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PubMed2006

Nitric oxide-induced fluorescence enhancement by displacement of dansylated ligands from cobalt.

The cobalt complexes [Co(Ds-AMP)(2)] (1) and [Co(Ds-AQ)(2)] (2), where Ds-AMP and Ds-AQ are the conjugate bases of dansyl aminomethylpyridine (Ds-HAMP) and dansyl aminoquinoline (Ds-HAQ), respectively, were synthesized in two steps as fluorescence-based nitric oxide (NO) sensors and characterized by X-ray crystallography. The fluorescence of the two complexes was significantly quenched in CH(3)CN or CH(3)OH compared to that of the free Ds-HAMP or Ds-HAQ ligands. Addition of NO to a CH(3)CN solution of 1 or 2 enhanced the integrated fluorescence emission by factors of 2.1(+/-0.3) or 3.6(+/-0.4) within 35 or 20 min, respectively. Introduction of NO to methanolic solutions of the complexes similarly increased the fluorescence by 1.4(+/-0.1) for 1 or 6.5(+/-1.4) for 2 within 1 h. These studies demonstrate that 1 and 2 can monitor the presence of NO with turn-on emission and that their fluorescence responses are more rapid than those of previously reported cobalt systems in coordinating solvents such as CH(3)CN and CH(3)OH. (1)H NMR and IR spectroscopic data revealed the formation of a [Co(NO)(2)](10) cobalt-dinitrosyl adduct, with concomitant dissociation of one ligand from the cobalt center, as the metal-containing product of the NO reactions, a result indicating NO-induced ligand release to be the cause of the fluorescence increase.

Cobalt↗
PubMed2004

A versatile polypeptide platform for integrated recognition and reporting: affinity arrays for protein-ligand interaction analysis.

A molecular platform for protein detection and quantification is reported in which recognition has been integrated with direct monitoring of target-protein binding. The platform is based on a versatile 42-residue helix-loop-helix polypeptide that dimerizes to form four-helix bundles and allows site-selective modification with recognition and reporter elements on the side chains of individually addressable lysine residues. The well-characterized interaction between the model target-protein carbonic anhydrase and its inhibitor benzenesulfonamide was used for a proof-of-concept demonstration. An affinity array was designed where benzenesulfonamide derivatives with aliphatic or oligoglycine spacers and a fluorescent dansyl reporter group were introduced into the scaffold. The affinities of the array members for human carbonic anhydrase II (HCAII) were determined by titration with the target protein and were found to be highly affected by the properties of the spacers (dissociation constant Kd=0.02-3 microM). The affinity of HCAII for acetazolamide (Kd=4 nM) was determined in a competition experiment with one of the benzenesulfonamide array members to address the possibility of screening substance libraries for new target-protein binders. Also, successful affinity discrimination between different carbonic anhydrase isozymes highlighted the possibility of performing future isoform-expression profiling. Our platform is predicted to become a flexible tool for a variety of biosensor and protein-microarray applications within biochemistry, diagnostics and pharmaceutical chemistry.

Acetazolamide↗
PubMed1989

Optical enrichment of dansyl-rac-amino acids by formation of crystalline inclusion complexes with cyclodextrins.

Optical enrichment from racemic dansyl-leucine, dansyl-norleucine, and dansyl-phenylalanine with both beta- and gamma-cyclodextrins in water is reported. Initial crystallization yielded the dansyl-L-Leucine isomer complexed in excess with beta-cyclodextrin with an optical purity of 62-78% depending on experimental conditions. The optical purities obtained for L-norleucine and L-phenylalanine were 71 and 64%, respectively. The optical purity can be increased with continued recrystallization. The dansyl-D-leucine isomer was obtained in the mother liquor with an optical purity of 54-93% depending on experimental conditions. The optical purities obtained for D-norleucine and D-phenylalanine were 72 and 58%. The optical purity of the isomer depended on the molar ratio of host:guest and the pH value of the solution. Optimum enrichment of both enantiomers was achieved with host:guest ratios of 2:1 and 3:1. Although maximum crystalline yield of the dansyl-leucine/CD inclusion complex was obtained at a pH of 3.5, optical purity of both enantiomers was less than that obtained at other pHs. The influence of the molar ratio of host:guest and the pH value of the solution are discussed. This method is suitable for large-scale enantiomeric separations.

