Determination of picomolar concentrations of metal ions using fluorescence anisotropy: biosensing with a "reagentless" enzyme transducer.
AuthorsThompson RB, Maliwal BP, Feliccia VL, Fierke CA, McCall K
JournalAnal Chem
PubMed ID9844569
Because of their high affinity and selectivity, metalloproteins can be used as transducers in novel sensors, i.e., biosensors, for the determination of trace levels of metal ions in solution. Here, we exploit carbonic anhydrase to determine picomolar to nanomolar concentrations of free transition metal ions by fluorescence anisotropy (polarization) in ... More
Endpoint fluorometric assays for determining activities of carnitine palmitoyltransferase and citrate synthase.
AuthorsHassett RP, Crockett EL
JournalAnal Biochem
PubMed ID11078597
4-(Aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole is not specific for labeling of sulfhydryl groups in proteins as it may also react with phenolic hydroxyl groups and amino groups.
AuthorsHusted LB, Sørensen ES, Sottrup-Jensen L
JournalAnal Biochem
PubMed ID12633619
A new HPLC micromethod to measure total plasma homocysteine in newborn.
AuthorsBartesaghi S, Accinni R, De Leo G, Cursano CF, Campolo J, Galluzzo C, Vegezzi PG, Parodi O
JournalJ Pharm Biomed Anal
PubMed ID11248510
'Total plasma homocysteine (tHcy) in children may be an useful biochemical marker for genetic risk of premature cardiovascular disease. We reported a rapid, isocratic HPLC method able to process very small amount of newborn plasma samples. A blood sample from heel capillary circulation was collected, using a heparinized capillary glass ... More
Selectivity and sensitivity of fluorescence lifetime-based metal ion biosensing using a carbonic anhydrase transducer.
AuthorsThompson RB, Maliwal BP, Fierke CA
JournalAnal Biochem
PubMed ID9918670
'A key performance criterion for metal ion determinations in complex media like serum, cytoplasm of the cell, and sea water is selectivity: the ability to determine the analyte(s) of interest, in the presence of relatively high concentrations of interferents. Cu(II), Zn(II), Cd(II), Co(II), and Ni(II) may be determined by changes ... More
Cysteine-reactive fluorescence probes of catalytic sites of ATP synthase.
'We searched for new fluorescent probes of catalytic-site nucleotide binding in F(1)F(0)-ATP synthase by introducing Cys mutations at positions in or close to catalytic sites and then reacting Cys-mutant F(1) with thiol-reactive fluorescent probes. Four suitable mutant/probe combinations were identified. beta F410C labeled by 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide (ABD-F) gave very large signal ... More
Fluorogenic reagent for thiols: 4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole.
AuthorsToyo'oka T, Suzuki T, Saito Y, Uzu S, Imai K
JournalAnalyst
PubMed ID2597282
'4-(N,N-Dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) was synthesised for use as a more reactive, thiol-specific fluorogenic reagent than 4-(aminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). The former had negligible fluorescence whereas its thiol derivatives fluoresced intensely at about 510 nm (excitation occurred at about 380 nm). The DBD-F reacted quantitatively with thiols after 10 min at 50 degrees C ... More
Determination of three disulfide bonds and one free sulfhydryl in the beta subunit of (Na,K)-ATPase.
AuthorsKirley TL
JournalJ Biol Chem
PubMed ID2540179
'The oxidation state of the 7 cyst(e)ine residues of the beta subunit of lamb kidney (Na,K)-ATPase was determined. The fluorescent sulfhydryl-reactive reagent 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole was utilized before and after reduction of disulfide bonds to determine the number and location of disulfide bonds present in the beta subunit. Treatment of tryptic peptides ... More
Antichymotrypsin interaction with chymotrypsin. Intermediates on the way to inhibited complex formation.
