Sharon G. Roscoe

Electrochemical oxidation reactions of tyrosine, tryptophan and related dipeptides

The electrochemical oxidation reactions of tyrosine, tryptophan, tyrosyl-glycine and tryptophyl-glycine were investigated at a platinum electrode in a phosphate buffer (pH 7.0). Cyclic voltammetry was used to examine the adsorption behaviour of these compounds. Strong adsorption was displayed by all four species at the electrode surface, and it appears that at low anodic potentials the amino acids are arranged predominantly in a horizontal position, but rearrange to a vertical configuration to allow for tighter packing at the higher potentials. Steady-state polarization measurements gave Tafel slopes in the range of 303–359 mV above the inflection point, considerably higher than those normally seen for oxygen evolution from a phosphate buffer solution. These results suggest that in the pH 7.0 buffer, the negatively charged carboxylate group preferentially adsorbs under these anodic potentials in a mechanism similar to the Kolbe reaction, with the decarboxylation step being rate determining. Cleavage of the dipeptide occurred in electrolysis experiments at potentials of 1.8–2.2 V (ESCE), as determined by HPLC product analysis. However, quantitative measurements indicated that cleavage of the dipeptide was not the predominant pathway in the anodic oxidation process.

An electrochemical study of the interfacial and conformational behaviour of cytochrome c and other heme proteins

Abstract An electrochemical investigation of the interfacial and conformational behaviour of microperoxidase and the heme proteins, cytochrome c , myoglobin and hemoglobin, has been made at the platinum electrode using cyclic voltammetry over the temperature range 273 to 363 K. Plateau values of surface charge density were obtained for increasing concentrations of protein in the bulk solution. The values were shown to increase with temperature until 333 K for cytochrome c and myoglobin, and 323 K for hemoglobin. At higher temperatures, the adsorption diminished due to denaturation and agglomeration of the protein. The absorbence measurements of the Soret band at 410 nm showed the same trends as the electrochemical results for cytochrome c and myoglobin. The absorbence of hemoglobin, however, decreased with increasing temperature from 273 K until it disappeared at temperatures above denaturation. The surface charge density was attributed to a flattening or denaturing of the protein at the electrode surface to allow adsorption of carboxylate groups accompanied by electron transfer at these anodic potentials. The number of carboxylate groups for each protein determined from a kinetic analysis agreed with the actual values and indicated that hemoglobin had dissociated into four polypeptide chains.

Electrochemical and PM-IRRAS a glycolipid-containing biomimetic membrane prepared using Langmuir-Blodgett/Langmuir-Schaefer deposition.

Differential capacitance, chronocoulometry, and polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) measurements were used to characterize the structure and orientation of a DMPC + cholesterol + GM 1 (60:30:10 mol %) bilayer supported at a Au(111) electrode surface prepared using combined Langmuir-Blodgett/Langmuir-Schaefer (LB/LS) deposition. The electrochemical measurements indicate that the incorporation of ganglioside GM 1 into the membrane significantly improves the quality of the bilayer, reflected in the very low capacitance value of approximately 0.8 microF cm (-2). An analysis of the infrared data suggests that the incorporation of the glycolipid into the membrane changes both the orientation of the lipid acyl chains in the membrane and the hydration of the membrane, particularly with respect to the interfacial region of the lipids.

Interfacial behavior of globular proteins at a platinum electrode

The surface adsorption behavior of the globular proteins, bovine β-lactoglobulin A, hen egg white lysozyme, and bovine pancreatic ribonuclease A have been investigated at a platinum electrode using cyclic voltammetry. A good correlation over the temperature range 273–343 K was obtained between the charge density due to protein adsorption and the accessibility of free sulfhydryl groups, which is an indication of the amount of conformational changes and/or dissociation of dimeric units of the protein. The surface charge density was attributed to a flattening or denaturing of the protein at the electrode surface to allow adsorption of carboxyl groups, accompanied by electron transfer at these anodic potentials. The calculated surface concentrations, Γ (mg m−2), based on the number of carboxyl groups and two-electron transfer processes/carboxyl group give good agreement with calculated values obtained by others using such techniques as radioactive labeling and ellipsometry. The present technique which measures the charge density due to adsorption of proteins at platinum electrode surfaces appears to be very sensitive to structural changes of the protein both in the bulk solution and at the metal/solution interface.

