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PubMed1983

High-performance liquid chromatography of methotrexate analogs containing terminal lysine or ornithine and their dansyl derivatives.

A procedure utilizing a reverse-phase semipreparative high-performance liquid chromatography column and a binary solvent system consisting of trifluoroacetic acid and 1-propanol has been developed for the semipreparative scale purification and analytical identification of four newly synthesized analogs of methotrexate. The methotrexate analogs containing a lysine or an ornithine residue in place of a terminal glutamate residue together with their respective dansyl derivatives were purified in milligram quantities by the procedures described.

Chromatography, High Pressure Liquid↗
PubMed1983

Synthesis and properties of fluorescent derivatives of atractyloside as potential probes of the mitochondrial ADP/ATP carrier protein.

The chemical synthesis of fluorescent derivatives of atractyloside (ATR), an inhibitor of the mitochondrial ADP/ATP carrier protein, is described. These derivatives are the following: 6'-O-dansyl ATR, 6'-O-dansyl-aminobutyryl ATR, and 6'-O-naphthoyl ATR. The spectral properties of these analogs were analyzed, and their biological features were compared to those of ATR. The fluorescence emission of the dansyl ATR derivatives was increased in organic solvents and that of naphthoyl ATR was decreased; for both analogs, solubilization in organic solvents resulted in a blue shift of the emission peak. The fluorescent dansyl and naphthoyl ATR derivatives were specifically recognized by the mitochondrial ADP/ATP carrier protein. Because of their spectral properties and their biochemical reactivities, the fluorescent analogs of ATR can be considered as potential probes to investigate the topography of the ADP/ATP carrier in the mitochondrial membrane and to monitor conformational changes of the ADP/ATP carrier protein associated with transport.

Adenosine Diphosphate↗
PubMed1983

A new substrate for porcine pepsin possessing cryptic fluorescence properties.

A fluorescent substrate for porcine pepsin, 50-dimethylaminonaphthalene-1-sulfonyl (Dns)-Ala-Ala-Phe-Phe-3-[4-(N-CH3)-pyridyl]propyl-1-oxy ester has been synthesized. It is stable, soluble from pH 1 to 7, and is readily hydrolyzed by pepsin with values of 288 (+/- 40) s-1 for kcat, 0.039 mM (+/- 0.005) for Km, and 7510 s-1 mM-1 (+/- 500) for kcat/Km in sodium formate, pH 3.1. Kinetic studies were carried out by following the increased fluorescence (300-nm excitation, 525-nm emission) as hydrolysis occurred. The products of hydrolysis were identified and established that the peptide bond between the phenylalanine residues is cleaved by pepsin. The inhibition of pepsin catalysis by pepsinogen (1-12) activation peptide was studied in order to compare the inhibition of the reaction of pepsin with Dns-Ala-Ala-Phe-Phe-OP4P-CH3+ with that obtained by the standard milk-clotting assay. The inhibition results were comparable. Dns-Ala-Ala-Phe-Phe-OP4P-CH3+ should be a valuable tool for studies of pepsin because of its solubility over an extended pH range, its excellent turnover rate, and the ease with which the hydrolysis can be followed.

Animals↗
PubMed1983

Protein N-terminal analysis using fast atom bombardment mass spectrometry.

Fast atom bombardment (FAB) mass spectrometry was employed to identify and quantitate dansyl amino acids obtained in the N-terminal analysis of proteins. FAB mass spectra of dansyl amino acids are characterized by intense quasimolecular ions formed by cationization of the molecular species and fragment ions produced by cleavage of the bonds on either side of the sulfanyl group. Investigation of the dansyl amino acid responses using dansyl aminobutyric acid as an internal standard showed that dansyl amino acids can be determined quantitatively at a level of 0.1 nmol. N-terminal residue analysis was performed on a number of proteins to substantiate the technique including bovine serum albumin, pepsinogen, trypsinogen, bromelain, ribonuclease A, and bacteriophage P-22 tail protein.

Amino Acids↗
PubMed1983

Determination of pipecolic acid in rat brain areas by high-performance liquid chromatography of dansyl derivatives with fluorimetric detection.

A method for the determination of pipecolic acid in the rat brain is reported. The identification and quantification of pipecolic acid is accomplished with reverse-phase high-performance liquid chromatography including precolumn dansylation procedure by using nipecotic acid, a regio-isomer of pipecolic acid, as an internal standard. The lower limit of quantification for the method is in the picomole range. Higher concentration of pipecolic acid in the rat brain regions were found in cerebellum, medulla oblongata, hypothalamus, and midbrain than the other regions. The method establishes the usefulness of dansyl chloride for the simple and sensitive detection of pipecolic acid, and is easily adapted to routine analysis of pipecolic acid in the rat brain regions.

