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81.
A method for fast in situ measurement of adsorption kinetics based on a finite bath was developed. We modified the conventional finite bath by replacing the external loop by a dip probe which enables in situ measurement of the concentration change in the contactor. Deposition of adsorbent particles on the reflection surface of the dip probe compromised measurements. Different membranes, a polyamide, a polypropylene and a nylon membrane were tested to protect the internal reflection surface of the dip probe from fouling with adsorbent particles. The nylon membrane provided efficient protection and high mass transfer evaluated by response time experiments. Unspecific adsorption of the model protein on the membrane could also be excluded. To corroborate the measurements of the dip probe the results were compared to a conventional finite bath and to a shallow-bed. The uptake curves for human polyclonal IgG at different concentrationes (0.1-3 g/l) on rProtein A Sepharose FF and MabSelect were used as model system. The effective diffusion coefficients were determined using a pore diffusion model. These values were in good agreement for all methods. 相似文献
82.
Rudnev AV Aleksenko SS Semenova O Hartinger CG Timerbaev AR Keppler BK 《Journal of separation science》2005,28(2):121-127
A CE method has been developed to evidence and quantitatively characterize the interaction between platinum-based antitumor drugs and human serum proteins. This method is a variant of affinity CE modified regarding both experimental setup and data treatment so as to measure the peaks (or vacancies) that correspond to the bound drug when it slowly binds to the protein. Using the formalism of the Hummel-Dreyer method and cisplatin and oxaliplatin as test compounds, a protocol for determining albumin and transferrin binding constants and stoichiometries, including (and distinguished by) 48 hours of incubation of the reaction mixture, was elaborated. Relative affinities of drugs toward different proteins in aqueous solution at physiological pH, chloride concentration, and temperature were compared in terms of overall binding constants and numbers of drug molecules attached to the protein. The results indicate that both platinum drugs bind to albumin more strongly than to transferrin, supporting the concept that the albumin fraction is a major drug supply route for chemotherapeutical needs. From a comparison with the binding parameters measured previously for cisplatin by other methods, conclusions were drawn about the validity of CE as a simple and convenient method for assaying protein-drug reactions with slow kinetics. 相似文献
83.
Interfacial layers have been widely applied to study the formation and stability of emulsion-based systems. However, the application of isolated interfaces to address digestibility of emulsions is often limited because of the complexity of experimental methods and results. This review summarizes the latest developments in analytical methods and literature data on effects of digestion on interfacial layers. Particular emphasis is given to understand the changes on interfacial magnitudes during oral, gastric, and duodenal digestion, either applied separately or sequentially. Limitations of interfacial aspects and key factors that influence emulsion microstructure in bulk and lipid digestion are identified. Understanding the behavior of interfacial layers upon gastrointestinal digestion promotes an accurate tracking of the physiological fate of emulsions. 相似文献
84.
Scoring functions: A view from the bench 总被引:2,自引:0,他引:2
Tame JR 《Journal of computer-aided molecular design》1999,13(2):99-108
Computational approaches to drug design are presently hindered by the complexity of the physical chemistry which underlies weak, non- covalent interactions between protein targets and small molecule ligands. Although a number of programs are now available for the design of novel potential ligands, it remains a key problem to rank these rapidly and reliably by estimated binding affinity. Such a step is necessary to select only the most promising candidates for synthesis and experimental characterisation. To calculate ligand affinity quickly and reliably is an extremely difficult problem, but it may well prove possible to estimate sufficiently accurately given an appropriate set of parameters to score individual protein–ligand interactions. Improvements in the situation will require a wider set of thermodynamically characterised systems than is currently available. 相似文献
85.
Deo SK 《Analytical and bioanalytical chemistry》2004,379(3):383-390
Reassembly of protein from its peptide fragments is a technique that can have many applications in the bioanalytical field. Typically, a reporter protein fragmented into its two peptides is employed as a label in this study. This fragments of peptide can reassemble yielding an active functional reporter. This reassembly of the protein can be assisted by non-covalently interacting peptides or proteins, which are attached to the fragmented reporter. This technique has been employed in several applications including study of protein–protein interactions, antibody screening, immunoassays, and high-throughput screening. This review focuses on different reporters employed in the study of reassembly of proteins and applications of this strategy in bioanalysis. 相似文献
86.