Electrochemical and FTIR studies of L-phenylalanine adsorption at the Au(111) electrode

Abstract The adsorption of l -phenylalanine (Phe) at the Au(111) electrode surface has been studied using electrochemical techniques and subtractively normalized interfacial Fourier transform infrared (SNIFTIR) techniques. The electrochemical measurements of cyclic voltammetry, differential capacity and chronocoulometry were used to determine Gibbs energies of adsorption and the reference (E1) and sample (E2) potentials to be used in the spectroscopic measurements. The vibrational spectra have been used to determine: (i) the orientation of the molecule at the surface as a function of potential; (ii) the dependence of the band intensity on the surface coverage; (iii) the character of surface coordination, and (iv) the oxidation of adsorbed Phe molecules at positive potentials. The adsorption of Phe is characterized by ΔG values ranging from −18 to −37 kJ mol−1 that are characteristic for a weak chemisorption of small aromatic molecules. The electrochemical and SNIFTIR measurements indicated that adsorbed Phe molecules change orientation as a function of applied potential. At the negatively charged surface Phe is predominantly adsorbed in the neutral form of the amino acid. At potentials positive to the pzc, adsorption occurs predominantly in the zwitterionic form with the COO− group directed towards the surface and the ammonium group towards the solution. At more positive potentials electrocatalytic oxidation of Phe occurs and is marked by the appearance of the CO2 asymmetric stretch band in the FTIR spectrum. Thus, relative to pzc, Phe is weakly chemisorbed at negative potentials, changes orientation at potentials close to the pzc and is oxidized at positive potentials.

FTIR studies of benzoate adsorption on the Au(111) electrode

Abstract Subtractively normalized interfacial Fourier transform infrared spectroscopy (SNIFTIRS) has been used to examine (i) the orientation and coordination of benzoate on the Au(111) electrode; (ii) the dependence of the IR band intensity on the surface coverage; and (iii) the chemical stability of benzoate at the Au(111) surface. The results indicate that orientation of adsorbed benzoate changes from a nearly flat co-ordination at a negatively charged surface to a tilted position at positive charge densities. The transition in the orientation of the adsorbed molecule occurs in the vicinity of the potential of zero charge in the solution investigated. The integrated IR band intensities correlate very well with the surface concentration of benzoate obtained from chronocoulometric measurements. An overall agreement between the spectroscopic and electrochemical results is excellent. The spectroscopic data demonstrate that no decomposition or oxidation of benzoate occurred at the Au(111) electrode surface over the potential range investigated.

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.

Surface electrochemistry of the oxidation reactions of α- and β-alanine at a platinum electrode

Abstract The electrochemical oxidation reactions of α- and β-alanine at a Pt electrode were investigated in aqueous solutions at pH 1, 7, and 13 using steady-state current-potential measurements, cyclic voltammetry, and open circuit potential decay. The capacitance behaviour and the high Tafel slopes suggest the production of free radicals at the surface of the electrode accompanied by a second reaction involving loss of CO 2 which is the rate determining step. In the surface electro-oxidation of α-alanine, it appears that the adsorbed intermediate species is either hydrolyzed anodically to acetaldehyde and ammonia, or is oxidized to a carbonium ion which is subsequently hydrolyzed to acetaldehyde and ammonia in solution, analogous to the behaviour observed for glycine [D.G. Marangoni, R.S. Smith and S.G. Roscoe, Can. J. Chem., 67 (1989) 921]. The mechanisms for β-alanine would be similar except carbonium ion formation would probably be accompanied by a hydride transfer to form acetaldehyde. No dimerized products were detected by gas chromatography. These mechanisms differ from the dimerization process typical of the radical reactions associated with the Kolbe mechanism.

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.

Electrochemical studies of the interfacial behaviour of α-lactalbumin and bovine serum albumin

Abstract Knowledge of the conformational behaviour of proteins as a function of temperature is important in the thermal processing of foods. Equally important is their interfacial behaviour with surfaces under these same processing conditions. This paper describes electrochemical techniques developed to study these structurally related properties of proteins. The interfacial behaviour of α-lactalbumin and bovine serum albumin was studied on a platinum electrode using cyclic voltammetry over a temperature range from 273 to 363 K. It was found that both proteins adsorbed strongly on the metal surface. As the temperature was increased up to the denaturation temperature of the protein, the surface charge density for protein adsorption also increased due to the conformational unfolding. Above the temperature for denaturation, the surface charge density continued to increase. Mixtures of the two proteins resulted in a substantial increase in surface charge density above the denaturation temperatures. A comparison of surface concentrations (Γ) of cyclic voltammetric measurements with those for ellipsometric measurements reported in the literature showed a similar behaviour with increasing adsorption with temperature. A value of 17 ± 4 carboxylate groups for the α-lactalbumin protein was determined from an analysis of the surface coverage which is consistent with the number of acidic residues on the protein.