Controlling protein transport in ultrafiltration using small charged ligands

Previous studies have demonstrated that protein transport during ultrafiltration can be strongly influenced by solution pH and ionic strength. The objective of this study was to examine the possibility of controlling protein transmission using a small, highly charged ligand that selectively binds to the protein of interest. Experiments were performed using bovine serum albumin and the dye Cibacron Blue. Protein sieving data were obtained with essentially neutral and negatively charged versions of a composite regenerated cellulose membrane to examine the effects of electrostatic interactions. The addition of only 1 g/L of Cibacron Blue to an 8 g/L BSA solution reduced the BSA sieving coefficient through the negatively-charged membrane by more than two orders of magnitude, with this effect being largely eliminated at high salt and with the neutral membrane. Protein sieving data were in good agreement with model calculations based on the partitioning of a charged sphere in a charged pore accounting for the change in net protein charge due to ligand binding and the increase in solution ionic strength due to the free ligand in solution.     

A Multidimensional Approach to Protein Characterization

When mass spectrometry (MS) is used to study protein primary structure, it is used in a static mode. That is, the information is derived from a single MS or MS-MS spectrum. Information about more complex protein structure or protein interactions can also be gained via MS. If a series of mass spectra is collected as something else in the experiment is changing, we increase the dimensionality of the MS data. For example, measuring mass spectra as a function of time after exposure of a protein to deuterated solvents can provide information about protein structure. Likewise, by measuring mass spectra of a protein as the concentration of a binding ligand is changed, one can infer the stoichiometry of the complex. Another important, but fundamentally different way of increasing the dimensionality of mass spectral data is by coupling the mass spectrometer to a one- or two-dimensional separation technique.     

Evaluation of π-acid chiral stationary phases deriving from tyrosine and related amino acids for the chromatographic resolution of racemates: Specific requirements for enantiorecognition ability

Chromatographic applications of three novel chiral stationary phases (CSPs) deriving from (S)-(N)-(3,5-dinitrobenzoyl)tyrosine are reported, under liquid chromatographic (LC) and subscritical fluid chromatographic (SubFC) conditions. Two grafting modes of the chiral moiety have been experimented starting either from γ-mercaptopropyl-silanized (type 1) or γ-aminopropyl-silanized (type 2) silica gels. For type 2 CSPs an evaluation of the stability of the amide linkage was achieved by means of SubFC; the relative contriution of ionic and covalent bindings to the ciral recognitio aility was then outlined. The chromatographic properties of these CSPs were compared with those of the corresponding CSPs deriving from phenylglycine, p-hydroxyphenylglycine, and phenylalanine for the resolution of some tertiary phosphine oxide, naphthoyl amide, and α-methylene γ-lactam enantiomers. Some simple requirements regarding the solute and CSP structures for chiral recognition ability can be inferred from these results. In addition, the resolutio of π-acid α-N-(3,5-dinitrobenzoyl)amino esters was investigated on these π-acid CSPs. An example of preparative scale chromatography is also presented.     

The stereochemical resolution of enantiomeric free and derivatized amino acids using an HPLC chiral stationary phase based on immobilized alpha-chymotrypsin: chiral separation due to solute structure or enzyme activity

The stereochemical separation of free and derivatized amino acids on active alpha-chymotrypsin bonded to silica is governed by two mechanisms based on the structure of the solutes or on the enzymatic activity of the enzyme. The deactivation of the hydrolytically active site of the enzyme demonstrated that a significant portion of the retention on this support is due to hydrophobic interactions at other sites. These sites appear to be stereoselective for the ester derivatives of amino acids but not for the other studied solutes.     

Protein chromatography in neat organic solvents

Pure formamide and ethylene glycol are used instead of water as processing media for protein chromatography. A number of common proteins are freely soluble in these solvents and most do not undergo irrersible inactivation in them. Batch adsorption studies reveal that proteins readily adsorbed to various ion-exchangers in formamide and ethyline glycol and subsequently can be completely desorbed by adding inorganic salts (LiCl and NH(4)NO(3)) to the solvents. The idea of protein separations in formamide and ethylene glycol is illustrated by column chromatography and preparative separation of mixtures of (i) oxidized A and B chains of insulin and (ii) lysozyme and ribonuclease on the anion-exchanger triethylaminoethycellulose and the cation-exchanger phosphocellulose, respectively.     