We adapted the genetic algorithm to minimize the AMBER potential energy function. We describe specific recombination and mutation operators for this task. Next we use our algorithm to locate low energy conformation of three polypeptides (AGAGAGAGA, A9, and [Met]-enkephalin) which are probably the global minimum conformations. Our potential energy minima are –94.71, –98.50, and –48.94 kcal/mol respectively. Next, we applied our algorithm to the 46 amino acid protein crambin and located a non-native conformation which had an AMBER potential energy 150 kcal/mol lower than the native conformation. This is not necessarily the global minimum conformation, but it does illustrate problems with the AMBER potential energy function. We believe this occurred because the AMBER potential energy function does not account for hydration. 相似文献
87.
C(alpha)-Formylglycine, the catalytic amino acid residue in the active site of sulfatases, is generated by post-translational modification of a cysteine or serine residue. We describe a highly sensitive procedure for the detection of C(alpha)-formylglycine-containing peptides in tryptic digests of sulfatase proteins. The protocol is based on the formation of hydrazone derivatives of C(alpha)-formylglycine-containing peptides when using dinitrophenylhydrazine as a matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The hydrazone derivatives desorb and ionize with high efficiency and can be detected in the sub-femtomole range. The presence of C(alpha)-formylglycine is indicated by a mass increment of 180.13 u, corresponding to the hydrazone moiety, and also by a unique C-terminal fragment ion, characteristic of sulfatases, that becomes prominent in MALDI post-source decay mass spectra of the hydrazone derivatives. 相似文献
88.
We have developed a process that significantly reduces the number of rotamers in computational protein design calculations. This process, which we call Vegas, results in dramatic computational performance increases when used with algorithms based on the dead-end elimination (DEE) theorem. Vegas estimates the energy of each rotamer at each position by fixing each rotamer in turn and utilizing various search algorithms to optimize the remaining positions. Algorithms used for this context specific optimization can include Monte Carlo, self-consistent mean field, and the evaluation of an expression that generates a lower bound energy for the fixed rotamer. Rotamers with energies above a user-defined cutoff value are eliminated. We found that using Vegas to preprocess rotamers significantly reduced the calculation time of subsequent DEE-based algorithms while retaining the global minimum energy conformation. For a full boundary design of a 51 amino acid fragment of engrailed homeodomain, the total calculation time was reduced by 12-fold. 相似文献
89.
Thereza Christina Vessoni Penna Marina Ishii Adalberto Pessoa Junior Laura de Oliveira Nascimento Luciana Cambricoli de Souza Olivia Cholewa 《Applied biochemistry and biotechnology》2004,114(1-3):453-468
To determine the influence of various culture conditions, transformed cells of Escherichia coli expressing recombinant green fluorescent protein (GFPuv) were grown in nine cultures with four variable conditions (storage
of inoculated broth at 4°C prior to incubation, agitation speed, isopropyl-β-d-thiogalactopyranoside [IPTG] concentration, and induction time). The pelleted cells were resuspended in extraction buffer
and subjected to the three-phase partitioning (TPP) extraction method. To determine the most appropriate purification resin,
protein extracts were eluted through one of four types of HiTrap hydrophobic interaction chromatography (HIC) columns prepacked
with methyl, butyl, octyl, or phenyl resins and analyzed further on a 12% sodium dodecylsulfatepolyacrylamidegel. With Coomassie
staining, a single band between 27 (standard GFPuv) and 29 kDa (molecular weight standard) was visualized for every HIC column
sample. TPP extraction with HIC elution provided about 90% of the GFPuv recovered and eight-fold GFPuv enrichment related
to the specific mass. Rotary speed and IPTG concentration showed, respectively, greater negative and positive influences on
GFPuv expression at the beginning of the logarithmic phase for the set culture conditions (37°C, 24-h incubation). 相似文献
90.
In this study we describe the activation with chloroformates of Trisacryl-GF-2000, a new synthetic gel support that is stable,
hydrophilic, and contains large amounts of hydroxyl groups available for activation.
Of all the reagents tested, the activation withN-hydroxysuccinimide-chloroformate andp-nitrophenylchloroformate in organic solvents provides the best activation yield and subsequent coupling. When Trisacryl was
activated in acetone with the chloroformates in the presence of 4-dimethylaminopyridine as base and catalyst, up to 30% of
the hydroxyl groups, (i.e., 1/repeating unit) could be activated. Amino-containing ligands and proteins could be coupled to
these carriers at pH 8 or higher. For better results in affinitychromatographic applications, spacers of ε-amino caproic acid
or diaminohexane were introduced. The efficacy of these columns was demonstrated by purification of enzymes, antibodies, and
antigens. The performance of these new columns were compared with that of Sepharose columns activated in various ways. In
every case, the properties of the Trisacryl support proved superior with particular reference to the purity of the product
obtained. 相似文献