A complete quantitative N-terminal analysis method.
AuthorsChang JY
JournalAnal Biochem
PubMed ID3134832
A complete quantitative N-terminal analysis (QNA) technique based on the application of dimethylaminoazobenzene isothiocyanate is described. The method allows recovery of all free N-terminal amino acids, including Asn, Gln, Trp, Ser, and Thr in quantitative yield. N-Termini of polypeptides as little as 5 pmol can be reliably and reproducibly determined ... More
Visualization of proteins by modification of lysines, cysteines, and phosphorylated serines facilitates sample preparation for microsequencing.
AuthorsHsi KL, O'Neill SA, Dupont DR, Yuan PM
JournalAnal Biochem
PubMed ID9527845
'A procedure for visualization and sensitive detection of protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent sample preparation for sequence analysis is described. This procedure utilizes either fluorescent or visible tags for certain amino acids in protein molecules, e.g., lysines modified with dansyl/dabsyl chloride and cystines/cysteines or phosphorylated ... More
Regulation of the rate and extent of phospholipase C beta 2 effector activation by the beta gamma subunits of heterotrimeric G proteins.
AuthorsRunnels LW, Scarlata SF
JournalBiochemistry
PubMed ID9799521
'The activity of mammalian phosphoinositide-specific phospholipase C beta 2 (PLC-beta 2) is regulated by the alpha q family of G proteins and by beta gamma subunits. We measured the affinity between the laterally associating PLC-beta 2 and G beta gamma on membrane surfaces by fluorescence resonance energy transfer. Using a ... More
Characterization of a cholecystokinin 8-generating endoprotease purified from rat brain synaptosomes.
AuthorsViereck JC, Beinfeld MC
JournalJ Biol Chem
PubMed ID1527068
'An endoproteolytic activity that specifically cleaves CCK 33, producing CCK 8, has been purified from a rat brain synaptosome preparation. The purification, which included anion exchange, chromatofocusing, hydroxyapatite, and gel filtration chromatography, resulted in a greater than 3000-fold increase in specific activity. This neutral endoprotease (pH optimum 8) exists as ... More
Determination of urinary amino acids by liquid chromatography with "dabsyl chloride".
AuthorsLin JK, Wang CH
JournalClin Chem
PubMed ID6790193
'We describe a "high-performance" liquid-chromatographic procedure for measuring amino acids in 0.5 mL of urine. The procedure includes direct derivatization of amino acids in urine samples with "dabsyl chloride" (4-dimethylaminoazobenzene-4''-sulfonyl chloride). An aliquot of this dabsylated amino acid solution is analyzed on a muBondapak C18 column with ethanol/sodium acetate (20 ... More
Evidence for assembly of small multidrug resistance proteins by a "two-faced" transmembrane helix.
AuthorsRath A, Melnyk RA, Deber CM
JournalJ Biol Chem
PubMed ID16574656
'Clinically significant bacterial resistance to drugs and cytotoxic compounds can be conferred by the energy-dependent efflux of toxicants, catalyzed by proteins embedded in the bacterial cell membrane. One such group of proteins, the small multidrug resistance family, are drug/proton antiporters that must oligomerize to function, a process that requires the ... More
Determination of total hydroxyproline and proline in human serum and urine by HPLC with fluorescence detection.
AuthorsInoue H, Date Y, Kohashi K, Yoshitomi H, Tsuruta Y
JournalBiol Pharm Bull
PubMed ID8850299
'A method to measure total hydroxyproline (Hyp) and proline (Pro) in human serum and urine by HPLC was developed. Hyp and Pro in acid hydrolysates of serum and urine were derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)phenysulfonyl chloride after treatment with o-phthaladehyde and cleanup on Bond Elut C18 column. The derivatives of imino acids ... More
Separation of 4-N,N-dimethylaminoazobenzene-4'-sulfonyl amino acids on polyamide sheets.
AuthorsChang JY, Creaser EH
JournalJ Chromatogr
PubMed ID812880
Glycophorin A helical transmembrane domains dimerize in phospholipid bilayers: a resonance energy transfer study.