AuthorsLuo Y, Zhou Y, Cooperman BS
JournalJ Biol Chem
PubMed ID10364215
'Serpins form enzymatically inactive covalent complexes (designated E*I*) with their target proteinases, corresponding most likely to the acyl enzyme that resembles the normal intermediate in substrate turnover. Formation of E*I* involves large changes in the conformation of the reactive center loop (residues P17 to P9'') and of the serpin molecule ... More
Differential fluorescence labeling of cysteinyl clusters uncovers high tissue levels of thionein.
AuthorsYang Y, Maret W, Vallee BL
JournalProc Natl Acad Sci U S A
PubMed ID11331777
'The isolation of thionein (T) from tissues has not been reported heretofore. T contains 20 cysteinyl residues that react with 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide to form fluorescent adducts. In metallothionein (MT) the cysteinyl residues, which are bound to zinc, do not react. However, they do react in the presence of a chelating agent ... More
Reduction and fluorescent labeling of cyst(e)ine-containing proteins for subsequent structural analyses.
AuthorsKirley TL
JournalAnal Biochem
PubMed ID2554752
'Procedures which allow rapid, quantitative, and selective fluorescent labeling of protein cyst(e)ine residues prior to electrophoresis by reaction with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) under mild conditions are described. After labeling, the protein(s) of interest is easily monitored throughout electrophoresis and subsequent electroblotting or electroelution procedures. The stoichiometry of labeling and therefore the ... More
Physicochemical properties of charge isomers of recombinant human superoxide dismutase.
AuthorsKajihara J, Enomoto M, Seya K, Sukenaga Y, Katoh K
JournalJ Biochem (Tokyo)
PubMed ID3241003
'Recombinant human Cu2Zn2SOD expressed in Escherichia coli consisted of mainly three isomers with isoelectric points of 5.14 (A), 5.06 (B), and 4.99 (C). Each isomer was isolated by DEAE-Toyopearl chromatography and the physiochemical properties were investigated. No significant differences in chemical and spectrophotometric properties, such as specific activity, metal contents, ... More
Reversibility of cysteine labeling by 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole.
AuthorsTreuheit MJ, Kirley TL
JournalAnal Biochem
PubMed ID8368486
'The fluorescent cysteine derivative formed upon alkylation of proteins with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) is shown to be unstable under certain conditions. Although previously thought to be an irreversible reaction, the fluorescence and absorbance associated with the ABD-cysteine can be released from the protein by heating at basic pH in the presence ... More
Solution structure of myosin-ADP-MgFn ternary complex by fluorescent probes and small-angle synchrotron X-ray scattering.
AuthorsMaruta S, Aihara T, Uyehara Y, Homma K, Sugimoto Y, Wakabayashi K
JournalJ Biochem (Tokyo)
PubMed ID11011152
'In the presence of excess amounts of fluorine, a physiological divalent cation, magnesium (Mg(2+)), forms a novel phosphate analogue, magnesium fluoride (MgFn). Park et al. [Biochim. Biophys. Acta 1430, 127-140 (1999)] previously demonstrated that MgADP. MgFn forms a complex with myosin subfragment-1 (S-1), and the S-1.ADP. MgFn ternary complex mimics ... More
Assay of plasma homocysteine: light sensitivity of the fluorescent 7-benzo-2-oxa-1, 3-diazole-4-sulfonic acid derivative, and use of appropriate calibrators.
'The recognition of homocysteine as a vascular risk factor has led to increased clinical interest in assaying plasma homocysteine concentrations. Our aim was to improve the reliability of a widely used assay based on HPLC of the fluorescent 7-benzo-2-oxa-1, 3-diazole-4-sulfonic acid (SBD) derivative. We found that SBD derivatives of homocysteine, ... More
Conformational changes at the highly reactive cystein and lysine regions of skeletal muscle myosin induced by formation of transition state analogues.
AuthorsMaruta S, Homma K, Ohki T
JournalJ Biochem (Tokyo)
PubMed ID9722668
'Myosin forms stable ternary complexes with Mg2+-ADP and phosphate analogues of aluminum fluoride (AlF4-), beryllium fluoride (BeFn), and scandium fluoride (ScFn). These complexes are distinct from each other and may mimic different transient states in the ATPase cycle [Maruta et al. (1993) J. Biol. Chem. 268, 7093-7100]. Regions of skeletal ... More
Derivatization of thiol-containing compounds.
