Donald G. Cornell

Circular dichroism of polypeptide monolayers

Abstract The circular dichroism (CD) spectra of Langmuir—Blodgett monolayers and collapsed films of poly-γ-methyl- l -glutamate (PMG) and poly- l -alanine (PA) collected from the air-water interface of a Langmuir film balance have been determined. The polypeptide films were formed by spreading from a variety of solvents, followed by transfer to quartz plates for CD spectrophotometry. When all plates were aligned identically in the light beam of the spectropolarimeter, films of PMG and PA exhibited linear dichroism which could be used to determine the orientation of the polymer chains on the surface of the film balance. True CD spectra, free of linear component, were obtained on films oriented at numerous angles around the axis of the incident light beam. The CD spectra of films of PA and PMG in the α-helical conformation were consistent with the optical properties expected of polypeptides which have the helix axis perpendicular to the direction of propagation of light. PMG films gave spectra characteristic of the α-helix or β-conformation, depending on the spreading solvent used to form the monolayers. The pressure-area and surface potential curves, as well as the interpretation of the CD spectra of PMG and PA films, were all consistent with the results of previous investigations employing infrared spectroscopy. Details of preparing and mounting polypeptide monolayers for CD spectroscopy are given.

Lipid—protein interactions in monolayers: Egg yolk phosphatidic acid and β-lactoglobulin

Abstract The interaction of β-lactoglobulin (β-1g) with phospholipids in monolayers was studied with a Langmuir film balance. Pressure—area curves of monolayers of β-1g mixed with either egg yolk phosphatidic acid (EYPA) or egg yolk phosphatidylcholine (EYPC) on pH 1.3 to 6 subphases were compared to curves calculated from the results obtained with the individual components. EYPA and β-1g exhibited electrostatic attaction for each other in mixed monlayers at the air—water interface when the pH of the subphase was 4 or 1.3. No interaction was observed on pH 6 subphase. Monolayers of egg yolk phosphatidylcholine-β-1g mixtures exhibited no interaction over the pH range studied. Calcium at 1 m M concentration in the subphase removed the evidence for interaction between EYPA and β-1g at pH 4, but not at pH 1.3. Circular dichroism spectroscopy suggested an increased amount of α helix and β sheet in the films as compared to solutions of β-1g.

Miscibility in lipid—protein monolayers

Abstract Lipid—protein monolayers of various lipids mixed with either β-lactoglobulin, bovine serum albumin, or β-casein were transferred from the air-water interface to mica substrates and examined by electron microscopy. Phospholipids and triglycerides which gave condensed films at the air—water interface did not mix with proteins in monolayers as shown by electron micrographs of the transferred films. Heterogeneous films were observed with as little as 5 mole (residue) percent phosphatidic acid or tristearin in the mixed films, suggesting little, if any, solubility of condensed lipids in protein before the onset of phase separation. Homogeneous lipid—protein films were observed when the mixed monolayers were prepared from phospholipids or triglycerides that exhibit expanded behavior at the air—water interface. Fatty acids did not always follow this behavior pattern. Stearic acid, which forms a condensed film at the air—water interface, did not separate from the protein in a mixed film until the lipid content exceeded 10–15 mole (residue) percent. Nervonic acid, which exhibits condensed behavior at high pressure and room temperature, also did not separate from protein in mixed films under these conditions. Film balance experiments suggest that lipid—protein interactions may be hindering segregation of nervonic acid from proteins in monolayers.

Microemulsions of triglyceride and non-ionic surfactant — effect of temperature and aqueous phase composition☆

Abstract The phase behavior of soybean oil, polyoxyethylene (40) sorbitol hexaoleate and water—ethanol was investigated. Regions of water-in-oil (W/O) microemulsions were determined and were found to be strongly dependent on temperature and water:alcohol ratios. At a water:ethanol ratio of 80/20 (wt.%), an oil:surfactant ratio of 2/3 and a temperature of 25°C, the microemulsion region extended continuously from the oil—surfactant axis to the phase diagram center. However, at the hydrophilic—lipophilic balance (HLB) temperature (20–22°C) and a water:ethanol ratio of 80/20 or 75/25 (wt.%), a single-phase area separated from the original microemulsion region. Conductivity measurements and dynamic light scattering intensifies at 25°C indicated that association structures were formed with increasing aqueous phase concentrations above 15 wt.%. At 20°C, the single-phase scattering intensifies increased sharply with increasing aqueous phase concentrations (38–46 wt.%) and a plateau in the conductivity was detected. Transmission electron microscopy results supported the finding that more particles are formed with increasing aqueous phase and form connected particles, resulting in constant conductance.