AuthorsAdair BD, Engelman DM
JournalBiochemistry
PubMed ID8180176
'Glycophorin A and its isolated transmembrane region (GpATM) are each known to form sequence-specific dimers in SDS micelles. Whether this behavior accurately reflects behavior in red cell membranes or lipid bilayers, however, has remained unclear. Resonance energy transfer between labeled GpATM peptides has been used to observe dimerization of GpATM ... More
High-throughput SNP genotyping by allele-specific PCR with universal energy-transfer-labeled primers.
AuthorsMyakishev MV, Khripin Y, Hu S, Hamer DH
JournalGenome Res
PubMed ID11156625
'We have developed a new method for high-throughput genotyping of single nucleotide polymorphisms (SNPs). The technique involves PCR amplification of genomic DNA with two tailed allele-specific primers that introduce priming sites for universal energy-transfer-labeled primers. The output of red and green light is conveniently scored using a fluorescence plate reader. ... More
Determination of dityrosine, phosphotyrosine, phosphothreonine, and phosphoserine by high-performance liquid chromatography.
AuthorsMalencik DA, Zhao ZZ, Anderson SR
JournalAnal Biochem
PubMed ID1691599
'4''-Dimethylaminoazobenzene-4-sulfonyl chloride is a chromophoric reagent commonly used to detect amino acids at picomole levels. This article describes a single-column reverse-phase high-performance liquid chromatography system which allows the resolution and analysis of the dabsyl chloride derivatives of several modified amino acids. Highly derivatized C18 (22 and 31%) columns from Phenomenex ... More
A determination of the radial coordinate of Tyr-69 in F-actin using fluorescence energy transfer.
AuthorsMiki M, dos Remedios CG
JournalBiochem Int
PubMed ID2126434
'Fluorescence energy transfer was measured between Tyr-69 residues in an F-actin filament using 5-dimethylaminonaphthalene-1-sulfonyl chloride (DNS-Cl) as a fluorescence energy donor and 4-dimethylaminoazobenzene-4-sulfonyl chloride (DABSYL-Cl) as the acceptor. Both labels are covalently attached to Tyr-69 residues in an F-actin filament. Taking the helical structure of the F-actin filament into consideration, ... More
Amino acid analysis in the picomole range by precolumn derivatization and high-performance liquid chromatography.
AuthorsChang JY, Knecht R, Braun DG
JournalMethods Enzymol
PubMed ID6406788
A sensitive, rapid method for monitoring sodium dodecyl sulfate-polyacrylamide gel electrophoresis by chromophoric labeling.
AuthorsTzeng MC
JournalAnal Biochem
PubMed ID6405657
Synthesis of dabsylhydrazine and its use in the chromatographic determination of monosaccharides by thin-layer and high-performance liquid chromatography.
AuthorsLin JK, Wu SS
JournalAnal Chem
PubMed ID3111300
Rapid thin-layer chromatographic semiquantification of morphine in urine via dabsylation: comparison with EMIT and thin-layer chromatographic-iodoplatinate methods.
AuthorsWang SY, Tham SY, Poon MK
JournalClin Chem
PubMed ID2644060
High-sensitivity amino acid analysis: measurement of amino acid neurotransmitter in mouse brain.
AuthorsChang JY, Martin P, Bernasconi R, Braun DG
JournalFEBS Lett
PubMed ID6117477
High performance liquid chromatographic determination of naturally occurring primary and secondary amines with dabsyl chloride.
AuthorsLin JK, Lai CC
JournalAnal Chem
PubMed ID7190362
Chromophoric labeling of amino acids with 4-dimethylaminoazobenzene-4'-sulfonyl chloride.
AuthorsLin JK, Chang JY
JournalAnal Chem
PubMed ID808149
Structure of actin observed by fluorescence resonance energy transfer spectroscopy.
