Journal of biochemical and biophysical methods最新文献

A new assay for biotin binding capacity of Streptavidin (SA) is presented in this work. The assay is based on the large decrease in the extinction coefficient at 493 nm that accompanies binding of biotin-4-fluorescein (B4F) to SA. This decrease is attributed to formation of a charge transfer complex between the B4F-donor and one or more SA residues. We show that one may observe the stoichiometric binding via monitoring the absorbance at 493 nm using either SA or B4F as the titrant. The sensitivity of the assay is at the lower end of similar fluorimetric and photometric assays. Though the sensitivity is not substantially lower than other comparable techniques, this assay allows one added flexibility in working range and instrumentation, since the same stock solutions may be used for this new photometric assay or the fluorescence assay for which this ligand was first developed.

The use of inorganic membranes in pollution treatment is actually limited by the cost of such membranes. Advantages of inorganic membranes are their chemical, thermal and pH properties. The purpose of this work was the development of microporous ceramic materials based on clay for liquid waste processing. The supports or ceramic filters having various compositions were prepared and thermally treated at 1100 °C. The results show that, at the temperature studied, porosity varied according to the support composition from 12% for the double-layered (ceramic) support to 47% for the activated carbon- filled support with a mean pore diameter between 0.8 and 1.3 μm, respectively. Volumes of 5 l of distilled water were filtered tangentially for 3 h under an applied pressure of 3.5 and 5.5 bar. The retention of tubular supports prepared was tested with molecules of varying size (Evans blue, NaCl and Sacharose). The study of the liquid filtration and flow through these supports showed that the retention rate depends on support composition and pore diameter, and solute molecular weight. The S1 support (mixture of barbotine and 1% (w/w) activated carbon) gave a flux for distilled water of 68 L/m² h while the double-layered support resulted in a flux of 8 L/m² h for the same solution at the pressure of 3.5 bar. At a pressure of 5.5 bar an increase in the distilled water flux through the various supports was observed. It was significant for the S1 support (230 L/m h).

A poly-o-phenylenediamine and multi-wall carbon nanotubes composite (PoPD/MWNTs) modified glassy carbon electrode (GCE) was prepared by in situ electropolymerization using an ionic surfactant as the supporting electrolyte. The morphology of the resulting PoPD/MWNTs composite was characterized by TEM and the electrochemical properties of the modified electrode were characterized by cyclic voltammetry. The electrochemical behavior of calcium dobesilate on PoPD/MWNTs modified electrode was also investigated. The large current response of calcium dobesilate on PoPD/MWNTs modified electrode is probably caused by the synergistic effect of the electrocatalytic property of PoPD and MWNTs. The reductive peak current increased linearly with the concentration of calcium dobesilate in the range of 0.1–1.0 μmol/L and 4.0–400 μmol/L by square wave adsorptive stripping voltammetry, respectively. The detection limit (three times the signal blank/slope) was 0.035 μmol/L. The modified electrode could eliminate the interference of dopamine, norepinephrine and epinephrine at 100-, 90- and 70-fold concentration of 1.0 μmol/L calcium dobesilate, respectively. The proposed modified electrode provides a new promising and alternative way to detect calcium dobesilate.

Most of the β-blocking drugs for treating diseases of the cardiovascular system are chiral aryloxy–propanolamine derivatives. Tipically, the S(−) enantiomers are more active than the R(+) enantiomers. Only some of them (for example timolol) are used as single enantiomers, the others are employed as racemates. For the determination of the enantiomeric purity of timolol European Pharmacopoeia prescribes an HPLC method using chiral stationary phase. However, the use of chiral capillary zone electrophoresis for the determination of the enantiomeric purity is of pharmaceutical interest. This study describes the application of various cyclodextrin derivatives, hydroxypropyl-β-cyclodextrin, randomly methylated β-cyclodextrin, sulphated β-cyclodextrin and sulphated α-cyclodextrin for the stereoselective analyses of β-blockers. Baseline separation was obtained for bopindolol, carvedilol, mepindolol, pindolol and alprenolol, while only partial separation was observed for sotalol, propranolol, oxprenolol, atenolol, bisoprolol, bupranolol, and metoprolol. The uneven molecular recognition of the enantiomers of the β-blockers, especially of the optical isomers of labetalol and nadolol, showed the importance of the chemical nature of the separators and the analytes.

The UV-absorption, fluorescence and CD spectra of aps 23 bp oligoduplexes were performed for potential diagnostic purpose. These oligonucleotide sequences were mimicked from natural mutations (mitochondrial genome) of human population (unpublished). This work was designed on the basis of hybridization of non-self complementary oligoduplexes (aps) containing no mismatch, one-mismatch and two-mismatches. Since melting temperature™ is dependent on concentration of the oligoduplex, various concentrations were used in this study protocol. The thermal spectra profiles (UV absorbance and fluorescence) of these oligoduplexes (aps) are different for a particular concentration, and can be implicated for mutations. − dF/dT (or dA/dT) vs T, lnK (or RlnK) vs T_M, ΔG vs T_M, ΔS vs T_M and ΔH vs T_M are also variable for those sequences. All these thermodynamic data were calculated from absorbance (at 260 nm) data. On the contrary to the 23 bp oligoduplexes (aps), the PCR products of 97 bp and 256 bp length were genotyped with ETBR (excitation 530 nm, emission 600 nm) fluorimetrically. But our attempts to genotype these PCR sequences with isothermal UV absorbance spectroscopy were unsuccessful. Isothermal UV absorbance spectra has a limitation of sequence length. However, the structural conformation (all B-type) of the oligoduplexes (aps) was determined using CD. The minor discrepancy in CD spectra of these oligoduplexes are not significant for mutational analysis. 97 bp nested PCR product was an amplicon having either GcT or AcC mutation of mitochondria of normal human population, whereas 256 bp PCR product was an amplicon of human BRCA2 gene (NCBI Accession No. AY151039) of chromosome 13 having either A or G mutation at position − 26.