The interaction of phospholipids in monolayers with bovine serum albumin and α-lactalbumin adsorbed from solution

Abstract The interaction of phospholipids in monolayers with bovine serum albumin (BSA) and bovine α-lactalbumin (ALA) adsorbed from solution has been studied by ultraviolet and circular dichroism spectroscopy of films transferred from the air—water interface of a Langmuir film balance. The lipid monolayers were mixtures of palmitoyloleoylphosphatidylcholine (POPC) and palmitoyloleoylphosphatidylglycerol (POPG). The subphase pHs studied (4.4–7.0) covered the range of acidities found in milk and other dairy products. Both BSA and ALA exhibited considerable adsorption to monolayers of POPC POPG ( 65 35 mol%) at pH 4.4, but only a small amount of either protein was detected in the mixed films when the subphase pH was 7. At a subphase pH of 4.4, a 50% reduction in BSA—lipid binding was observed with about 0.5 mM calcium in the subphase, whereas less than 25 μM calcium was required when the subphase pH was 5.2. At pH 4.4, reduction of ALA—lipid binding by 50% required only 25 μM calcium. The results with BSA suggest an electrostatic mechanism for lipid—protein interaction where the protein is tightly bound to the lipid at pHs below the isoionic point of the protein while the lipid—protein interaction is weaker at the isoionic pH and essentially negligible at pHs above the isoionic point. The results with ALA are consistent with this mechanism.

Ultraviolet spectroscopy of β-lactoglobulin films

Abstract β-Lactoglobulin transferred from a film balance to the surface of quartz plates exhibits a molar (residue) absorptivity of 11,950 liters mole −1 cm −1 at 193–194 nm compared to 9,200 liters mole −1 cm −1 observed with isotropic solutions of the labeled protein and 9,600 liters mole −1 cm −1 observed with solutions of unlabeled protein. It is proposed that the main factor responsible for the enhanced absorptivity is the effect of orienting a planar sample in the light beam of the spectrophotometer. Solvent effects and changes in the conformation of the protein are unable to account for the increased absorptivity observed in films.

Electron microscopy of lipid—protein monolayers

Abstract Mixtures of phospholipids with β-lactoglobulin (β-lg) were spread from acidic chloroform: methanol onto the surface of a film balance, then transferred to freshly cleaved mica at 15 mN/m for examination under an electron microscope. Dipalmitoylphosphatidylcholine and dipalmitoylphosphatidic acid did not mix with β-lg and were visible as separate phases in the electron micrographs of the transferred films. Mixed monolayers containing either unsaturated lipids or dimyristoylphosphatidylcholine mixed with β-lg gave completely homogeneous micrographs with no evidence of phase separation. The results suggest that phase separation occurs in monolayers of phospholipid-β-lg when the films are prepared under conditions where the pure lipid exhibits liquid condensed behavior at the aim—water interface. Homogeneous lipid—protein films apparently result when the monolayers are prepared under conditions where the lipid exhibits expanded behavior.

NMR relaxation measurements of sucrose in aqueous ethanol solutions

Abstract The molecular motion of sucrose in water/alcohol/sucrose/casein solutions was studied by 13 C-NMR spin-lattice relaxation measurements. The results suggest that the conformation of sucrose is unaffected by concentration, temperature, or the presence of alcohol or casein. There is no evidence for the interaction of sucrose with either alcohol or protein. Changes in the hydrodynamic radius of sucrose from relaxation measurement using solution or water viscosities were inconclusive.

Cobaltothiocyanate colorimetric analysis for homologous polyoxyethylated alkyl amides

Cobaltothiocyanate colorimetry has been used for quantitative analysis of polyethoxylates in biodegradation and environmental studies. Usually, the calibration parameters (slope and intercept of a calibration line) for the cobaltothiocyanate colorimetry are dependent on ethylene oxide chain length. To make the colorimetric method useful for polyoxyethylated surfactant mixtures, where the average chain length is polydispersed, a condition was found yielding parameters independent of chain length in a homologous series of polyethoxylates within a limited chain length range. The dependency can be correlated to the thermodynamics of the ethylene oxide-cobaltothiocyanate complexation and the solvent extraction.