Amino Acids↗
PubMed1989

Studies on the heterogeneity of anti-hapten antibodies by means of two-dimensional affinity electrophoresis.

The molecular heterogeneity of rabbit anti-hapten antibodies has been investigated by two-dimensional affinity electrophoresis (2D-AEP). Anti-dansyl and anti-arsanilic diazo-antibodies were separated into several hundred IgG spots as in the case of anti-DNP antibodies. They were grouped into a number of monoclonal IgG families. At the beginning of immunization, IgG spots having low pI and low affinity were predominant but one or two weeks after immunization the IgG spots with high affinity and high pI increased. After the second or third immunization, the 2D-AEP patterns became stable and constant. Anti-arsanilic diazo and anti-DNP antibodies exhibited only weak cross-reactivity with other aromatic haptens. In contrast, anti-dansyl antibodies cross-reacted to a considerable degree with DNP-hapten. A few anti-dansyl IgG families which have cross-reactivity with DNP-hapten were separated. Their apparent dissociation constants to the haptens and their affinity ratios were calculated from their 2D-AEP patterns, according to the affinity theory.

Animals↗
PubMed1990

Enantiomer resolution in immobilized pH gradient gels via inclusion of a chiral discriminator.

The separation of enantiomeric forms of dansylated amino acids by isoelectric focusing in immobilized pH gradients (IPG) is demonstrated for the first time. Separations occur in a pH 3.0-4.0 IPG interval, in presence of 7Murea, 10% methanol and 60 mM beta-cyclodextrin (CD) as chiral discriminator. It is found that the inclusion complex formed between the D-form and CD has a lower pI than the uncomplexed form (delta pI = 0.05 for DL-Phe and delta pI = 0.025 for DL-Trp); from this, it is calculated that the pK of the tertiary amino group in the dansyl moiety is lowered by 0.1 pH unit in the former case (D-Phe) and by 0.05 in the case of D-Trp (both values referring to 60 mM CD gels). For some racemates (e.g., DL-Phe) the separation mechanism is still operative with CD concentrations as low as 20 mM. In our system 60 mM CD appears to be the solubility limit of CD. As the complex is stable in the electric field for at least 15 h, this separation mechanism could be exploited for purifying large quantities of pure D and L forms from racemates in multicompartment electrolyzers with isoelectric Immobiline membranes.

Amino Acids↗
PubMed1991

Barley cultivar discrimination: I. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and glycoprotein blotting.

Two different methods of detecting electroblotted glycoproteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Tris-buffer soluble barley seed proteins were examined for their applicability for barley cultivar discrimination. These are the highly specific, lectin-based concanavalin A/peroxidase method and the more general periodate/danyslhydrazine method. The results of the periodate/dansylhydrazine method enabled us to divide the 20 examined cultivars into three groups, whereas the more sensitive concanavalin A/peroxidase method revealed six different glycoprotein patterns. In comparison, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the alcohol-soluble barley seed proteins (hordeins) gave nine different banding patterns. A combination of hordein electrophoresis together with glycoprotein staining by the concanavalin A/peroxidase method made it possible to classify the cultivars into twelve groups, the largest of which contained four cultivars. The qualitative expression of the glycoprotein patterns seemed to be independent of growth conditions, whereas the band intensities obviously were not. As a whole, glycoprotein blotting is a valuable supplement to sodium dodecyl sulfate-polyacrylamide gel electrophoresis of hordeins in barley cultivar discrimination.

Concanavalin A↗
PubMed1991

A new multiphasic buffer system for sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins and peptides with molecular masses 100,000-1000, and their detection with picomolar sensitivity.