AuthorsShimada K, Mitamura K
JournalJ Chromatogr B Biomed Appl
PubMed ID7820279
'The determination of thiol-containing compounds in biological fluids is important in biochemistry and clinical chemistry. In this paper, derivatization reagents for thiols are reviewed with respect to their reactivity, selectivity, spectroscopic characteristics and their applicability especially to high-performance liquid chromatography. Derivatization used in ultraviolet and electrochemical detection. The derivatization reagents ... More
Derivatization with fluorogenic benzofurazan reagents of amino acid enantiomers and their separation on a Pirkle type column.
AuthorsImai K, Fukushima T
JournalBiomed Chromatogr
PubMed ID8305859
'L- and D-Amino acids (Leu or Phe) were derivatized with fluorogenic reagents, 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F), 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (ABD-F) and 5-(N,N-dimethylamino)naphthalene-1-sulfonylchloride (DNS-CI), and separated on a Pirkle type column, Sumichiral OA 2500 (S) ((S)-1-naphthylglycyl-3,5-dinitrophenylamide silica gel) with a mobile phase of 20 mM ammonium acetate in methanol. The fluorometric detection of ... More
Structural studies of the contractile tail sheath protein of bacteriophage T4. 1. Conformational change of the tail sheath upon contraction as probed by differential chemical modification.
AuthorsTakeda S, Arisaka F, Ishii S, Kyogoku Y
JournalBiochemistry
PubMed ID2143079
'Differential chemical modifications of tyrosine residues of the tail sheath protein, gp18, were performed to elucidate the structural change of the tail sheath upon contraction. Tyrosine residues of monomeric gp18, extended tail sheath, and contracted tail sheath were nitrated by tetranitromethane, and the modified tyrosine residues in each state of ... More
Fluorescent peptide probes for high-throughput measurement of protein phosphatases.
AuthorsNoble JE, Ganju P, Cass AE
JournalAnal Chem
PubMed ID12720338
'A homogeneous microplate assay for the serine/threonine protein phosphatases PP1 and PP2A, employing fluorescent-labeled phosphopeptides, has been developed. Phosphopeptides derived from a phosphoacceptor site in myelin basic protein were designed with a cysteine adjacent to the phosphoresidue, allowing site-selective labeling with dyes. The fluorescence emission from the environmentally sensitive fluorophore ... More
Production of functionalized single-chain Fv antibody fragments binding to the ED-B domain of the B-isoform of fibronectin in Pichia pastoris.
AuthorsMarty C, Scheidegger P, Ballmer-Hofer K, Klemenz R, Schwendener RA
JournalProtein Expr Purif
PubMed ID11162401
'The Pichia pastoris expression system was used to produce functionalized single-chain antibody fragments (scFv) directed against the ED-B domain of the B-fibronectin (B-Fn) isoform which was found to be present only in newly formed blood vessels during tumor angiogenesis. Therefore, scFv antibody fragments recognizing the ED-B domain are potential markers ... More
Glutathione depletion induces giant DNA and high-molecular-weight DNA fragmentation associated with apoptosis through lipid peroxidation and protein kinase C activation in C6 glioma cells.
AuthorsHiguchi Y, Matsukawa S
JournalArch Biochem Biophys
PubMed ID10049497
'Glutathione (GSH) depletion caused by l-buthionine-(S,R)-sulfoximine (BSO) induced apoptosis that was recognized by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick endo-labeling (TUNEL), nuclear DNA staining with fluorescence dye, and internucleosomal DNA fragmentation in C6 rat glioma cells. The BSO-induced cell death was associated with caspase-3 activation. Lipid peroxidation and protein kinase C ... More
Removal of the fluorescent 4-(aminosulfonyl)-2,1,3-benzoxadiazole label from cysteine-containing peptides.
