Thermodynamic studies on the adsorption of fibronectin adhesion-promoting peptide on nanothin films of poly(2-vinylpyridine) by SPR

This paper describes a methodology for preparing uniform, nanothin polymer films for the study of biomolecule adsorption by surface plasmon resonance spectroscopy (SPR). The methodology combines molecular self-assembly of alkanethiols on gold with surface-confined atom transfer radical polymerization (ATRP). Poly(2-vinylpyridine) was chosen to demonstrate the methodology, and growth kinetics were studied by ex situ ellipsometry. Atomic force microscopy (AFM) indicated that the polymer films were uniform with RMS roughness of approximately 0.5 nm. Subsequent SPR measurements were done to determine thermodynamic adsorption properties (deltaG, deltaH, and deltaS) between fibronectin adhesion-promoting peptide and the surface-confined poly(2-vinylpyridine) at 15, 20, and 25 degrees C. The flexibility in synthesis conditions and the opportunities for manipulating film thicknesses and graft densities that ATRP provides to grow polymer films from gold surfaces holds advantages over conventional spin-coating and grafting to approaches in the design of model polymer films for biomolecule adsorption studies. These advantages are described.     

Folding of aminosuccinyl peptides: Thermodynamic data from temperature dependent circular dichroism measurements

The conformational equilibrium of aminosuccinyl peptides between extended conformations and an intramolecularly hydrogen bonded type II′ β-turn conformation has been studied on the peptide Boc-L -Asu-Gly-L -Ala-OMe (Asu = aminosuccinyl residue) by means of temperature dependence of circular dichroism spectra. Owing to the peculiar chiroptical and conformational properties of the Asu residue, this technique proved to be very useful for deriving thermodynamic data for the above folding process. The value of ΔH⁰ (?6.6 kJ mol^?1), obtained for the peptide studied in a chloroformacetonitrile mixture, shows that the lower energy of the folded conformer is primarily due to the characteristic intramolecular hydrogen bond of the β turns. © 1995 Wiley-Liss, Inc.     

NMR study of the structure of short-chain peptides in solution.

The electron-mediated spin–spin coupling constant J between the amide NH and the α-CH protons in the dipeptide fragment C^α? CO(NH? C^αH)R? C′ONH? C^α is dependent on the dihedral angle of rotation (Φ) around the N? C bond. Measurement of J in a series of zwitterionic dipeptides H₃N⁺? CHR₁? CONH? CHR₂? CO₂^? (which is conformationally similar to the dipeptide fragment) in TFA solution shows that J is independent of R₁, but dependent on the steric bulk of R₂. The data are interpreted in terms of a model that assumes that what we measure is an average value of J? a thermal average over all the possible rotamers. The groups R₁ and R₂ are, in most cases, sterically kept apart by the trans and planar amide bonds, and hence the independence of J of R₁. This model is consistent with the theoretical calculations done on the dipeptide fragment. The effect of the structural characteristics of the side chains (e.g., the effect of lengthening and branching the side chains) on the J values in dipeptides is discussed in the light of the existing results of theoretical calculations. Study of 〈J〉 values in tripeptides (C₆H₅CH₂OCONH? CHR₁? CONH? CHR₂? CO₂CH₃, essentially three linked peptide units) shows that electrostatic interaction between the two amide bonds modifies the potential energy surface and the 〈J〉 value of a dipeptide subunit in the tripeptides. Also in some cases, direct steric interaction between the two side chains in the two adjacent dipeptide subunits in the tripeptide affects the potential energy surfaces of the individual dipeptide subunits and hence the 〈J〉 values. The influence of the structural characteristics of the side chains of individual amino acids on structure formation at or beyond the dipeptide level is discussed at various points. The J(NH? ^αCH) values of CH₃CONH? CHR? CONH₂ and CH₃CONH? CHR? CO₂CH₃ with the same R are quite different for R = valine, leucine, phenylalanine, methionine, but equal for R = glycine. This, coupled with the fact that one of the carboxamide NH resonances has a chemical shift different from its counterpart in simple amides like CH₃CONH₂ and the other carboxamide NH has the same chemical shift as its counterpart in CH₃CONH₂, suggest the presence of a hydrogen bond in dipeptide CH₃CONH? CHR? CONH₂ with carboxamide NH as the donor. Theoretical evidence for two seven-membered hydrogen-bonded rings with the carboxamide NH as donor and the acetyl oxygen as acceptor is summarized. Our data cannot suggest the number of such hydrogen-bonded rings, nor can they conclude the relative proportion of these rings in a particular dipeptide. A discussion of the difficulty of interpretation is presented and the data are discussed under certain simplifying assumptions.     