Animals↗
PubMed1984

A fluorometric assay for angiotensin-converting enzyme activity.

A simple and sensitive assay for angiotensin-converting enzyme (ACE; EC 3.4.15.1) activity has been developed which employs fluorescently labeled tripeptides. ACE hydrolyzes dansylphenylalanyl-arginyl-tryptophan or dansyl-phenylalanyl-arginyl-phenylalanine, liberating dansyl-phenylalanine and a dipeptide. Dansyl-phenylalanine partitions quantitatively into chloroform, whereas the substrates are virtually insoluble in chloroform. This allows rapid measurement of ACE activity with high signal-to-noise ratios even when microliter aliquots of human serum are assayed. Inhibition studies of the dansyl-tripeptide cleaving activity of human serum and rat lung, the identity of the products of enzyme action, and the regional distribution of enzyme activity among rat tissues demonstrate that only ACE cleaves these substrates under the conditions employed here. This assay may be useful for the clinical measurement of human serum ACE activity and for research investigations of ACE from a variety of tissues.

Animals↗
PubMed1984

Preparation of eight ovalbumin subfractions by combined lectin affinity chromatography.

Ovalbumin was fractionated by successive lectin affinity chromatography using concanavalin A/Sepharose and wheat germ agglutinin/Sepharose. Eight glycoprotein fractions, all behaving as ovalbumin on polyacrylamide gel electrophoresis, were obtained. To characterize the carbohydrate chains, the asparaginyl-carbohydrates were prepared from the Pronase digests of the ovalbumin fractions and their dansyl derivatives were analyzed by high-performance liquid chromatography. The elution profile of the dansylated asparaginyl-carbohydrates from each subfraction was compared with that from the unfractionated ovalbumin. The results indicated that the above eight subfractions could be separated from each other according to their carbohydrate chains and that three of the subfractions were homogeneous with respect to their carbohydrate chains.

Carbohydrate Sequence↗
PubMed1985

Separation of dansyl hydrazine-derivatized oligosaccharides by liquid chromatography.

A sensitive method has been developed for the rapid separation of neutral oligosaccharides derivatized with dansyl hydrazine by liquid chromatography on silica gel modified with 1,4-diaminobutane. Malto-oligosaccharides from corn syrup in amounts ranging from 7 to 280 nmol and ovalbumin-derived oligosaccharides in amounts ranging from 0.25 to 250 nmol were easily detectable and gave linear responses over the respective ranges.

Chemical Phenomena↗
PubMed1985

Influences of metal ions on the reaction of amino and imino acids with fluorogenic reagents.

When a twofold excess of metal ions (Cu2+, Ni2+, Zn2+, Mg2+, Ca2+, Fe3+, and Al3+) was added to a reaction medium of amino acids (1-50 microM each) with the fluorogenic reagents NBD-F (4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole), DNS-Cl (5-N,N-dimethylaminonaphthalenesulfonyl chloride), OPA (o-phthalaldehyde), and fluorescamine (4-phenylspiro[furan-2(3H),1'-phthalan]-3,3'-dione), a reduction of derivatization was observed. The effect was especially marked on the reaction with NBD-F. The derivatization of His and Asp with DNS-Cl and fluorescamine and of His with OPA was suppressed by Cu2+ and/or Ni2+. The suppression by the metal ions can be overcome by the addition of EDTA to the reaction medium. For determination of a small amount of amino and imino acids (less than microM) the addition of a chelating reagent to biological samples is recommended before derivatization with these electrophilic reagents.

4-Chloro-7-nitrobenzofurazan↗
PubMed1986

Rapid sulfopropyl-disk chromatographic purification of bovine and human thrombin.

Thrombin, from either a crude commercial preparation (bovine) or a prothrombin activation mixture (human), was purified by sulfopropyl-disk chromatography to homogeneity in a rapid and convenient single-step procedure. The yield of both proteinases was greater than 85%. Purified bovine and human thrombin had sp act of 2500 and 3000 "NIH" units/mg, respectively. Human thrombin was more reactive than bovine thrombin with these active site-directed probes: phenylmethylsulfonyl fluoride, dansyl-Glu-Gly-Arg-chloromethylketone, and human heparin cofactor II. The sulfopropyl-disk chromatographic method is a useful and rapid technique to prepare milligram quantities of highly purified bovine and human thrombin.