AuthorsLauren SL, Treuheit MJ
JournalJ Chromatogr A
PubMed ID9542125
'The complete removal of the fluorescent cysteine derivative 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) from an intact protein has not been demonstrated even after extended treatment with a reducing agent. It has been suggested that this may be due to incomplete denaturation under the conditions employed. We were interested in investigating this phenomenon utilizing ... More
Inactivation of (Na+,K+)-ATPase by beta-mercaptoethanol. Differential sensitivity to reduction of the three beta subunit disulfide bonds.
AuthorsKirley TL
JournalJ Biol Chem
PubMed ID2155215
'The relative rates of reduction of disulfide bonds in sheep kidney (Na+,K+)-ATPase were determined in order to test the hypothesis that cleavage of a single disulfide bond in the beta subunit is responsible for loss of ATPase activity upon treatment with high concentrations of beta-mercaptoethanol at elevated temperatures (Kawamura, M., ... More
Gating charge displacement in voltage-gated ion channels involves limited transmembrane movement.
AuthorsChanda B, Asamoah OK, Blunck R, Roux B, Bezanilla F
JournalNature
PubMed ID16094369
'Voltage-gated ion channels are responsible for generating electrical impulses in nerves and other excitable cells. The fourth transmembrane helix (S4) in voltage-gated channels is the primary voltage-sensing unit that mediates the response to a changing membrane electric field. The molecular mechanism of voltage sensing, particularly with respect to the magnitude ... More
An introduction to methods for analyzing thiols and disulfides: Reactions, reagents, and practical considerations.
AuthorsHansen RE, Winther JR,
JournalAnal Biochem
PubMed ID19664585
'This review outlines the basic issues to consider when dealing with biochemical and cellular aspects of thiol–disulfide chemistry. The overall focus is on practical aspects, including typical biochemical experimental conditions and caveats to consider in interpreting results. Reagents for thiol derivatization and disulfide reduction are evaluated and compared, and we ... More
Evaluation and intermethod comparison of the Bio-Rad high-performance liquid chromatographic method for plasma total homocysteine.
Fluorometric assay of thiols with fluorobenzoxadiazoles.
AuthorsImai K, Toyo'oka T
JournalMethods Enzymol
PubMed ID3155390
Isolation and characterization of cysteine-containing regions of proteins using 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole and high-performance liquid chromatography.
AuthorsToyo'oka T, Imai K
JournalAnal Chem
PubMed ID4037347
Site-specific reactivities of cysteine residues in horse L-apoferritin.
AuthorsTakeda S, Yamaki M, Ebina S, Nagayama K
JournalJ Biochem (Tokyo)
PubMed ID7608110
Recombinant horse L-apoferritin and its mutants were used to compare the reactivities of two different cysteinyl residues with 7-fluoro-4-sulfamoyl-2,1,3-benzoxadiazole (ABD-F), p-chloromercuribenzoic acid (PCMB), N-(9-acridinyl)maleimide (NAM), and 4-maleimido-2,2,6,6-tetramethylpiperidine-N-oxyl (NEM-TEMPO). ABD-F selectively reacted with cysteine 52 (C52), which is located on the inner surface of the peptide shell of apoferritin. In contrast, ... More
Ultraviolet and fluorescence derivatization reagents for carboxylic acids suitable for high performance liquid chromatography: a review.
AuthorsMukherjee PS, Karnes HT
JournalBiomed Chromatogr
PubMed ID8879524
Pre- and postcolumn derivatization with a suitable chromophore or fluorophore have often been utilized to enhance the sensitivity and selectivity of detection for analytes possessing a carboxyl group. The major classes of UV and fluorescent derivatization reagents include the coumarin analogues, alkyl halides, diazoalkanes, and amines. The derivatization reaction conditions, ... More
Location of disulfide bonds in the Na+, K(+)-ATPase alpha subunit.
AuthorsGevondyan NM, Gevondyan VS, Modyanov NN
JournalBiochem Mol Biol Int
PubMed ID8395926
For localizing S-S bonds in the pig kidney Na+,K(+)-ATPase alpha subunit, cystine-containing peptides (V-1, VII-1, and VII-2), obtained in our previous study from the enzyme's tryptic digest, were analysed. Chemical modification of the cystine-containing peptides performed at cysteine residues involved successive alkylation, first with radioactive iodoacetic acid and then with ... More
Chiral separation of homocysteine by derivatization with 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole followed by capillary electrophoresis using gamma-cyclodextrin.