A novel multiphasic buffer system for high resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of dansylated and nondansylated proteins/peptides in the relative molecular mass (Mr) range of 100,000-1000 is described. The system, based on Jovin's theory of multiphasic zone electrophoresis, allows complete stacking and destacking of proteins/peptides within the above Mr range. The buffer system uses Bicine and sulfate as trailing and leading ion, respectively, and Bistris and Tris as counter ions in the stacking and separating phase, respectively. Through selection of two different counter ions--the characteristic feature of the present ionic system--the stacking limits of a multiphasic buffer system can be further widened, thus making it applicable to gel electrophoresis of a larger spectrum of rapidly migrating species, such as sodium dodecyl sulfate-proteins/peptides and nucleic acids, than has been possible previously. Highly sensitive detection methods for proteins as well as for polypeptides down to approximately Mr 1000 are described. Dansylated proteins/peptides were detected by their fluorescence either directly within the gel or following electroblotting into anion-exchange or polyvinylidene difluoride membranes. The latter procedure resulted in detection sensitivities of approximately 1 ng. Nondansylated proteins/peptides were either detected within the gel by colloidal Coomassie staining or by electroblotting into polyvinylidene difluoride membranes, followed by colloidal gold staining. Prior to both staining procedures the proteins/peptides were pretreated with glutardialdehyde in the presence of borate at near neutral pH values to generate protein/peptide polymers of poor solubility. For a given pH the efficiency of the latter procedure was significantly influenced by the nature of the buffer ion used in the fixation buffer. In contrast to conventional fixation procedures even small polypeptides (Mr 1000) were immobilized and approximately 15 ng and 0.75 ng could be detected after colloidal Coomassie and colloidal gold staining, respectively.

Buffers↗
PubMed1994

Enhanced chiral separation of dansylated amino acids with cyclodextrin-dextran polymer network by capillary electrophoresis.

A method for the chiral separation of dansyl-DL-amino acid mixtures using a dextran (M(r) 2,000,000) polymer network containing beta-cyclodextrin (beta-CD) in HPCE was developed. Mixtures of up to seven amino acids could be baseline resolved by this method under neutral pH conditions. Resolution was dependent on the dextran concentration in the polymer network. Temperature effects on the chiral separation were studied. Optimal efficiency and resolution of DL-enantiomeric pairs of amino acid samples were obtained at 25 degrees C. The resolution of different amino acids used in this work decreased with an increase in temperature.

Amino Acids↗
PubMed1995

Microchip electrophoresis with sample stacking.

A fused quartz microchip with a serpentine column geometry is fabricated to perform rapid microchip electrophoresis of dansylated amino acids. A 67 mm separation column is constructed in a 7 x 10 mm area on a quartz substrate using standard photolithographic, etching and deposition techniques. Buffer and sample flows within the channel manifold are precisely controlled through potentials applied to the reservoirs. To enhance the detection limits, a stacking injection technique is used to concentrate the sample at the inlet of the separation column. The stacked injections exhibit high reproducibility (2.1% relative standard deviation in peak area). Using a separation length of 67 mm and a separation field strength of 1100 V/cm, separations are performed in < or = 15 s generating approximately 40,000 theoretical plates.

Amino Acids↗
PubMed1996

Determination of carbohydrates as their dansylhydrazine derivatives by capillary electrophoresis with laser-induced fluorescence detection.

A mixture of mono- and disaccharides was derivatized with dansylhydrazine in a relatively fast chemical reaction (15 min) which is selective toward aldehydes and ketones. The products of the derivatization reaction were then separated by capillary electrophoresis (CE) using laser-induced fluorescence (LIF) as the detection mode. The smallest amount of carbohydrate derivatized and determined by CE/LIF was 10 pmol contained in 100 microL of the final reaction mixture. The CE/LIF method provided a linear response for the carbohydrates tested (over 2-3 orders of magnitude), with a limit of detection of 100 amol. Separation efficiencies over 250,000 theoretical plates were obtained. The procedure was used to analyze a tear fluid sample to demonstrate the applicability of the method for the glucose determination in small volumes of biological samples.