AuthorsMiki M, O'Donoghue SI, Dos Remedios CG
JournalJ Muscle Res Cell Motil
PubMed ID1534564
Derivatization of posttranslationally modified amino acids.
AuthorsTeerlink T
JournalJ Chromatogr B Biomed Appl
PubMed ID7820276
After a brief overview of posttranslational modifications of protein amino acids, the use of various derivatizing reagents for amino acid analysis is discussed. Derivatization and chromatographic separation of hydroxyproline, methylhistidine, and phosphorylated amino acids are discussed in detail to illustrate some of the strategies that can be applied to the ... More
Quencher as leaving group: efficient detection of DNA-joining reactions.
AuthorsSando S, Kool ET
JournalJ Am Chem Soc
PubMed ID11878946
We describe a new fluorescence reporting strategy in which dabsyl, a well-known quencher, activates a hydroxyl group in a probe to convert it to a leaving group. When a nucleophilic phosphorothioate probe binds adjacent to a dabsyl quenched probe, autoligation occurs, releasing the quencher, and lighting up the probes, This ... More
Analysis of polyamines as their dabsyl derivatives by reversed-phase high-performance liquid chromatography.
AuthorsKoski P, Helander IM, Sarvas M, Vaara M
JournalAnal Biochem
PubMed ID3118735
The polyamines putrescine, cadaverine, spermidine, and spermine and the corresponding mono-N-acetylpolyamines can be separated as their dimethylaminoazobenzenesulfonyl derivatives in a single analysis in less than 22 min. The method employs reversed-phase high-performance liquid chromatography (Spherisorb S5 ODS2 column) with an acetonitrile/acetate buffer gradient elution system and detection in the visible ... More
Fluorogenic bimane substrates with dabsyl group for endopeptidases; chymotrypsin, collagenase and thermolysin.
AuthorsSato E, Hattori H, Kanaoka Y
JournalJ Pharmacobiodyn
PubMed ID1667927
It was found that the fluorescence of 9,10-dioxa-syn-3,4,6,7-tetramethylbimane (bimane) can be quenched in the presence of dimethylaminoazobenzensulfonyl (Dabsyl) group. New combination of bimane (fluorophor) and dabsyl group (quencher) was applied to the syntheses of intramolecularly quenched fluorogenic substrates for hydrolytic enzymes. Bimane peptides containing dabsyl group were prepared, and were ... More
Measurement of glutamate, aspartate and glycine and its potential precursors in human brain using high-performance liquid chromatography by pre-column derivatization with dimethylaminoazobenzene [correction of diethylaminoazobenzene] sulphonyl chloride.
AuthorsWatanabe A, Semba J, Kurumaji A, Kumashiro S, Toru M
JournalJ Chromatogr
PubMed ID1362201
This paper describes a high-performance liquid chromatographic technique, with dimethylaminoazobenzene sulphonyl chloride derivatization, for the measurement of glutamate, aspartate and glycine and its potential precursors in human brain tissue. The derivatization procedure is simple, sensitive and highly reproducible. The derivatized amino acids are stable and can be analysed by reversed-phase ... More
Analysis of reducing sugars as their chromophoric hydrazones by high-performance liquid chromatography.
AuthorsMuramoto K, Goto R, Kamiya H
JournalAnal Biochem
PubMed ID3111299
A chromophoric hydrazide, 4'-N,N-dimethylamino-4-azobenzene sulfonyl hydrazide (DABS-hydrazide), was prepared from 4'-N,N-dimethylamino-4-azobenzene sulfonyl chloride by reaction with hydrazine. Reducing sugars were derivatized with DABS-hydrazide at 50 degrees C for 120 min. The chromophoric hydrazones were separated by reversed-phase HPLC isocratically using a short column (4.6 X 50 mm) and 0.08 M ... More
Sphingosine determination at the picomole level using dimethylaminoazobenzene sulphonyl chloride.
