DOI: 10.1021/acs.langmuir.8b01110 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir
points on the same surface. θ primarily depends on interfacial tensions at the air−liquid, solid−liquid, and solid−air interfaces and is related through Young’s equation
research that should be carefully studied. Hence, in this work, we attempt to gain mechanistic insight into the competitive adsorption behavior of three most commonly occurring proteins in serum i.e., BSA, IgG, and FB and the changes in their secondary structure upon adsorption. Self-assembled monolayers of amino silanes and octyl silanes were used to generate five different surfaces with varying nanoroughnesses and surface energies (SEs). Adsorption from single and binary solutions of proteins was analyzed on the above modified surfaces, and the conformations of the adsorbed proteins were investigated using Fourier transform infrared-attenuated total reflection (FTIR-ATR) spectroscopy. The interprotein interactions in a binary mixture were analyzed using isothermal titration calorimetry (ITC).
γSV − γSL = γLV cos θ
(1)
where γSV, γSL, and γLV are interfacial tensions between solid and vapor (SV), solid and liquid (SL), and liquid and vapor (LV) phases, respectively. The surface energy (γSV) was calculated on the basis of contact angles of both the liquids, using the geometric mean expression (shown below) reported previously by our group.6 γij = γi + γj − 2⌀[(γidγjd)1/2 + (γiPγjP)1/2 ] γdi
(2)
γPi
and are the dispersive and polar components of liquid where surface energy, whereas γdj and γPj are the dispersive and polar components of solid surface energy, respectively. ⌀ is the interaction parameter, whose value is taken as 1 for most of the similar types of molecules. 2.3.3. Surface Roughness and Morphology Analyses. Surface features such as roughness and topology can be analyzed using atomic force microscopy (AFM) that can detect the dynamic events of the adsorption process at the molecular level. It was carried out at room temperature under non-contact mode using an Innova AFM system (Bruker) fitted with a silicon nitride tip of
