Biomacromolecules 2005, 6, 9-13
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Communications Thermodynamic Measurements and Predictions of the Adsorption of Short-Chain Peptides on Nanothin Polymer Films Nripen Singh and Scott M. Husson* Department of Chemical Engineering, Clemson University, Clemson, South Carolina 29634-0909 Received November 12, 2004
This contribution describes experimental measurements of submolecular-level interaction energies involved in the process of peptide adsorption on polymer films. The objective of this study was to use surface plasmon resonance (SPR) spectroscopy to measure the Gibbs energy change on adsorption (∆Gad) for pairs of various homopeptides on highly uniform, nanothin polymer films and to use these data, along with the principle of additivity, to predict ∆Gad for homologous homopeptides, as well as for a mixed-residue peptide. By using a graft polymerization methodology, a nanothin poly(2-vinylpyridine) film was prepared and adsorption energies were measured first for a homologous series of tyrosine (Y) homopeptides on this film to determine submolecular-level interaction energies. By using SPR, adsorption isotherms were measured for YY and YYY peptides; analysis of these isotherms provided ∆Gad data for a midchain tyrosine unit and a set of chain-end tyrosine units; values were -0.75 ( 0.07 kcal/mol and -2.12 ( 0.04 kcal/mol, respectively. Combining the thermodynamic contributions for adsorption of individual tyrosine units allowed a predictive estimate of -5.12 ( 0.32 kcal/mol for the adsorption energy for YYYYYY; this estimate deviated by only 2.3% from its measured value of -5.24 ( 0.06 kcal/mol. Similarly, adsorption energies were found for phenylalanine, glycine, and tyrosine-leucine peptides. Combining the thermodynamic contributions for adsorption of individual residue units allowed a predictive estimate of -3.24 ( 0.38 kcal/mol for a pentapeptide, leucine enkephalin; this estimate deviated by only 3.0% from its measured value of -3.34 ( 0.11 kcal/mol. Introduction Much has been learned and written about biomolecule adsorption to materials surfaces,1-6 and we and others have used surface plasmon resonance (SPR) spectroscopy to study peptide and protein adsorption onto polymer films.7-14 Adsorption of these compounds involves noncovalent interactions (e.g., hydrophobic, electrostatic, hydrogen bonding, van der Waals)6 that depend, in part, on their structure, size, and stability; surface properties such as surface energy, roughness, and chemistry; and environmental conditions (e.g., temperature, pH, ionic strength). For applied purposes, the scientific challenges include understanding how to predict under what conditions biomolecules will adsorb and, in many cases, how to control or to minimize their adsorption.15 For example, the adsorption of proteins on the surfaces of biomedical devices and heat exchangers in the food and dairy industries disrupts their function. Therefore, materials designed to resist the adsorption of proteins would lead to improved performances of these devices. This contribution describes an experimental procedure for measuring submolecular-level interaction energies involved in the process of short-chain peptide adsorption on polymer * To whom correspondence should be addressed. Tel.: (864) 656-4502. Fax: (864) 656-0784. E-mail:
[email protected].
films. The objective of this study was to use SPR to measure the Gibbs free energy change on adsorption (∆Gad) for pairs of homopeptides on highly uniform, nanothin polymer films and to use these data, along with the principle of additivity, to predict ∆Gad for homologous homopeptides, as well as a mixed-residue peptide. We espouse the hypothesis of Latour and Rini,16 that, at a fundamental level, adsorption depends on interactions between individual amino acid residues on the biomolecule surface and the functional groups of the material surface. Because van der Waals and electrostatic interactions are generally additive for proteins adsorbing on surfaces17,18 and because adsorption of proteins onto synthetic surfaces is a thermodynamically driven process,6 it should be possible, theoretically, to predict adsorption behavior for a short-chain peptide that does not undergo surface-induced conformation changes by knowing values of ∆Had and ∆Sad for the residues that comprise the peptide. Lacking temperature-dependent adsorption data, constant-temperature predictions should be possible simply by knowing values of ∆Gad for these residues.
∑∆Had,residue - T∑∆Sad,residue ∆Gad,protein ) ∑∆Gad,residue
∆Gad,protein )
10.1021/bm049281j CCC: $30.25 © 2005 American Chemical Society Published on Web 12/02/2004
(1) (2)
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Biomacromolecules, Vol. 6, No. 1, 2005
Using our previously described7,19,20 polymerization methodology, we prepared a highly uniform, nanothin film of poly(2-vinylpyridine) on a gold biosensor chip and measured values of ∆Gad for individual amino acid residues adsorbing onto this film surface from solutions that mimic physiological conditions. The SPR analysis method allowed us to check whether adsorption hysteresis existed and to what extent, if any, were the model short-chain peptides bound irreversibly to the surface. On the basis of results here and previously,7 adsorption hysteresis does not appear to be problematic for these systems under the conditions of low concentration used to extract thermodynamic adsorption data. Thus, the use of reversible thermodynamic principles seems justified. Materials and Methods Materials. All peptides were used as received from Sigma Chemical, unless noted otherwise; they were leucine enkephalin (g95%), tyrosine (three units), tyrosine (six units), phenylalanine (two units), phenylalanine (four units), glycine (four units), and glycine (five units). Tyrosine (two units), phenylalanine (three units), glycine (six units), and leucinetyrosine peptides were used as received from Spectrum Chemical. Gold substrates were used as received from BIAcore, Inc. (SIA Au kit, BR-1004-05). All chemicals were purchased from Aldrich and used as received, unless noted otherwise; they were 11-mercapto-1-undecanol (MUD; 97%), (4-chloromethyl)benzoyl chloride (97%), 2-vinylpyridine (97%), copper(I) bromide (99.999%), and tris-(2-aminoethyl)amine (TREN; 96%). N-2-Hydroxyethylpiperazine-N′2-ethanesulfonic acid (HEPES) buffer was used as received from Sigma. Solvents were purchased from Aldrich as ACS reagent grade; they were ethyl alcohol (99.5%), anhydrous toluene (99.8%), and acetonitrile (99.9+%). All percentages are in weight percent. 2-Vinylpyridine was purified by vacuum distillation at 25 mmHg before use to remove the inhibitor (p-tert-butyl catechol). Tris-[2-(dimethylamino)ethyl]amine (Me6TREN) was prepared from TREN.21 Preparation of Poly(2-vinylpyridine) Surface Films. Details of the film synthesis and characterization methods, including characterization of the growth kinetics, are given in a previous paper.7 Prior to use, the gold-coated glass substrates (1 cm × 1 cm) were cleaned in a UV cleaner (Boekel, Inc., model 135500) and then rinsed with deionized water. Next, a self-assembled monolayer (SAM) of MUD was formed on the gold substrate by contacting it with a 2 mM MUD solution in ethanol for at least 16 h. The surfaces were washed in ethanol using an Aquasonic ultrasonic cleaner for 10 s, rinsed with ethanol and deionized water, and dried in a stream of nitrogen. An ellipsometric thickness of 1.1 ( 0.2 nm and the appearance of aliphatic -CH2stretch peaks at 2921 and 2853 cm-1 in the reflectance Fourier transform infrared (FTIR) spectrum of the film confirmed SAM formation. To functionalize the surface with polymerization initiator groups, gold substrates with the SAM layer were incubated in a 2 mM solution of (4-chloromethyl)benzoyl chloride in toluene for 12-16 h at room temperature in a water-free (