Thermodynamic regulation of human short-chain acyl-CoA dehydrogenase by substrate and product binding

Human short-chain acyl-CoA dehydrogenase (hSCAD) catalyzes the first matrix step in the mitochondrial beta-oxidation cycle for substrates with four and six carbons. Previous studies have shown that the act of substrate/product binding induces a large enzyme potential shift in acyl-CoA dehydrogenases. The objective of this work was to examine the thermodynamic regulation of this process through direct characterization of the electrochemical properties of hSCAD using spectroelectrochemical methodology. A large amount of substrate activation was observed in the enzymatic reaction of hSCAD (+33 mV), the greatest magnitude measured in any acyl-CoA dehydrogenase to date. To examine the role of the substrate as well as the product in electron transfer by hSCAD, a catalytic base mutation (E368Q) was constructed. The E368Q mutation inactivates the reductive and oxidative pathways such that the individual effects of substrate and product binding on the redox potential can be investigated. Optimal substrate (butyryl-CoA) was seen to shift the flavin redox potential slightly more positive (+38 mV) than did optimal product (crotonyl-CoA) (+31 mV), a finding opposite of that observed in another short-chain enzyme, bacterial SCAD. These results indicate that substrate redox activation occurs in hSCAD leading to a large enzyme midpoint potential shift. Substrate binding in hSCAD appears to make a larger contribution than does product to thermodynamic modulation.     

Studies on adsorption of dyes on beta-cyclodextrin polymer

Beta-cyclodextrin (beta-CD) polymers are used for the removal of various dyes from aqueous solutions. Three insoluble polymers with different degrees of beta-CD were used. Results of adsorption experiments showed that these polymers exhibited high sorption capacities toward dyes. The mechanism of adsorption was both physical adsorption and hydrogen bonding due to the polymer and the formation of an inclusion complex due to the beta-CD molecules through host-guest interactions.     

Reliability of upper and lower extremity anthropometric measurements and the effect on tissue mass predictions

Accurate modeling of soft tissue motion effects relative to bone during impact requires knowledge of the mass of soft and rigid tissues in living people. Holmes et al., [2005. Predicting in vivo soft tissue masses of the lower extremity using segment anthropometric measures and DXA. Journal of Applied Biomechanics, 21, 371–382] developed and validated regression equations to predict the individual tissue masses of lower extremity segments of young healthy adults, based on simple anthropometric measurements. However, the reliability of these measurements and the effect on predicted tissue mass estimates from the equations has yet to be determined. In the current study, two measurers were responsible for collecting two sets of unilateral measurements (25 male and 25 female subjects) for the right upper and lower extremities. These included 6 lengths, 6 circumferences, 8 breadths, and 4 skinfold thicknesses. Significant differences were found between measurers and between sexes, but these differences were relatively small in general (75–80% of between-measurer differences were <1 cm). Within-measurer measurement differences were smaller and more consistent than those between measurers in most cases. Good to excellent reliability was demonstrated for all measurement types, with intra-class correlation coefficients of 0.79, 0.86, 0.85 and 0.86 for lengths, circumferences, breadth and skinfolds, respectively. Predicted tissue mass magnitudes were moderately affected by the measurement differences. The maximum mean errors between measurers ranged from 3.2% to 24.2% for bone mineral content and fat mass, for the leg and foot, and the leg segments, respectively.     

Effect of cosolvents on the adsorption of peptides at the solid-liquid interface

The adsorption of a peptide at solid surfaces is the result of a complex interplay of interactions between the peptide, solvent, and surface. In this work, Monte Carlo simulations were performed to evaluate the effect of the solvent hydrogen bonding ability on the adsorption of the peptide ASP(1)-ASP(2)-ILE(3)-ILE(4)-ASP(5)-ASP(6)-ILE(7)-ILE(8) at a charged surface consisting of CH(2) atoms with a fixed lattice arrangement. Various water-alcohol mixtures were used as solvent because alcohols are known to alter the dielectric constant, hydrophobicity, and hydrogen bonding capacity of water. Solvent-solvent, solvent-surface, solvent-peptide, and peptide-surface interactions were studied independently and correlated with the observed peptide behavior at the solvent-surface interface. We concluded that the behavior (and orientation) of the peptide at the surface is directly related to changes in water-water hydrogen bonding properties in water-alcohol mixtures. In the presence of increasing concentrations of methanol, the strength of solvent-peptide and solvent-surface interactions was reduced, and as a result, a stronger interaction between the peptide and the surface was observed. Stronger solvent-peptide and solvent-surface interactions were responsible for a weaker interaction of the peptide with the surface in the presence of increasing concentrations of glycerol. These results suggest that by changing solvent conditions it is possible to finely tune the orientation of a macromolecule at solid/liquid interfaces.     