AuthorsRosenfelder G, Chang JY, Braun DG
JournalJ Chromatogr
PubMed ID6404916
A sensitive method (lower detection limit 5 picomoles) is proposed for the determination of sphingoid bases liberated from mammalian glycosphingolipids by acid hydrolysis. The azo dye 4-dimethylaminoazobenzene-4'-sulfonyl chloride reacts with the primary amino group of the sphingosine bases, forming a stable derivative. Excess reagent, which is degraded during the derivatization, ... More
Visible labeling of proteins for polyacrylamide gel electrophoresis with dabsyl chloride.
AuthorsParkinson D, Redshaw JD
JournalAnal Biochem
PubMed ID6437269
Several proteins, which are used as molecular weight markers in polyacrylamide gel electrophoresis, were reacted with dabsyl chloride. This labeled them deep orange and the chromophore attachment was stable throughout the electrophoretic procedure and fixation. Small amounts (10-50 micrograms) of the labeled proteins could be loaded onto gels and seen ... More
Derivatization reactions for neurotransmitters and their automation.
AuthorsBovingdon ME, Webster RA
JournalJ Chromatogr B Biomed Appl
PubMed ID7820275
Many reagents suitable for the derivatization of neurotransmitters are selective for the amino function. Others, however, are selective for the carboxyl-, thiol- and hydroxyl function, and recently, reagents selective for more than one function have been produced. Interest persists in the established reagents, with their well understood behaviour which assists ... More
Chromophoric labeling of cannabinoids with 4-dimethylaminoazobenzene-4'-sulfonyl chloride.
AuthorsMaseda C, Fukui Y, Kimura K, Matsubara K
JournalJ Forensic Sci
PubMed ID6415216
A simple and sensitive assay for the cannabinoids is presented using a dabsylation procedure. Dabsyl derivatives of delta 9-tetrahydrocannabinol (delta 9-THC) and cannabinol (CBN) were prepared by reacting with 4-dimethylaminoazobenzene-4'-sulfonyl chloride (dabsyl chloride) in acetone in the presence of sodium carbonate-sodium bicarbonate buffer (pH 10). Crystalline dabsylcannabinoids gave intense absorption ... More
Determination of amino sugars in synthetic glycopeptides during the conditions of amino acid analysis utilizing precolumn derivatization and high-performance liquid chromatographic analysis.
AuthorsGorbics L, Urge L, Otvos-Papp E, Otvos L
JournalJ Chromatogr
PubMed ID8514837
The growing number of synthetic glycopeptides required an in-house method for the analysis of the final products. A reversed-phase high-performance liquid chromatographic protocol was developed to verify the presence of N-acetylglucosamine and N-acetylgalactosamine in synthetic glycopeptides after hydrolysis and derivatization with 4-dimethylaminoazobenzene-4'-sulphonyl chloride. Different sugar and glycoamino acid standards were ... More
The pleckstrin homology domain of phospholipase C-beta(2) links the binding of gbetagamma to activation of the catalytic core.
AuthorsWang T, Dowal L, El-Maghrabi MR, Rebecchi M, Scarlata S
JournalJ Biol Chem
PubMed ID10713048
Pleckstrin homology (PH) domains are membrane tethering devices found in many signal transducing proteins. These domains also couple to the betagamma subunits of GTP binding proteins (G proteins), but whether this association transmits allosteric information to the catalytic core is unclear. To address this question, we constructed protein chimeras in ... More
Determination of the unusual amino acid hypusine at the lower picomole level by derivatization with 4-dimethylaminoazobenzene-4'-sulphonyl chloride and reversed-phase high-performance or medium-pressure liquid chromatography.
AuthorsBartig D, Klink F
JournalJ Chromatogr
PubMed ID1500466
Hypusine, an unusual amino acid formed by post-translational modification of lysine, is normally determined by specific metabolic labelling followed by measurement of released radioactivity after protein hydrolysis. This paper describes a sensitive non-radioactive method for the determination of hypusine, involving complete protein hydrolysis and precolumn derivatization of the released amino ... More
Chemical modification of rat liver microsomal cytochrome P-450: study of enzymic properties and membrane topology.
