Electrochemical and PM-IRRAS characterization of cholera toxin binding at a model biological membrane.

A mixed phospholipid-cholestrol bilayer, with cholera toxin B (CTB) units attached to the monosialotetrahexosylganglioside (GM1) binding sites in the distal leaflet, was deposited on a Au(111) electrode surface. Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) measurements were used to characterize structural and orientational changes in this model biological membrane upon binding CTB and the application of the electrode potential. The data presented in this article show that binding cholera toxin to the membrane leads to an overall increase in the tilt angle of the fatty acid chains; however, the conformation of the bilayer remains relatively constant as indicated by the small decrease in the total number of gauche conformers of acyl tails. In addition, the bound toxin caused a significant decrease in the hydration of the ester group contained within the lipid bilayer. Furthermore, changes in the applied potential had a minimal effect on the overall structure of the membrane. In contrast, our results showed significant voltage-dependent changes in the average orientation of the protein α-helices that may correspond to the voltage-gated opening and closing of the central pore that resides within the B subunit of cholera toxin.

Direct visualization of the enzymatic digestion of a single fiber of native cellulose in an aqueous environment by atomic force microscopy.

Atomic force microscopy (AFM) was used to study native cellulose films prepared from a bacterial cellulose source, Acetobacter xylinum, using a novel application of the Langmuir-Blodgett technique. These films allowed high-resolution AFM images of single fibers and their microfibril structure to be obtained. Two types of experiments were performed. First, the fibers were characterized using samples that were dried after LB deposition. Next, novel protocols that allowed us to image single fibers of cellulose in films that were never dried were developed. This procedure allowed us to perform in situ AFM imaging studies of the enzymatic hydrolysis of single cellulose fibers in solution using cellulolytic enzymes. The in situ degradation of cellulose fibers was monitored over a 9 h period using AFM. These studies provided the first direct, real-time images of the enzymatic degradation of a single cellulose fiber. We have demonstrated the tremendous potential of AFM to study the mechanism of the enzymatic digestion of cellulose and to identify the most effective enzymes for the digestion of various cellulose structures or isomorphs.

Adsorption of N-Decyl-N,N,N-trimethylammonium triflate (DeTATf), a cationic surfactant, on the Au(111) electrode surface.

The adsorption behavior of the cationic surfactant N-decyl-N,N,N-trimethylammonium triflate (DeTATf) on the Au(111) electrode surface was characterized using cyclic voltammetry, differential capacity, and chronocoulometry. The thermodynamics of the ideally polarized electrode have been employed to determine the Gibbs excess and the Gibbs energy of adsorption. The results show that the adsorption of DeTATf has a multistate character. At low bulk DeTATf concentrations, the adsorption state is consistent with the formation of an adsorbed film of nearly flat molecules. At higher concentrations this film may represent a three-dimensional aggregated state. At negative potentials and charge densities close to 0 microC cm-2, the data suggest the formation of a film of tilted molecules oriented with the hydrocarbon tail toward the metal surface and the polar head toward the solution. A surprising result of this study is that DeTATf displays adsorption characteristics of a zwitterionic rather than a cationic surfactant. This behavior indicates that the adsorbed species is an ion pair.

Corrosion behavior of titanium in the presence of calcium phosphate and serum proteins.

The effect of calcium phosphate surface deposit and the surface adsorption of the serum proteins, bovine serum albumin (BSA) and fibrinogen, on the corrosion resistance and electrochemical behavior of (cp)titanium in phosphate buffer saline solution (pH 7.4) was investigated at physiological temperature, 37 degrees C, using electrochemical impedance spectroscopy and dc electrochemical polarization techniques. The formation of calcium phosphate deposit on the Ti surface decreased both the corrosion rate at the open circuit potential (OCP) and the anodic reaction current in the high anodic potential range (>2.6 V). Addition of BSA significantly moved the OCP towards a more negative (cathodic) potential and inhibited the cathodic corrosion reaction, but did not significantly change the corrosion resistance at the OCP. Addition of fibrinogen showed a similar, but less pronounced effect than BSA. The possible mechanisms leading to these observed effects are discussed.

Electrochemical quartz crystal nanobalance to detect solvent displacement by pH-induced conformational changes of proteins at Pt.

The electrochemical quartz crystal nanobalance (EQCN) techniques of simultaneous measurements of frequency and cyclic voltammetry (CV) were used to investigate protein adsorption behavior resulting from pH-induced conformational changes at the Pt electrode at 298 K. The adsorption behavior of holo- and apo-alpha-lactalbumin was studied in electrolyte solutions of pH < 2, 7.4, and 11. The EQCN frequency measurements did not directly monitor the mass of the adsorbed protein at anodic potentials, but instead, at a potential characteristic of the double layer for platinum, gave a measure of the extent of solvent displacement by the adsorbed protein (i.e., a "footprint"), which correlated well with known pH-induced conformational changes of the protein. Simultaneous CV charge transfer measurements provided information on the number of layers of protein adsorbed to the surface. This ability of the EQCN to detect solvent displacement by protein adsorption is potentially useful for biosensors to detect and to monitor protein conformational changes in the bulk and during the adsorption process. The Langmuir adsorption isotherm provided the Gibbs energy of adsorption, DeltaG(ADS), and showed excellent agreement between the CV and EQCN frequency measurements.

Electrochemical quartz crystal nanobalance study of the adsorption/displacement phenomena of proteins and lipids on Pt.

The electrochemical quartz crystal nanobalance (EQCN) was used to measure the adsorption behavior of a series of lipids (stearate, oleate, linoleate, and gamma-linolenate) on a Pt surface from a phosphate buffer pH 7.0 solution at 295 K and to investigate their adsorption/displacement behavior with the proteins, beta-lactoglobulin and alpha-lactalbumin, which are known to cause fouling during milk processing. The EQCN technique and the complementary technique of cyclic voltammetry measured simultaneously provided information on the efficiency of solubilization of the proteins by these lipids. Excellent agreement was obtained for the surface concentration of adsorbed lipid from the surface charge density from cyclic voltammetry measurements and the change in mass from the EQCN frequency measurements. The Gibbs energy of adsorption showed the lipids to have a strong affinity for the platinum surface. Addition of protein to a preadsorbed lipid layer showed alpha-lactalbumin to be able to coadsorb with the lipids, while beta-lactoglobulin was able to desorb some of the unsaturated lipids but appeared to coadsorb with the saturated lipid, stearate. Addition of lipid to a preadsorbed protein layer showed the unsaturated lipids to be able to displace some of the protein. A comparison of the desorption ability of the lipids showed stearate to be very inefficient at removing protein, while the other three lipids were able to remove each of the proteins, with the order of efficiency for protein desorption being oleate > linoleate > gamma-linolenate.

Electrochemically assisted co-precipitation of protein with calcium phosphate coatings on titanium alloy.

A bovine serum albumin protein-containing calcium phosphate coating (BSA/brushite) was prepared by electrochemically assisted co-precipitation onto a hydroxyapatite (HA) coated Ti-6Al-4V surface. Electrochemically assisted co-precipitation of BSA/brushite coatings onto HA resulted in a 70-fold increase in BSA inclusion compared to simple adsorption, and was subsequently released by a slower mechanism (15% loss over 70 h). Thus, this electrochemically assisted co-precipitation technique provides an efficient method of protein incorporation at physiological temperature, with a potential for sustained release of therapeutic agents as may be required for metallic implant fixation.