Thermodynamic effects of the hydrophobic surfactant proteins on the early adsorption of pulmonary surfactant

We determined the influence of the two hydrophobic proteins, SP-B and SP-C, on the thermodynamic barriers that limit adsorption of pulmonary surfactant to the air-water interface. We compared the temperature and concentration dependence of adsorption, measured by monitoring surface tension, between calf lung surfactant extract (CLSE) and the complete set of neutral and phospholipids (N&PL) without the proteins. Three stages generally characterized the various adsorption isotherms: an initial delay during which surface tension remained constant, a fall in surface tension at decreasing rates, and, for experiments that reached approximately 40 mN/m, a late acceleration of the fall in surface tension to approximately 25 mN/m. For the initial change in surface tension, the surfactant proteins accelerated adsorption for CLSE relative to N&PL by more than ten-fold, reducing the Gibbs free energy of transition (DeltaG(O)) from 119 to 112 kJ/mole. For the lipids alone in N&PL, the enthalpy of transition (DeltaH(O), 54 kJ/mole) and entropy (-T. DeltaS, 65 kJ/mole at 37 degrees C) made roughly equal contributions to DeltaG(O). The proteins in CLSE had little effect on -T. DeltaS(O) (68 kJ/mole), but lowered DeltaG(O) for CLSE by reducing DeltaH(O) (44 kJ/mole). Models of the detailed mechanisms by which the proteins facilitate adsorption must meet these thermodynamic constraints.     

Root surface area measurements based on adsorption and desorption of nitrite

A method, based on the negative adsorption of NO₂ ^-, has been developed to determine surface area of roots. Young roots of 3–4 year old plants ofAcacia catechu, Eucalyptus camaldulensis andLeucaena leucocephala were up-rooted and cut into 18 cylindrical pieces. Each root piece was immersed individually for 10 sec in 0.05M, 0.10M and 0.15M aqueous NaNO₂ solution and the excess solution on the root surface allowed to drain off. It was then transferred to conical flask containing distilled water and shaken for 15 min for desorption of nitrite. A known quantity of this aliquot was reacted with 1% acidic sulphanilamide and 0.02% NED HCl. A pink colour developed, and its optical density was read at 540 nm. A positive linear correlation was noted between colour density and root surface area. The respective correlation coefficientvalues for 0.05M, 0.10M and 0.15M NaNO₂ solutions were 0.973, 0.963 and 0.964 forAcacia catechu, 0.933, 0.903 and 0.898 forEucalyptus camaldulensis and 0.968, 0.976 and 0.972 forLeucaena leucocephala (significant atp<0.001). The method was successfully adopted to determine the root surface area of seedlings ofAlbizia lebbek, A. procera, Acacia auriculiformis, A. nilotica, Dalbergia latifolia andD. sissoo.     

Physical polymer matrices based on affinity interactions between peptides and polysaccharides

A rapidly forming polymer matrix with affinity-based controlled release properties was developed based upon interactions between heparin-binding peptides and heparin. Dynamic mechanical testing of 10% (w/v) compositions consisting of a 3:1 molar ratio of poly(ethylene glycol)-co-peptide (approximately 18,000 g/mol) to heparin (approximately 18,000 g/mol) revealed a viscoelastic profile similar to that of concentrated, large molecular weight polymer solutions and melts. In addition, the biopolymer mixtures recovered quickly following thermal denaturation and mechanical insult. These gel-like materials were able to sequester exogenous heparin-binding peptides and could release these peptides over several days at rates dependent on relative heparin affinity. The initial release rates ranged from 3.3% per hour for a peptide with low heparin affinity to 0.025% per hour for a peptide with strong heparin affinity. By altering the affinity of peptides to heparin, a series of peptides can be developed to yield a range of release profiles useful for controlled in vivo delivery of therapeutics.     

Thermodynamic and dynamic characteristics of hydroxypropylmethylcellulose adsorbed films at the air-water interface

Surface pressure isotherms and structural and surface dilatational properties of three hydroxypropylmethycelluloses (HPMCs, called E4M, E50LV, and F4M) adsorbed films at the air-water interface were determined. In this work we present evidence that HPMC molecules are able to diffuse and saturate the air-water interface at very low concentrations in the bulk phase. As bulk concentration increased, structural changes at a molecular level occurred at the interface. These changes corresponded to transition from an expanded structure (structure I) to a condensed one (structure II). When the surface concentration of HPMC was high enough, the collapse of the monolayer was observed. The three HPMCs formed very elastic films at the air-water interface, even at low surface pressures. E4M showed features that make it unique. For instance it showed the highest surface activity, mainly at low bulk concentrations (<10(-4) wt %). The differences observed in surface activity may be attributed to differences in the hydroxypropyl molar substitution and molecular weight of HPMC. All three HPMCs formed films of similar viscoelasticity and elastic dilatational modulus, which can be accounted for by their similar degree of methyl substitution.     

