ovalbumin and alpha-chymotrypsin

A novel magnetic solid-phase extraction (MSPE) method based on 1-hexyl-3-methyl imidazolium chloride ionic liquid (IL) modified magnetic Fe

Topics: Animals; Cattle; Chymotrypsin; Complex Mixtures; Enzyme Assays; Equipment Reuse; Ferrosoferric Oxide; Hemoglobins; Ionic Liquids; Magnetite Nanoparticles; Metal-Organic Frameworks; Nanotubes, Carbon; Osmolar Concentration; Ovalbumin; Pancreas; Sensitivity and Specificity; Serum Albumin, Bovine; Solid Phase Extraction; Swine; Zeolites

Refractive index matching was used to create optically transparent polyaphrons to enable proteins adsorbed to the aphron surface to be characterized. Due to the significant light scattering created by polyaphrons, refractive index matching allowed for representative circular dichroism (CD) spectra and acceptable structural characterization. The method utilized n-hexane as the solvent phase, a mixture of glycerol and phosphate buffer (30% [w/v]) as the aqueous phase, and the non-ionic surfactants, Laureth-4 and Kolliphor P-188. Deconvolution of CD spectra revealed that the immobilized protein adapted its native conformation, showing that the adsorbed protein interacted only with the bound water layer ("soapy shell") of the aphron. Isothermal calorimetry further demonstrated that non-ionic surfactant interactions were virtually non-existent, even at the high concentrations used (5% [w/v]), proving that non-ionic surfactants can preserve protein conformation.

Topics: Animals; Buffers; Cattle; Chickens; Chymotrypsin; Circular Dichroism; Glycerol; Hexanes; Immobilized Proteins; Muramidase; Ovalbumin; Polidocanol; Polyethylene Glycols; Protein Conformation; Refractometry; Serum Albumin, Bovine; Solutions; Solvents

Despite the prevalence of active smoking in asthmatics, data on the short-term effect of acute mainstream tobacco smoke exposure on airway responsiveness are very scarce. The aim of this study was to assess the immediate effect of acute exposure to mainstream cigarette smoke on airway reactivity to subsequent nonspecific and allergenic challenges in healthy control (n = 5) and ovalbumin-sensitized rabbits (n = 6). We combined low-frequency forced oscillations and synchrotron radiation CT imaging to differentiate central airway and peripheral airway and lung parenchymal components of the response to airway provocation. Acute exposure to smoke generated by four successive cigarettes (CS) strongly inhibited the central airway response to subsequent IV methacholine (MCh) challenge. In the sensitized animals, although the response to ovalbumin was also inhibited in the central airways, mainstream CS did not blunt the peripheral airway response in this group. In additional groups of experiments, exposure to HEPA-filtered CS (n = 6) similarly inhibited the MCh response, whereas CO (10,000 ppm for 4 min, n = 6) or nitric oxide inhalation instead of CS (240 ppm, 4 x 7 min, n = 5) failed to blunt nonspecific airway responsiveness. Pretreatment with alpha-chymotrypsin to inhibit endogenous VIP before CS exposure had no effect (n = 4). Based on these observations, the gas phase of mainstream cigarette smoke may contain one or more short-term inhibitory components acting primarily on central airways and inhibiting the response to both specific and nonspecific airway provocation, but not on the lung periphery where both lung mechanical parameters, and synchrotron-imaging derived parameters, showed large changes in response to allergen challenge in sensitized animals.