In our previous studies, we demonstrated that expression levels of genes determined by Agilent oligoarray system, code G4130A, could be quantitatively evaluated by spike-in of synthetic full-length mRNAs as standards [Kakuhata R, Watanabe M, Yamamoto T, Akamine R, Yamazaki N, Kataoka M, et al. Possible utilization of in vitro synthesized mRNAs specifically expressed in certain tissues as standards for quantitative evaluation of the results of microarray analysis, J Biochem Biophys Methods 2007;70:755–60]. However, in its successor version (Agilent oligo array system, code G4131F), which was established to enable gene expression analysis over a much wider dynamic range, multiple probes are often utilized for evaluation of expression levels of individual genes; and they showed markedly distinct signal intensities. This result indicates that the observed signal intensities in this new version seemed not to simply reflect the transcript levels of individual genes. To understand the factors influencing the signal intensities of probes, we characterized the properties of the probes used in this new array system and the cRNAs formed during the labeling process. Analysis of cRNAs in the reaction mixture, which were hybridized with the arrays, revealed that the cRNAs were not fully transcribed under the conditions used. For this reason, probes located at the 5′ side of the message were found to give lower signals than those at the 3′ end; and the observed signal intensities were dependent upon the location of probes in the mRNA. Analysis of the correlation between signal intensities and locations on mRNAs for larger numbers of probes also supported the idea that probe location is one of the major determinants of signal intensities of probes in microarray analysis.

The catalytic ability of Ricin Toxin A-Chain (RTA) to create an abasic site in a 14-mer stem-tetraloop RNA is exploited for its detection. RTA catalyzes the hydrolysis of the N-glycosidic bond of a specific adenosine in the GAGA tetraloop of stem-loop RNA. Thus, a 14-mer stem-loop RNA substrate containing an intact “GAGA” sequence can be discriminated from the product containing an abasic “GabGA” sequence by hybridization with a 14-mer DNA stem-loop probe sequence and following the fluorescent response of the heteroduplexes. Three DNA beacon probe designs are described. Beacon 1 probe is a stem-loop structure and has a fluorophore and a quencher covalently linked to the 5′- and 3′-ends. In this format the probe–substrate heteroduplex gives a fluorescent signal while the probe–product one remains quenched. Beacon 2 is a modified version of 1 and incorporates a pyrene deoxynucleoside for recognition of the abasic site. In this format both the substrate and product heteroduplexes give a fluorescent response. Beacon 3 utilizes a design where the fluorophore is on the substrate RNA sequence at its 5′-end while the quencher is on the probe DNA sequence at its 3′-end. In this format the fluorescence of the substrate–probe heteroduplex is quenched while that of the product–probe one is enhanced. The lower limit of detection with beacons is 14 ng/mL of RTA.

Labelling of carbohydrates with fluorescent dyes is of importance in the analysis of complex oligo- or polysaccharides. The complexity of the hydrolization mixtures of the oligosaccharides can be studied with separation methods after labelling. However, the procedures might provide unexpected phenomena. The experimental circumstances for labelling carbohydrates with 8-aminopyrene-1,3,6-trisulfonic acid were studied and the labelling efficiency of mono and oligosaccharides present in endotoxins was followed by capillary electrophoresis using LIF detection. Significant differences were observed in the labelling of the different sugar molecules, and the lowest reactivity with the dye was observed in the cases of amino-sugars. The results provide a basis for studies in determining the structures of the core parts and the O-chains of bacterial endotoxins.

Development and testing of protein classification algorithms are hampered by the fact that the protein universe is characterized by groups vastly different in the number of members, in average protein size, similarity within group, etc. Datasets based on traditional cross-validation (k-fold, leave-one-out, etc.) may not give reliable estimates on how an algorithm will generalize to novel, distantly related subtypes of the known protein classes. Supervised cross-validation, i.e., selection of test and train sets according to the known subtypes within a database has been successfully used earlier in conjunction with the SCOP database. Our goal was to extend this principle to other databases and to design standardized benchmark datasets for protein classification. Hierarchical classification trees of protein categories provide a simple and general framework for designing supervised cross-validation strategies for protein classification. Benchmark datasets can be designed at various levels of the concept hierarchy using a simple graph-theoretic distance. A combination of supervised and random sampling was selected to construct reduced size model datasets, suitable for algorithm comparison. Over 3000 new classification tasks were added to our recently established protein classification benchmark collection that currently includes protein sequence (including protein domains and entire proteins), protein structure and reading frame DNA sequence data. We carried out an extensive evaluation based on various machine-learning algorithms such as nearest neighbor, support vector machines, artificial neural networks, random forests and logistic regression, used in conjunction with comparison algorithms, BLAST, Smith-Waterman, Needleman-Wunsch, as well as 3D comparison methods DALI and PRIDE. The resulting datasets provide lower, and in our opinion more realistic estimates of the classifier performance than do random cross-validation schemes. A combination of supervised and random sampling was used to construct model datasets, suitable for algorithm comparison.

The datasets are available at http://hydra.icgeb.trieste.it/benchmark.