AuthorsKim IJ, Park SJ, Kim HJ
JournalJ Chromatogr A
PubMed ID10845800
Homocysteine was derivatized with 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (ABD-F) to form an inclusion complex with cyclodextrin and to facilitate UV detection. ABD-homocysteine showed interaction with beta- and gamma-cyclodextrin in capillary electrophoresis at pH 2.25 as indicated by the decreased migration time. However, chiral separation of D,L-ABD-homocysteine was observed using gamma-CD only. Optimal separation ... More
Characterization of cysteine residues of glutathione S-transferase P: evidence for steric hindrance of substrate binding by a bulky adduct to cysteine 47.
AuthorsNishihira J, Ishibashi T, Sakai M, Nishi S, Kumazaki T, Hatanaka Y, Tsuda S, Hikichi K
JournalBiochem Biophys Res Commun
PubMed ID1417864
Glutathione S-transferase P (GST-P) lost the enzymatic activity by 7-fluoro-4-sulfamoyl-2, 1, 3-benzodiazole (ABD-F), a thiol-group chemical modifier, but did not by methylmethanethiol-sulfonate. Both ABD-F and methylmethanethiolsulfonate reacted with Cys47 and Cys101. These two cysteine residues were site-directedly mutated with serine residues. Only the Cys101Ser lost the enzymatic activity by the ... More
Application of micro-scale liquid chromatography with fluorescence detection to the determination of thiols.
AuthorsLing BL, Dewaele C, Baeyens WR
JournalJ Chromatogr
PubMed ID2258395
Different thiol compounds of biological and pharmacological interest were separated on a packed reversed-phase fused-silica capillary column and determined with fluorescence detection. Conventional inexpensive liquid chromatographic equipment could be adapted for such purposes, providing a highly efficient analytical system. The different compounds were derivatized with the fluorogenic reagents SBD-F and ... More
The occurrence of a mutant dimerizable histone H5 in Japanese quail erythrocytes.
An allelic variant of linker histone H5 has been found in the erythrocytes of Japanese quail (Coturnix japonica) descended from a small group of feral birds captured on the island of Hawaii. This variant spontaneously forms protein dimers in vitro in the absence of reducing agents. That this depends upon ... More
An enzymatic assay of chloramphenicol coupled with fluorescence reaction.
AuthorsYamato S, Sugihara H, Shimada K
JournalChem Pharm Bull (Tokyo)
PubMed ID2279294
An alternative method for the assay of chloramphenicol using an enzymatic reaction coupled with a fluorescence detection system has been developed. Chloramphenicol was enzymatically acetylated by chloramphenicol acetyltransferase in the presence of acetyl-coenzyme A (acetyl-CoA) as the acetyl-donor, after which the liberated CoA-SH was derivatized with a fluorogenic reagent, 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole. ... More
On-column derivatization for the analysis of homocysteine and other thiols by capillary electrophoresis with laser-induced fluorescence detection.
AuthorsKang SH, Wei W, Yeung ES
JournalJ Chromatogr B Biomed Sci Appl
PubMed ID10985576
On-column derivatization and capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection has been developed for the fully automated assay of homocysteine and other thiols. The unique feature of this CE technique comes from the direct injection of a sample including homocysteine, enabling the derivatization with 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadizole (ABD-F) to be accomplished ... More
Determination of thiols of biological and pharmacological interest by high-performance thin-layer chromatography and fluorescence scanning densitometry.