AuthorsKunz BC, Vergères G, Winterhalter KH, Richter C
JournalBiochim Biophys Acta
PubMed ID2012820
Isolated rat liver cytochrome P-450IIB1 was alkylated and acetylated at primary amino groups, and the position of the modified amino acids in the protein was identified. Alkylation of up to nine amino groups did not disturb the interaction of reconstituted P-450 and NADPH-cytochrome-P-450 reductase in a way that hydroxylation of ... More
The hepatocyte primary culture/DNA repair assay using mouse or hamster hepatocytes.
AuthorsMcQueen CA, Kreiser DM, Williams GM
JournalEnviron Mutagen
PubMed ID6299721
The hepatocyte primary culture (HPC)/DNA repair assay using rat hepatocytes was developed to identify genotoxic chemicals. Since there are species differences in susceptibility to chemical carcinogens, it was desirable to extend the assay to other species. Carcinogens and noncarcinogens from six structural classes were tested with hepatocytes from B6C3F1 mice ... More
Electroblotting onto activated glass. High efficiency preparation of proteins from analytical sodium dodecyl sulfate-polyacrylamide gels for direct sequence analysis.
AuthorsAebersold RH, Teplow DB, Hood LE, Kent SB
JournalJ Biol Chem
PubMed ID3949810
We have developed a new method for the isolation of proteins for microsequencing. It consists of electrophoretic transfer (electroblotting) of proteins or their cleavage fragments onto activated glass filter paper sheets immediately after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins are immobilized on the glass fiber sheets by ... More
Functional methionines in the collagen/gelatin binding domain of plasma fibronectin: effects of chemical modification by chloramine T.
AuthorsMiles AM, Smith RL
JournalBiochemistry
PubMed ID8347617
Chemical modification of plasma fibronectin (pFn) or its 40-kDa collagen/gelatin binding (CGB) domain by low concentrations of chloramine T (CT), a methionine-specific oxidant, caused decreased binding affinity between pFn or the isolated CGB domain and Sepharose-immobilized denatured collagen or a Texas Red-labeled CNBr fragment CB7 from the alpha 1 chain ... More
Quenched auto-ligating DNAs: multicolor identification of nucleic acids at single nucleotide resolution.
AuthorsSando S, Abe H, Kool ET
JournalJ Am Chem Soc
PubMed ID14746476
We describe the synthesis and study of multicolor quenched autoligating (QUAL) probes for identification and discrimination of closely related RNA and DNA sequences in solution and in bacteria. In these probes, a dabsyl quencher doubles as an activator in the oligonucleotide-joining reaction. The oligonucleotides remain dark until they bind at ... More
Differing reactivities of human and guinea-pig complement on haptenized liposomes via the alternative pathway.
AuthorsOkada N, Yasuda T, Tsumita T, Okada H
JournalMol Immunol
PubMed ID6194429
Several haptens coupled to dipalmitoylphosphatidylethanolamine (DPPE) were inserted into the liposome membrane with a base composition of an equimolecular mixture of dimyristoylphosphatidylcholine (DMPC) and cholesterol (Chol). Haptens used were trinitrophenyl (TNP)-DPPE, TNP-aminocaproyl (TNP-Cap)-DPPE, dinitrophenyl (DNP)-DPPE, DNP-aminocaproyl (DNP-Cap)-DPPE, fluoresceinthiocarbamyl (Fl)-DPPE, azobenzenarsonate-tyrosyl (ABA-Tyr)-DPPE, dansyl (DNS)-DPPE, dabsyl (DABS)-DPPE, dithiopyridyl (DTP)-DPPE and maleimidobenzoyl (MB)-DPPE. ... More
Novel fluorescent phospholipids for assays of lipid mixing between membranes.