Evaluation of the Edman degradation product of vancomycin bonded to core‐shell particles as a new HPLC chiral stationary phase

A modified macrocyclic glycopeptide‐based chiral stationary phase (CSP), prepared via Edman degradation of vancomycin, was evaluated as a chiral selector for the first time. Its applicability was compared with other macrocyclic glycopeptide‐based CSPs: TeicoShell and VancoShell. In addition, another modified macrocyclic glycopeptide‐based CSP, NicoShell, was further examined. Initial evaluation was focused on the complementary behavior with these glycopeptides. A screening procedure was used based on previous work for the enantiomeric separation of 50 chiral compounds including amino acids, pesticides, stimulants, and a variety of pharmaceuticals. Fast and efficient chiral separations resulted by using superficially porous (core‐shell) particle supports. Overall, the vancomycin Edman degradation product (EDP) resembled TeicoShell with high enantioselectivity for acidic compounds in the polar ionic mode. The simultaneous enantiomeric separation of 5 racemic profens using liquid chromatography‐mass spectrometry with EDP was performed in approximately 3 minutes. Other highlights include simultaneous liquid chromatography separations of rac‐amphetamine and rac‐methamphetamine with VancoShell, rac‐pseudoephedrine and rac‐ephedrine with NicoShell, and rac‐dichlorprop and rac‐haloxyfop with TeicoShell.     

L-谷氨酰胺生产及分离纯化研究进展

谷氨酰胺是一种条件性必需氨基酸,具有重要的生理作用,是一种极有开发前途的新药.本文对谷氨酰胺的生理作用、应用、生产及分离纯化工艺进行了综述.     

Supramolecular dynamics of thalidomide and its derivatives in water‐sediment system

The contamination of drug residues, including chiral ones, is not acceptable in earth's ecosystem. The dynamicity of enantiomers of thalidomide and its derivatives (3‐methyl thalidomide, 3‐ethyl thalidomide, and 3‐butyl thalidomide) was ascertained at supramolecular level in water‐sediment system using solid phase extraction (SPE) and stereoselective HPLC. Enantiomeric separation of these drugs was carried out on Ceramosphere RU‐2 (25 cm × 0.46 cm, particle size 50 μm) chiral column using pure ethanol (1.0 ml/min) as eluent at 230 nm detection. Retention times, capacity, separation, and resolution factors of the enantiomers of these drugs were in the range of 20.0–36.0, 2.08–3.93, 1.35–1.57, and 1.0–2.0 min, respectively. Percentage recoveries of the enantiomers in SPE were in the range of 90.0 to 95.0 in water‐sediment system. Langmuir and Freundlich model were best fitted for dynamic equilibrium concentrations at different experimental parameters. Thalidomide and its derivatives follow first‐order kinetics at dynamic equilibrium. The rate constants of chiral interconversions were 0.390 and 0.385 days^?1 for S‐ and R‐enantiomers, respectively. The uptake of thalidomide by sediment is quite good and of endothermic nature indicating good self‐purification capacity of the nature for such toxic species. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Principles of membrane stability and phase behavior under extreme conditions

Biological membranes consist of a complex assortment of lipids and proteins. The arrangement of the components, particularly in regard to their lateral disposition in the plane of the membrane under physiological conditions, is dependent on the phase behavior of the different membrane lipids and the way that this behavior is modified by interaction with other membrane components and electrolytes in the aqueous medium. Irreversible phase separation of components within the membrane may result from exposure to extreme environmental conditions including temperature, pressure, or electrolyte concentration. The principles underlying the phase-mixing behavior of model membrane systems can be used to provide useful information about the factors that determine the stability of biomembranes under physiological and non-physiological conditions. These data are reviewed and used to predict events that take place when membranes are exposed to environmental stress.