AuthorsLing BL, Baeyens WR, Del Castillo B, Imai K, De Moerloose P, Stragier K
JournalJ Pharm Biomed Anal
PubMed ID2490555
The application of high-performance thin-layer chromatography (HPTLC) with fluorescence scanning densitometry provides a simple, rapid and reliable system for the qualitative and quantitative determination of several thiols of biological and pharmacological interest. The determination of a mixture of thiols (captopril, coenzyme A, cysteamine, cysteine and glutathione), together with their disulphides ... More
N-methyl-D-glucamine improves the laser-induced fluorescence capillary electrophoresis performance in the total plasma thiols measurement.
AuthorsZinellu A, Carru C, Galistu F, Usai MF, Pes GM, Baggio G, Federici G, Deiana L
JournalElectrophoresis
PubMed ID12929176
We describe an ultrarapid capillary electrophoresis with laser-induced fluorescence (CE-LIF) method for total plasma thiols measurement. Reduced thiols by 10% tri-n-butylphosphine (TBP) were derivatized in 10 min at room temperature with 5-iodoacetamidofluorescein (5-IAF) as fluorescent reagent. We show that CE-LIF allows a baseline separation of total plasma cysteinylglycine, homocysteine, cysteine, ... More
Determination of homocysteine and other thiols in human plasma by capillary electrophoresis.
AuthorsKang SH, Kim JW, Chung DS
JournalJ Pharm Biomed Anal
PubMed ID9226573
A new capillary electrophoresis (CE) assay for thiols in human plasma, including homocysteine, which is an indicator of several clinical states has been developed. The thiols were derivatized quantitatively at 50 degrees C, pH 8.0 with a fluorogenic reagent, ADB-F (4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole), which is about 30 times faster compared to the ... More
Use of enhancers in the HPTLC fluorescence analysis of thiols.
AuthorsLing BL, Baeyens WR, Del Castillo B, Stragier K, Marysael H, De Moerloose P
JournalJ Pharm Biomed Anal
PubMed ID2490556
Several thiols of biological and pharmacological interest, including glutathione, coenzyme A, acetylcysteine and captopril were derivatized with the fluorogenic reagents SBD-F and ABD-F and analysed by high-performance thin-layer chromatography (HPTLC)-fluorodensitometry on silica gel 60 plates, using isopropyl ether-methanol-water-acetic acid (9:8:2:1, v/v/v/v) as the developing solvent. The luminescence was considerably increased ... More
Analytical methods to investigate glutathione and related compounds in biological and pathological processes.
AuthorsCamera E, Picardo M
JournalJ Chromatogr B Analyt Technol Biomed Life Sci
PubMed ID12450659
Reduced glutathione (GSH, gamma-L-glutamyl-L-cysteinylglycine) is a fundamental low-molecular mass antioxidant that serves several biological functions. Upon enzymatic and non-enzymatic oxidation, GSH forms glutathione disulfide (GSSG) and, under particular conditions, may generate other oxidative products. The determination of GSH, its precursors, and metabolites in several bio-matrices is a useful tool in ... More
Fluorescence microscopy of stimulated Zn(II) release from organotypic cultures of mammalian hippocampus using a carbonic anhydrase-based biosensor system.
AuthorsThompson RB, Whetsell WO, Maliwal BP, Fierke CA, Frederickson CJ
JournalJ Neurosci Methods
PubMed ID10704669
We demonstrate here that electrical stimulation of organotypic cultures of rat hippocampus results in the prompt release of significant amounts of Zn(II) by a fluorescence microscopic method. The fluorescence imaging of free Zn(II) is achieved using a highly selective biosensing indicator system consisting of human apo-carbonic anhydrase II (apoCAII) and ... More
Determination of thiols and disulfides in normal rat tissues and hamster pancreas treated with N-nitrosobis(2-oxopropyl)amine using 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole and ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate.
AuthorsToyo'oka T, Furukawa F, Suzuki T, Saito Y, Takahashi M, Hayashi Y, Uzu S, Imai K
JournalBiomed Chromatogr
PubMed ID2590728
Biological thiols and disulfides in rat and hamster tissues were simultaneously determined by HPLC-fluorescence detection using 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) and ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F). The coefficients of variation (CV) of the method for reduced glutathione (GSH) and oxidized glutathione (GSSG) in liver and for cysteine (CySH) and cystine (CySSCy) in kidney were ... More
Measurement of thiols in human plasma using liquid chromatography with precolumn derivatization and fluorescence detection.