Amino Acid Chloromethyl Ketones↗
PubMed1986

Optimization of erythrocyte membrane glycoprotein fluorescent labeling with dansylhydrazine after polyacrylamide gel electrophoresis.

An improved procedure for the labeling of glycoproteins with dansylhydrazine subsequent to electrophoresis in polyacrylamide gels is reported. This procedure is derived from the work of Eckhardt et al. (1976, Anal. Biochem. 73, 192-197) and Weber and Hof (1975, Biochem. Biophys. Res. Commun. 65, 1298-1302) who showed that dansylhydrazine may be condensed with the aldehyde groups of oxidized glycoprotein carbohydrates and the resulting hydrazones reduced with dimethylamine borane and/or sodium borohydride. Using the known distribution of erythrocyte membrane glycoproteins as a benchmark the effect of variation of a number of process parameters was investigated and an optimal procedure identified. The procedure is shown to be relatively insensitive to moderate variations in reagent composition, pH, and time of incubation with dansylhydrazine solution or reducing agents. It is also shown that labeling patterns may be preserved in dried gels if dimethylsulfoxide is replaced or omitted from all of the process solutions and destaining is effected with 1 M sodium acetate, pH 5.6. While specifically developed for the labeling of erythrocyte membrane proteins, the procedure is demonstrated to be applicable to other glycoprotein containing preparations.

Dansyl Compounds↗
PubMed1986

High-performance liquid chromatographic determination of urinary catecholamines by pre-column solid-phase dansylation on alumina.

Sensitive and selective high-performance liquid chromatographic determination of catecholamines by pre-column solid-phase dansylation is described. After catecholamines are adsorbed on alumina, the amino groups not responsible for adsorption are dansylated by a solid-phase reaction. The excess reagent and fluorescent contaminants are washed out, and the dansylated catecholamines are eluted and separated by reversed-phase high-performance liquid chromatography. The four catecholamine derivatives can be separated within 10 min and no major interfering peak is observed on chromatograms. The response of each catecholamine is linear from 10 to 500 pmol per sample and the detection limit is 0.5 pmol. This method was applied to determination of catecholamines in human urine.

Adult↗
PubMed1986

Analysis of S-methylmethionine and S-adenosylmethionine in plant tissue by a dansylation, dual-isotope method.

A method is presented for determining the levels of S-methylmethionine (MeMet) and S-adenosylmethionine (AdoMet) in the same plant tissue sample, utilizing readily available equipment. The bottom limit of sensitivity, ca. 100 pmol, can be lowered if required. A trichloracetic acid homogenate of the tissue is supplemented with [carboxyl-14C]MeMet and [carboxyl-14C]AdoMet. After separation of MeMet and AdoMet from each other and from endogenous homoserine on a phosphocellulose column, the two fractions are heat treated at appropriate pH values to liberate [14C]homoserine. Quantitation is via the 3H/14C ratio of [3H]dansyl-[14C]homoserine isolated by thin-layer chromatography. The method is validated with pea cotyledon, corn root, and cauliflower leaf.

Dansyl Compounds↗
PubMed1987

Fluorescent inhibitor probes of enzyme active site conformation: anion binding to angiotensin-converting enzyme.

Dansylated tight-binding inhibitors are effective fluorophoric probes for detecting conformational changes of enzyme active sites. In this study they have been employed to examine the effect of anions on the conformation of angiotensin-converting enzyme. The efficiency of radiationless energy transfer between enzyme tryptophan residues and an active site-bound dansyl inhibitor has been shown to be enhanced by the addition of chloride. Half-maximal fluorescence enhancement occurs at about 2 mM chloride and is the same for both N-(1-carboxyl-5-dansylamino-pentyl)-glycyl-L-phenylalanine [Ki,app = 50 nM (pH 7.5, 300 mM NaCl)] and N-(1-carboxyl-5-dansylamino-pentyl)-glycyl-L-lysine (Ki,app = 5.7 nM). Other activating anions also evoke similar increases in enzyme-inhibitor energy transfer. Fluorescence changes are not due to binding additional inhibitor molecules but rather to an anion-induced change in protein conformation.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1987

Methods for in situ visualization and assay of sulfurtransferases.