AuthorsSilvius JR, Leventis R, Brown PM, Zuckermann M
JournalBiochemistry
PubMed ID3663589
A series of fluorescent phospholipids has been synthesized, by a general and versatile procedure, with various fluorescent groups attached to the methyl-terminal half of one acyl chain in an otherwise normal phospholipid structure. Phospholipids labeled with (dialkylamino)coumarin moieties, and to a slightly lesser extent those labeled with a bimane group, ... More
Thioacetylation method of protein sequencing: derivatization of 2-methyl-5(4H)-thiazolones for high-performance liquid chromatographic detection.
AuthorsStolowitz ML, Paape BA, Dixit VM
JournalAnal Biochem
PubMed ID2683860
A reinvestigation of the thioacetylation method of protein sequencing (G. A. Mross and R. F. Doolittle (1971) Fed. Proc. 30, 1241. G. A. Mross and R. F. Doolittle (1977) in Advanced Methods in Protein Sequence Determination (Needleman, S. B., Ed.), pp. 1-20, Springer, Berlin) has revealed that 2-methyl-5(4H)-thiazolones, prepared by ... More
A fluorescence energy transfer method for analyzing protein oligomeric structure: application to phospholamban.
AuthorsLi M, Reddy LG, Bennett R, Silva ND, Jones LR, Thomas DD
JournalBiophys J
PubMed ID10233073
We have developed a method using fluorescence energy transfer (FET) to analyze protein oligomeric structure. Two populations of a protein are labeled with fluorescent donor and acceptor, respectively, then mixed at a defined donor/acceptor ratio. A theoretical simulation, assuming random mixing and association among protein subunits in a ring-shaped homo-oligomer, ... More
Reversed-phase high-performance liquid chromatography separation of dimethylaminoazobenzene sulfonyl- and dimethylaminoazobenzene thiohydantoin-amino acid derivatives for amino acid analysis and microsequencing studies at the picomole level.
AuthorsStocchi V, Piccoli G, Magnani M, Palma F, Biagiarelli B, Cucchiarini L
JournalAnal Biochem
PubMed ID2499218
A simple and fast reversed-phase high-performance liquid chromatographic method has been developed for the complete separation of 35 dimethylaminoazobenzene sulfonyl (DABS)-amino acids and by-products. This method allows simultaneous determination of primary and secondary amino acids which can be present in protein and peptide hydrolysates and also detects the presence of ... More
Semiautomated clone verification by real-time PCR using molecular beacons.
Authorsvan Schie RC, Marras SA, Conroy JM, Nowak NJ, Catanese JJ, de Jong PJ
JournalBiotechniques
PubMed ID11126133
Conventional, high-throughput PCR analysis of common elements utilizing numerous primer sets and template DNA requires multiple rounds of PCR to ensure optimal conditions. Laborious gel electrophoresis and staining is then necessary to visualize amplification products. We propose novel multicolor molecular beacons, to establish a high-throughput, PCR-based sequence tagged site (STS) ... More
Depolymerization of phospholamban in the presence of calcium pump: a fluorescence energy transfer study.
AuthorsReddy LG, Jones LR, Thomas DD
JournalBiochemistry
PubMed ID10194307
Phospholamban (PLB), a 52-amino acid protein, regulates the Ca-ATPase (calcium pump) in cardiac sarcoplasmic reticulum (SR) through PLB phosphorylation mediated by beta-adrenergic stimulation. The mobility of PLB on SDS-PAGE indicates a homopentamer, and it has been proposed that the pentameric structure of PLB is important for its regulatory function. However, ... More
Site-directed mutagenesis probing the catalytic role of arginines 165 and 166 of human cytomegalovirus protease.
AuthorsLiang PH, Brun KA, Feild JA, O'Donnell K, Doyle ML, Green SM, Baker AE, Blackburn MN, Abdel-Meguid SS
JournalBiochemistry
PubMed ID9558326
Human cytomegalovirus (CMV) is a member of the Herpesviridae family of viruses that also includes herpes simplex viruses (HSV-1 and HSV-2), varicella-zoster virus (VZV), human herpes virus-6, 7, and 8 (HHV-6, HHV-7, and HHV-8), and Epstein-Barr virus (EBV). Each member of this family encodes a serine protease that is a ... More