A liquid chromatography (LC) method for the simultaneous measurement of the main low molecular mass thiols (i.e., cysteine, cysteinylglycine, homocysteine, and glutathione) in human plasma is described. The sample treatment consists of the reduction of disulfide bounds with tri-n-butylphosphine and protein precipitation with trichloroacetic acid followed by precolumn derivatization with ... More
The use of tributylphosphine and 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole in the study of protein sulfhydryls and disulfides.
AuthorsChin CC, Wold F
JournalAnal Biochem
PubMed ID8250214
The use of the reagent tributyl phosphine (Bu3P) to reduce disulfides (Ruegg, U.T., and Rudinger, J., Methods Enzymol. 47, 111-116, 1977) and of 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) to block free sulphydryl groups (Toyo'oka, T., and Imai, K. Anal. Chem. 56, 2461-2464, 1984) is well established in the literature. Since the two reagents ... More
Determination of total homocysteine in dried blood spots using high performance liquid chromatography for homocystinuria newborn screening.
AuthorsFebriani AD, Sakamoto A, Ono H, Sakura N, Ueda K, Yoshii C, Kubota M, Yanagawa J
JournalPediatr Int
PubMed ID15043656
BACKGROUND: The most widely used method for newborn screening for homocystinuria (HCU) is a semi-quantitative bacterial inhibition assay for measuring methionine concentration in dried blood spots (DBS). Because this method has resulted in a number of missed cases due to many factors, we developed a high performance liquid chromatography (HPLC) ... More
Behavior of Cys-707 (SH1) in myosin associated with ATP hydrolysis revealed with a fluorescent probe linked directly to the sulfur atom.
AuthorsHiratsuka T
JournalJ Biol Chem
PubMed ID8227033
4-Fluoro-7-sulfamoylbenzofurazan (ABDF) has a rather small fluorophore that is linked directly to the sulfur atom of thiols without a flexible alkyl chain (Imai, K., and Toyo'oka, T. (1987) Methods Enzymol. 143, 67-75). In the present study I examined the fluorescent and chemical properties of ABDF as an environmentally sensitive probe ... More
Identification of the disulfide bonds in human plasma protein SP-40,40 (apolipoprotein-J).
AuthorsChoi-Miura NH, Takahashi Y, Nakano Y, Tobe T, Tomita M
JournalJ Biochem (Tokyo)
PubMed ID1491011
SP-40,40, a human plasma protein, is a modulator of the membrane attack complex formation of the complement system as well as a subcomponent of high-density lipoproteins. In the present study, the positions of the disulfide bonds in SP-40,40 were determined. SP-40,40 was purified from human seminal plasma by affinity chromatography ... More
Site-specific conjugation of diethylenetriaminepentaacetic acid to recombinant human granulocyte-colony-stimulating factor: preservation of protein structure and function.
AuthorsRalph LD, Ransone CM, Treuheit M, Lauren S, Katta V, Litzinger DC
JournalBiochemistry
PubMed ID7536471
The chelating agent diethylenetriaminepentaacetic acid (DTPA) was conjugated site-specifically to the N-terminus of recombinant human granulocyte-colony-stimulating factor (rhG-CSF) by reaction of the protein with DTPA dianhydride at an initial pH of 6.0. The reaction was efficient in that 84% of the starting rhG-CSF was N-terminally modified and could be purified ... More
Zinc-buffering capacity of a eukaryotic cell at physiological pZn.