Dansyl Compounds↗
PubMed1996

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of human parotid salivary proteins: comparison of dansylation, coomassie blue R-250 and silver detection methods.

Detection of human parotid salivary proteins by dansylation and UV-transillumination after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) has been compared with Coomassie Blue R-250 and silver staining procedures. Dansylation gives superior results in terms of both resolution and sensitivity, especially with basic proline-rich proteins (PRPs).

Dansyl Compounds↗
PubMed1996

Electrokinetic chromatography in suppressed electroosmotic flow environment: use of a charged cyclodextrin for the separation of enantiomers and geometric isomers.

Electrokinetic chromatography (EKC), with negatively-charged cyclodextrins (NCDs) added to the buffer, was conducted in polyacrylamide-coated columns under suppression of electroosmotic flow. The equations of migration and resolution for neutral solutes in this mode of chromatography, which for brevity we term NCD-EKC, are presented. The chiral sulfated cyclodextrin, beta-CD-SBE (IV), used in this study is anionic over the entire pH range accessible to capillary electrophoresis, and the coated columns are stable and provide reproducible performance in the pH range 2.5-8.8. Optimum separation was obtained in the pH range where the solutes are neutral. The incorporation of an alkyl spacer between the sulfate ion and the rim of the cyclodextrin allows an unhindered approach and inclusion of neutral solutes in the cyclodextrin cavity. Solute migration time is inversely proportional to the concentration of the chiral selector. Separation (relative migration time difference) increases with decreasing chiral selector concentration and approaches a maximum, beyond which further decreases in chiral selector concentration result in broad peaks and loss of resolution. A chiral selector concentration of 1% in a 10 mM phosphate buffer produced excellent separation of amino acids and dipeptide enantiomers. In addition to being chiral selectors, cyclodextrins are also known as shape selectors. NCD-EKC is particularly suited for the separation of positional isomers of hydrophobic solutes. The separation of aflatoxin isomers and chlorophenol congeners is presented. In the separation of chlorophenols the more hydrophobic trichlorophenols eluted first and the least hydrophobic, phenol, eluted last.

Amino Acids↗
PubMed1996

Enantioseparation of amino acids and dipeptides using vancomycin as chiral selector in capillary electrophoresis.

Vancomycin was applied as chiral selector for the enantiomeric separation of derivatized amino acids and dipeptides. The influence of vancomycin concentration, pH and presence of 2-propanol in the buffer were examined in order to find optimal separation conditions. Optimization was by factorial design. Further, chiral separation of derivatives prepared with three different reagents was compared. These reagents were 9-fluorenylmethyl chloroformate (FMOC), 2-(9-anthryl)ethyl chloroformate (AEOC) and dansyl chloride (dansyl). Optimum resolution was at high vancomycin concentrations, while optimum efficiency was at low vancomycin concentrations. As a consequence of the very high enantioselectivity of vancomycin, the vancomycin concentration below the amount necessary for maximal resolution can be used. Separation efficiency was relatively low, and this could be attributed to adsorption of the selector at the capillary wall. Three factors led to decreased adsorption: application of a pH above the zero mobility pH value, low vancomycin concentrations and the presence of 2-propanol. For amino acids, the resolutions of the different derivatives were: dansyl > AEOC > FMOC, while for dipeptides, the highest selectivity was with AEOC.

1-Propanol↗
PubMed1997

Histamine-modified beta-cyclodextrins for the enantiomeric separation of dansyl-amino acids in capillary electrophoresis.