Topics: Administration, Inhalation; Allergens; Animals; Carbon Monoxide; Chymotrypsin; Lung; Male; Methacholine Chloride; Ovalbumin; Rabbits; Smoking; Tomography, X-Ray Computed

Using an experimental technique recently developed in this laboratory (Fernández C. and A. P. Minton. 2008. Anal. Biochem. 381:254-257), the Rayleigh light scattering of solutions of bovine serum albumin, hen egg white ovalbumin, hen egg white ovomucoid, and binary mixtures of these three proteins was measured as a function of concentration at concentrations up to 125 g/L. The measured concentration dependence of scattering of both pure proteins and binary mixtures is accounted for nearly quantitatively by an effective hard particle model (Minton A. P. 2007. Biophys. J. 93:1321-1328) in which each protein species is represented by an equivalent hard sphere, the size of which is determined by the nature of repulsive interactions between like molecules under a given set of experimental conditions. The light scattering of solutions of chymotrypsin A was measured as a function of concentration at concentrations up to 70 g/L at pH 4.1, 5.4, and 7.2. At each pH, the measured concentration dependence is accounted for quantitatively by an effective hard particle model, according to which monomeric protein may self-associate to form an equilibrium dimer and, depending upon pH, an equilibrium pentamer or hexamer.

Topics: Animals; Cattle; Chickens; Chymotrypsin; Female; Hydrogen-Ion Concentration; Light; Models, Chemical; Ovalbumin; Ovomucin; Scattering, Radiation; Serum Albumin, Bovine

Vibrational circular dichroism (VCD) spectra in the 1800-1400 cm(-)(1) region have been measured for the first time for protein films prepared from aqueous buffer solutions. These measurements demonstrate several advantages of significant importance. First, the interference from infrared absorption of water in the amide I region, which is a serious limitation for measurements in water solutions, is eliminated. Second, the amounts of protein samples required for VCD measurements on films are approximately 2 orders of magnitude smaller than those required for the same in water solutions. In addition, the amide I absorption and VCD bands of protein films are found to be independent of film orientation. Furthermore, characteristic VCD patterns have been observed for protein films whose secondary structure is dominated by alpha-helix, beta-sheet, and alpha + beta combinations. These results demonstrate that VCD can be used to study the structure of proteins in the film state.

Topics: Animals; Chymotrypsin; Circular Dichroism; Humans; Ovalbumin; Pepsin A; Protein Structure, Secondary; Proteins; Serum Albumin, Bovine; Trypsin

A kinetic study was conducted on the effect of heating in the temperature range of 50-92 degrees C, on the susceptibility of ovalbumin and albumen solutions to enzymatic hydrolysis by a mixture of trypsin and alpha-chymotrypsin at 37 degrees C and pH 8.0. Heat treatment resulted in an increase in degree of hydrolysis after 10 min of enzymatic reaction of both ovalbumin and albumen, as measured using the pH-stat method. The time-dependent change in the susceptibility to enzymatic hydrolysis after heat treatment was described by a fractional conversion model (based on an apparent first-order reaction kinetic model). Different end levels of degree of hydrolysis were obtained after heating for a long time at different temperatures, which suggests that the final degree of unfolding of the protein is temperature dependent.

Topics: Chymotrypsin; Egg Proteins; Hot Temperature; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Ovalbumin; Protein Denaturation; Substrate Specificity; Trypsin

Lambda coli phage is not inactivated by chymotrypsin, trypsin, or ficin. T(2) phage is slowly inactivated by high concentrations of (alpha-, beta-, gamma-, or Delta-chymotrypsin, but not by trypsin or ficin. P(1) phage is slowly inactivated by alpha-, beta-, or gamma-chymotrypsin, or ficin, more rapidly by Delta-chymotrypsin, and much more rapidly by trypsin. Crystalline egg albumin, crystalline serum albumin, E. coli nucleoprotein, and yeast nucleoprotein are hydrolyzed slowly by alpha-chymotrypsin. Yeast nucleoprotein, like P(1) phage, is hydrolyzed more rapidly by Delta-chymotrypsin than by alpha-chymotrypsin, but not by trypsin or ficin. Neither phages nor native proteins were attacked by papain, carboxypeptidase, deoxyribonuclease, or ribonuclease.

Topics: Bacteriophages; Carboxypeptidases; Chymotrypsin; Coliphages; Deoxyribonucleases; DNA; Endopeptidases; Escherichia coli; Kinetics; Nucleoproteins; Ovalbumin; Papain; Peptide Hydrolases; Proteins; Research; Ribonucleases; Serum Albumin; Trypsin; Yeasts