AuthorsKrezel A, Maret W
JournalJ Biol Inorg Chem
PubMed ID16924557
In spite of the paramount importance of zinc in biology, dynamic aspects of cellular zinc metabolism remain poorly defined at the molecular level. Investigations with human colon cancer (HT-29) cells establish a total cellular zinc concentration of 264 microM. Remarkably, about 10% of the potential high-affinity zinc-binding sites are not ... More
[Determination of total plasma homocysteine and other aminothiols by liquid chromatography coupled to the detection by fluorescence]
AuthorsJacob N, Guillaume L, Garçon L, Foglietti MJ
JournalAnn Biol Clin (Paris)
PubMed ID9499919
Plasma concentrations of homocysteine, cysteine, cysteinylglycine and glutathione were measured using a HPLC technique with fluorescence detection of the derivatives obtained with 7-fluoro-2,1,3-benzoxadiazole-4-sulfonamide (ABD-F). Blood was drawn into chilled EDTA-evacuated tubes. After centrifugation at 4 degrees C without delay, plasma samples were kept frozen at -20 degrees C until analysis. ... More
Enantiomeric separation and sensitive determination of D,L-amino acids derivatized with fluorogenic benzofurazan reagents on Pirkle type stationary phases.
AuthorsFukushima T, Kato M, Santa T, Imai K
JournalBiomed Chromatogr
PubMed ID7734928
The enantiomeric separations of D,L-amino acids derivatized with fluorogenic reagents, 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), 4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) and 4-aminosulphonyl-7-fluoro-2,1,3-benzoxadiazole (ABD-F) by high-performance liquid chromatography (HPLC) on various Pirkle type chiral stationary phases (CSPs, Sumichiral OA series) with citric acid in methanol as a mobile phase were studied. Since the least retention and no ... More
Studies on the mechanism of the UVA light-dependent loss of glutathione reductase activity in human lenses.
AuthorsLinetsky M, Hill JM, Chemoganskiy VG, Hu F, Ortwerth BJ
JournalInvest Ophthalmol Vis Sci
PubMed ID12939310
PURPOSE: To determine the mechanism that leads to the UVA light-dependent loss of glutathione reductase (GR) activity in human lens (HL). METHODS: Both the HL water-soluble (WS) fraction and yeast GR were irradiated with UVA light (200 mW/(cm(2). h) for 1 hour at +20 degrees C, and the specific activity ... More
Characterization of the disulfide bonds and the N-glycosylation sites in the glycoprotein from Rathke's gland secretions of Kemp's ridley sea turtle (Lepidochelys kempi).
AuthorsChin CC, Krishna RG, Weldon PJ, Wold F
JournalAnal Biochem
PubMed ID8789716
The disulfide bonds and N-glycosylation sites in a glycoprotein from the Rathke's gland secretion of the Kemp's ridley turtle (Lepidochelys kempi) have been characterized with respect to peptide sequences and glycan structures. The glycoprotein constitutes about 70% of the total protein in the secretion, and based on partial sequence information, ... More
Affinity probing of flavin binding sites. 2. Identification of a reactive cysteine in the flavin domain of Escherichia coli DNA photolyase.
AuthorsRaibekas AA, Jorns MS
JournalBiochemistry
PubMed ID7918492
8-(Methylsulfonyl)FAD reacts with a single cysteine residue (Cys293) in the flavin domain of Escherichia coli DNA photolyase to form an 8-(cysteinyl)FAD derivative covalently bound to the protein. About 80% protection against covalent attachment with 8-(methylsulfonyl)FAD was observed in the presence of an equimolar amount of FAD. Flavinylated photolyase retains the ... More
Mutations of either or both Cys876 and Cys888 residues of sarcoplasmic reticulum Ca2+-ATPase result in a complete loss of Ca2+ transport activity without a loss of Ca2+-dependent ATPase activity. Role of the CYS876-CYS888 disulfide bond.
AuthorsDaiho T, Yamasaki K, Saino T, Kamidochi M, Satoh K, Iizuka H, Suzuki H
JournalJ Biol Chem
PubMed ID11438520
Disulfide-containing peptides in pepsin digest of sarcoplasmic reticulum vesicles were identified by using a fluorogenic thiol-specific reagent 4-fluoro-7-sulfamoylbenzofurazan and a reductant tributylphosphine. Sequencing of the purified peptides revealed the presence of a Cys(876)-Cys(888) disulfide bond on the luminal loop connecting the 7th and 8th transmembrane helices (loop 7-8) of the ... More