Two novel monosubstituted beta-cyclodextrins (CD) bearing the histamine moiety linked to the upper CD rim, either through the amino group or the imidazole nitrogen 1N, CD-hm and CD-mh, were successfully used as chiral selectors in capillary electrophoresis for the enantiomeric discrimination of dansyl (Dns)-amino acids. Good results were obtained by using low concentrations of the selectors (1-3 mM). The effect of pH on the chiral discrimination was studied in order to modulate the number and the position of the positive charges. By increasing the pH from 5 to 7.5, chiral discrimination decreased along with the deprotonation of the imidazolyl moiety. Inversion of the migration order was observed with the two CDs, depending on the relative position of the charged moieties on the upper rim. Ion pair interaction coupled to inclusion complexation seems to account for the discrimination process. The effects of the temperature, CD concentration and capillary length on chiral resolution were also examined.

Carbohydrate Sequence↗
PubMed1997

Comparison of alkylglycoside surfactants in enantioseparation by capillary electrophoresis.

Three alkylglycoside surfactants, namely n-octyl-beta-D-glucopyranoside (OG), n-nonyl-beta-D-glucopyranoside (NG), and n-octyl-beta-D-maltopyranoside (OM), were compared in the enantiomeric separation of dansyl amino acids, binaphthyl phosphate, bupivacaine and warfarin. While only OM exhibited an enantioselectivity toward warfarin, bupivacaine, and dansyl tryptophan, all three surfactants were effective in the enantiomeric resolution of napthyl phosphate and other dansyl amino acids. With the exception of naphthyl phosphate, which could be resolved enantiomerically with OM at surfactant concentrations below the CMC, all solutes required surfactant concentrations greater than the CMC value. This was attributed to the strong hydrophobic association of napthyl phosphate with the OM monomers and to the presence of maltoside residue in the OM surfactant. In general, the optimum surfactant concentration needed for maximum enantiomeric resolution was an inverse function of the hydrophobic character of the solute. Under a given set of conditions, the enantiomeric resolution exhibited by the alkylglycoside surfactants was largely influenced by the extent and loci of solute solubilization into the micelle, and by the nature of the chiral sugar head group of the surfactant.

Amino Acids↗
PubMed1997

Determination of association constants of dansyl-amino acids and beta-cyclodextrin in N-methylformamide by capillary electrophoresis.

The use of nonaqueous background electrolytes in capillary electrophoresis (CE) is a promising new trend which should widen the scope of this technique. We demonstrate the chiral separation of dansyl-amino acids (Dns-AAs) in N-methylformamide (NMF) using beta-cyclodextrin (beta-CD) as chiral selector. The solubility of beta-CD is much better in NMF than in water, allowing high concentration of the chiral selector and successful enantioseparation despite the weak host-guest interaction between the Dns-AAs and beta-CD. The association constants for the complexation between Dns-AAs and beta-CD could be calculated from the electrophoretic mobilities, with attention paid to the change in viscosity of the electrolyte upon addition of beta-CD. The association constants ranged between 2 and 13 M(-1).

Amino Acids↗
PubMed1997

Capillary electrophoretic separation of structurally similar solutes in noncross-linked poly(acrylamide-co-N-isopropylacrylamide) solution.

Noncross-linked acrylamide (AA)-N-isopropylacrylamide (IPAAm) copolymers have been used as a buffer additive in capillary electrophoretic separation of structurally similar small solutes. Seven kinds of barbiturates and five kinds of dansylated (Dns) amino acids, which have different hydrophobic side chains, were separated on poly(AA-co-IPAAm)-filled columns and on AA polymer-filled columns under the condition of totally eliminating the contribution of electroosmotic flow (EOF). It is known that the copolymer containing IPAAm has thermosensitive properties, and the hydrophobicity of its surface changes with surrounding temperature. In this investigation, therefore, the effects of an isopropyl group in the copolymer on the electrophoretic separation of the small solutes were studied by comparing the two polymer-filled columns at ambient temperature and at elevated temperature. Although slight differences in migration behavior were observed at ambient temperature between the columns filled with these two polymer solutions, obvious differences in the separation of the solutes were observed at elevated temperature. The observed changes on the migration behavior might be caused by the interaction between copolymer chains exhibiting hydrophobic property and the solute.

Acrylamides↗