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PubMed1977

Mutagenic effectiveness and efficiency of sodium azide versus ethyl methanesulfonate in maize: induction of somatic mutations at the yg2 locus by treatment of seeds differing in metabolic state and cell population.

This study was conducted to compare the effectiveness and efficiency of sodium azide tNaN3) and ethyl methanesulfonate (EMS) for inducing somatic mutations at the yg2 locus in maize seeds of two different metabolic states and cell populations. Dormant or presoaked (72 h at 20 degrees C) seeds heterozygous for yg2 locus were treated with different concentrations of either EMS or NaN3. The cell populations with respect to the percentage of cells in G1, S, G2, and M were also determined for seeds of the two metabolic states. Dormant seeds possessed a higher percentage of cells in G1 and the presoaked seeds a higher percentage of cells in S, G2, and M. The frequency of yg2 sectors in leaves 4 and 5 increased with increasing concentration of both mutagens in both dormant and presoaked seeds. Both mutagens were more effective and efficient in the presoaked seeds. NaN3 was more effective than EMS in terms of number of sectors induced per unit of dose. However, EMS was more efficient as determined by sectors induced per unit of seedling injury and clearly had the ability to induce much higher sector frequencies (more than 10 times greater) than NaN3. The low ability of NaN3 (compared to EMS) to induce mutant sectors may be related to the cells not being treated at the optimum time during the cell cycle, but it is more likely due to its low effectiveness for inducing chromosome aberrations.

Azides↗
PubMed1987

Photolabeling of the adipocyte hexose carrier with an aryl azide derivative of maltose.

A nitrophenyl azide derivative of maltose, N-(4-azido-2-nitrophenyl)-maltosylamine (NAP-maltosylamine), was synthesized as a potential photoaffinity label for the hexose carrier of the rat adipocyte. This derivative inhibited 3-O-methylglucose uptake with a Ki of 1.3 mM in the dark, while that of maltose was 10.0 mM. Carbon-14-labeled maltose and NAP-maltosylamine entered adipocytes via the hexose carrier, the latter in a concentrative fashion. Photolysis of NAP-[14C]maltosylamine in the presence of an adipocyte low density microsomal membrane fraction labeled several electrophoretic bands. Among these are a 45 kDa band which showed features expected of the hexose carrier: its labeling was decreased 40% by D- but not L-glucose and pretreatment of intact adipocytes with insulin decreased labeling of the 45 kDa band by 10-40%, as predicted by the translocation theory of insulin-stimulated transport activation. These studies show the suitability of using carbon-1-modified sugar photoaffinity labels as probes for the hexose carrier and possibly of its regulation in rat adipocytes.

Adipose Tissue↗
PubMed1993

Simultaneous determination of cholesterol and cholestanol in human serum by high-performance liquid chromatography using 3-(5,6-methylenedioxy-2-phthalimidyl)benzoyl azide as precolumn fluorescent labelling reagent.

A fluorescent labelling reagent, 3-(5,6-methylenedioxy-2-phthalimidyl) benzoyl azide, designed for the determination of alcohols by precolumn high-performance liquid chromatography, has been applied to the simultaneous determination of cholesterol and cholestanol in human serum. The reagent reacts with cholesterol and cholestanol at 140 degrees C for 10 min to produce the fluorescent derivatives, which can be separated on a reversed-phase column with acetonitrile-ethanol-water (60:35:7.5, v/v) as eluent. The detection limits for cholesterol and cholestanol were 45 and 50 fmol per injection (20 microliters), respectively. The values of cholesterol and cholestanol in normal human sera were 135-212 mg/dl and 137-928 micrograms/dl, respectively.

Azides↗
PubMed2004

Azide binding to yeast cytochrome c peroxidase and horse metmyoglobin: comparative thermodynamic investigation using isothermal titration calorimetry.

Yeast cytochrome c peroxidase (CcP) and horse metmyoglobin (Mb) bind HN3 with similar affinities at 25 degrees C. The pH-independent equilibrium association constants for formation of the CcP.HN3 and Mb.HN3 complexes are (1.05 +/- 0.06)x10(5) and (1.6 +/- 0.8)x10(5) M(-1), respectively. However, the thermodynamic parameters for formation of the two complexes are quite different. The DeltaH0 values for formation of CcP.HN3 and Mb.HN3 are -16.4 +/- 0.7 and -9.0 +/- 0.5 kcal/mol, respectively, and the Delta S0 values are -32 +/- 2 and -16 +/- 2 cal/deg mol, respectively. The proton associated with HN3 is retained in both protein complexes at low pH but dissociates with apparent pKA values of 5.5 +/- 0.2 and > or =8.2 for the Mb.HN3 and CcP.HN3 complexes, respectively. CcP and Mb differ significantly in their reactivity toward the azide anion, N3-. CcP binds N3- very weakly, if at all, and only an upper-limit of 18 +/-5 M(-1) for the pH-independent equilibrium association constant for the CcP.N3- complex can be determined. Mb binds N3- with an association constant of (1.8 +/- 0.1)x10(4) M(-1). The ratio of the equilibrium association constants for HN3 and N3- binding provides a discrimination factor between the neutral and charged forms of the ligand. The discrimination factor is greater than 5800 for CcP but only nine for Mb. Protonation of the distal histidines in the two proteins influences binding of HN3. Protonation of His-64 in Mb enhances HN3 binding due to a gating mechanism while protonation of His-52 in CcP decreases the affinity for HN3 due to loss of base-assisted association of the ligand to the heme iron.