The dansyl derivative 5-dimethylamino-1-naphthalene thiosulfonate (DANTS) can serve as a sulfane sulfur-donor substrate for several of the sulfurtransferases, the reaction being dependent on the acceptor substrates supplied. Enzymatic cleavage of the sulfur-sulfur bond of DANTS releases the intrinsic fluorescence of the molecule, with an emission maximum of 500-510 nm (excitation at 325 nm). This process permits selective visualization of active sulfurtransferase enzymes separated in nondenaturing polyacrylamide gels, even from impure preparations. This technique was used to locate rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1), thiosulfate reductase (EC unassigned), and a recently isolated prokaryotic enzyme that has been called sulfane sulfurtransferase. In addition, a refinement of the thiosulfate reductase assay technique is reported.

Dansyl Compounds↗
PubMed1987

The use of reverse-phase high-performance liquid chromatography and precolumn derivatization with dansyl chloride for quantitation of specific amino acids in collagen and elastin.

A rapid and accurate reverse-phase high-performance liquid chromatography procedure for amino acid analysis of connective tissue proteins has been optimized. The method is based on quantitative dansyl chloride precolumn derivatization of protein hydrolyzates and chromatographic separation of the dansyl derivatives on Ultrasphere ODS C18 column. High molar uv absorption of Pro and Hyp derivatives, quantitation of low Des and Ide amounts (60 pmol), and good separation of all the amino acid derivatives (CV mostly less than 0.1%) including Hyl overcome the difficulties of other methods in producing reliable single run amino acid analysis of collagen and elastin. The use of fluorescence detector and the possibility of concentrating derivatized samples would give even higher sensitivity to the system. The procedure appeared to be suitable for single run analysis of other proteins, particularly those having an unbalanced amino acid composition.

Amino Acids↗
PubMed1988

Fluorophore-labeled ether lipids: substrates for enzymes of the platelet-activating factor cycle in peritoneal polymorphonuclear leukocytes.

Cell-free preparations of ionophore-stimulated peritoneal rat polymorphonuclear neutrophils (PMNs) incubated with 1-(N-dansyl-11-amino-1-undecyl)-sn-glycerol-3-phosphorylcholine (dansyllyso-PAF) converted this fluorescent lyso ether lipid into two different classes of products. In the absence of acetyl-CoA 1-(N-dansyl-11-amino-1-undecyl)-2-long chain acyl-sn-glycerol-3-phosphorylcholine (dansylalkyl-2-acyl-GPC) was the only identified new fluorescent phospholipid. In the presence of acetyl-CoA an additional new product, 1-(N-dansyl-11-amino-1-undecyl)-2-acetyl-sn-glycerol-3-phosphorylcholine (dansyl-PAF), was formed. The formation of dansyl-PAF in PMN homogenates was only transient with a maximum after about 4 min. When PMN homogenates were incubated with dansyl-PAF the formation of dansyllyso-PAF was observed prior to the formation of dansyl-2-acyl-GPC. Thus, our data indicate that enzymatically formed dansyl-PAF is completely remodeled into dansylalkyl-2-acyl-GPC by the sequential action of PAF acetylhydrolase and CoA-independent transacylase. These results demonstrate that peritoneal rat PMNs contain lyso-PAF acetyltransferase, PAF acetylhydrolase, and CoA-independent transacylase and that fluorophore-labeled ether lipids provide an easy means to assay enzymes which catalyze important enzymatic reactions involved in the biosynthesis and remodeling of platelet-activating factor.

Animals↗
PubMed1988

Fluorescent labeling of nitrocellulose-bound proteins at the nanogram level without changes in immunoreactivity.

Proteins blotted on nitrocellulose were stained with either 5-dimethylamino-1-naphthalene-sulfonylchloride (dansyl chloride) or fluorescein isothiocyanate. In both cases the staining procedure can be completed in less than 30 min. The sensitivity for detecting fluorescent-labeled proteins on nitrocellulose was 0.5 ng using a dot test. This was accomplished by transparentizing the nitrocellulose with either immersion oil or toluene. Dansylated proteins were successfully utilized for optimizing the electroblotting procedure. In the presence of 0.2% sodium dodecyl sulfate and 20% methanol the distribution of proteins on the nitrocellulose was an exact replica of the protein pattern seen in the polyacrylamide gel. The fluorescent labeling did not affect the antigenic properties of proteins allowing the subsequent probing with antisera. For this procedure, only one set of samples is needed to obtain accurate photographic records of the gel, the nitrocellulose blot, and the probed blot.

Animals↗