Biosensor based on enzyme-catalysed degradation of thin polymer films

A biosensor based on the enzyme-catalysed dissolution of biodegradable polymer films has been developed. Three polymer-enzyme systems were investigated for use in the sensor: a poly(ester amide), which is degraded by the proteolytic enzyme alpha-chymotrypsin; a dextran hydrogel, which is degraded by dextranase; and poly(trimethylene) succinate, which is degraded by a lipase. Dissolution of the polymer films was monitored by Surface Plasmon Resonance (SPR). The rate of degradation was directly related to enzyme concentration for each polymer/enzyme couple. The poly(ester amide)/alpha-chymotrypsin couple proved to be the most sensitive over a concentration range from 4 x 10(-11) to 4 x 10(-7) mol l(-1) of enzyme. The rate of degradation was shown to be independent of the thickness of the poly(ester amide) films. The dextran hydrogel/dextranase couple was less sensitive than the poly(ester amide)/alpha-chymotrypsin couple but showed greater degradation rates at low enzyme concentrations. Enzyme concentrations as low as 2 x 10(-11) mol l(-1) were detected in less than 20 min. Potential fields of application of such a sensor system are the detection of enzyme concentrations and the construction of disposable enzyme based immunosensors, which employ the polymer-degrading enzyme as an enzyme label.     

Lipid mono- and bilayer supported on polymer films: composite polymer-lipid films on solid substrates.

We report the deposition of lipid monolayers and bilayers on polyacrylamide films deposited by radical chain reaction onto solid substrates in aqueous solutions. Polymer films of various degrees of monomer density and cross-linking are prepared. Lateral diffusion and fluorescent probe permeation measurements yield insight into the continuity of the lipid layers and show that monolayers exposed to air are much less sensitive towards polymer heterogeneities than bilayers below water, which is explained in terms of the wetting laws. The diffusion studies of lipid and lipopeptide probes yield absolute values of the frictional coefficients between the lipid layer and the polymer films and allow one to estimate the surface viscosity of the polymer film. The potential applications of supported membranes on soft thin polymer films for the preparation of biofunctionalized surfaces or biocompatible receptive surfaces for biosensors are discussed.     

Thermodynamic aspects of plant cell adhesion to polymer surfaces

Summary A thermodynamic model of particle adhesion from a suspension onto a solid surface is used to predict the extent of adhesion of suspension-cultured Catharanthus roseus cells to the following polymer substrates: fluorinated ethylene-propylene (FEP), polystyrene (PS), polyethylene terephthalate (PET), sulphonated polystyrene (SPS), and glass. According to this model, the extent of adhesion is determined by the surface tensions of the plant cells, the polymer substrates, and the suspending liquid medium. Experimentally, adhesion of the washed plant cells was found to decrease with increasing substrate surface tension, following the sequence FEP>PS>PET>SPS>glass, when the surface tension of the liquid was greater than that of the plant cells, in agreement with the model. However, adhesion increased with increasing substrate surface tension when the liquid surface tension was lower than the cellular surface tension, also in agreement with the model. When the liquid and cellular tensions were equal the extent of adhesion was independent of the substrate surface tension. This also agrees with model predictions and leads to a value for the surface tension of C. roseus cells of approximately 54 ergs/cm² which is in agreement with a value obtained from contact angle measurements on layers of cells and sedimentation volume analysis. The cellular surface tension determined by the sedimentation volume method showed a biphasic alteration during growth cycles of C. roseus cell cultures. These variations (between 55 and 58 ergs/cm²) agree with the pattern of adhesion previously described.     

The molecular mechanism of adsorption immobilization of inulinase on polymer matrices

The conditions and mechanisms of the immobilization of inulinase on polymeric carriers were studied using the VION KN-1 and KU-2 cation-exchangers, VION AN-1 and AV-17-2P anion-exchangers, and the ampholyte KOPAN-90. The calculated data showed a significant role of van der Waals interactions and hydrogen bonding in the formation of virtually all inulinase complexes with the immobilization matrices. The AV-17-2P anion-exchanger was the only one of the studied polymer matrices that was unable to form hydrogen bonds with inulinase. The mechanisms of the interaction between inulinase and various ampholytes and cation and anion exchange resins differ from each other. The strongest differences are observed in mechanisms of the sorption of inulinase on VION KN-1 and chitosan matrices. Approximately 87% of the identical amino-acid residues are involved in the interaction of the enzyme with the KU-2 and AV-17-2P resins and the VION AN-1 and KOPAN-90 fibers.     

Thermodynamic interpretation of protein dynamics from NMR relaxation measurements

Protein dynamics and thermodynamics can be characterized through measurements of relaxation rates of side chain (2)H and (13)C, and backbone (15)N nuclei using NMR spectroscopy. The rates reflect protein motions on timescales from picoseconds to milliseconds. Backbone and methyl side chain NMR relaxation measurements for several proteins are beginning to reveal the role of protein dynamics in protein stability and ligand binding.