Animals↗
PubMed2006

Application of azide-alkyne cycloaddition 'click chemistry' for the synthesis of Grb2 SH2 domain-binding macrocycles.

Copper (I) promoted [3+2] Huisgen cycloaddition of azides with terminal alkynes was used to prepare triazole-containing macrocycles based on the Grb2 SH2 domain-binding motif, 'Pmp-Ac(6)c-Asn', where Pmp and Ac(6)c stand for 4-phosphonomethylphenylalanine and 1-aminocyclohexanecarboxylic acid, respectively. When cycloaddition reactions were conducted at 1mM substrate concentrations, cyclization of monomeric units occurred. At 2mM substrate concentrations the predominant products were macrocyclic dimers. In Grb2 SH2 domain-binding assays the monomeric (S)-Pmp-containing macrocycle exhibited a K(d) value of 0.23microM, while the corresponding dimeric macrocycle was found to have greater than 50-fold higher affinity. The open-chain dimer was also found to have affinity equal to the dimeric macrocycle. This work represents the first application of 'click chemistry' to the synthesis of SH2 domain-binding inhibitors and indicates its potential utility.

Alkynes↗
PubMed1997

5-iodonaphthyl-1-azide labeling of plasma membrane proteins adjacent to specific sites via energy transfer.

We have examined conditions optimal for 5-iodonaphthyl-1-azide (INA4) labeling of membrane proteins proximal to known membrane sites. Membrane-bound INA can be indirectly activated by energy transfer from visible chromophores. We demonstrate that the efficiency of this sensitized activation is enhanced by use of triplet-forming chromophores such as eosin and by deoxygenation. Variation of sensitized activation efficiency with INA concentration indicates that the critical distance for eosin-INA energy transfer in solution is 8-14 A. We suggest that photosensitization occurs through triplet exchange and present an improved labeling protocol based on these findings. This protocol was used to examine whether different accessory proteins are associated with isolated and crosslinked Type I Fc epsilon receptors on 2H3 rat basophilic leukemia cells. 2H3 cells were incubated with eosin-conjugated IgE and irradiated at 514 nm yielding [125I]INA derivatized peptides at 53, 38, 34, and 29 kDa. Crosslinking IgE with mouse anti-rat IgE prior to irradiation labeled three additional proteins at 60, 54, and 43 kDa. These results demonstrate the utility of sensitized INA labeling in characterizing protein-protein interactions in membranes of intact cells and indicate the importance of considering photophysical factors when selecting sensitizers and reaction conditions. We discuss estimation of the size of the membrane region surrounding a sensitizing chromophore within which INA labeling of membrane proteins occurs.

Animals↗
PubMed1989

A yeast strain with mutated beta-subunits of mitochondrial ATPase-ATPsynthase: high azide and bicarbonate sensitivity of the ATPase activity.

A phenotypic revertant with modified beta-subunits of mitochondrial ATPase-ATP synthase has been obtained for the first time by selection from a beta-less mutant of the yeast Schizosaccharomyces pombe. Contrary to the parental mutant, the phenotypic revertant grows on glycerol, has normal respiratory activity and shows immunodetectable beta-subunits. However the kinetic properties of its submitochondrial particles ATPase activity differ markedly from those of the wild strain. The optimal pH is increased by about one unit. The maximal rate of the revertant ATPase activity at pH 8.5 is 4 to 5-fold lower than that of the wild strain, but it can be greatly increased upon addition of bicarbonate whereas the wild strain is completely insensitive to this anion. Furthermore the revertant ATPase activity is much more sensitive to azide inhibition. The results suggest that ADP dissociation is the rate-limiting step of ATP hydrolysis by the revertant.

Azides↗
PubMed2002

The effect of anions on azide binding to myoglobin: an unusual functional modulation.

The effect of increasing concentrations of several anions on the azide (N(-)(3)) binding properties of sperm whale and horse ferric myoglobin has been studied. Surprisingly, a number of anions may act as heterotropic effectors, decreasing the affinity of myoglobins for N(-)(3), in the following order: ClO(-)(4)=I(-)>Br(-)>Cl(-) and SO(2-)(4), which mirrors the increase in their charge density. The largest effects were measured using ClO(-)(4) and I(-), which produce a 4-fold and 8-fold reduction of the N(-)(3) binding affinity in horse and sperm whale myoglobins, respectively. A dissociation equilibrium constant (K(d)) ranging from 150 to 250 mM was estimated for ClO(-)(4) and I(-) binding to myoglobins. In order to analyse the molecular mechanism producing the reduction of the N(-)(3) binding affinity to ferric myoglobin, the potential anionic binding sites within ferric myoglobin were investigated by a molecular modelling study using the program Grid. Analysis of the theoretical results suggests two particularly favourable binding sites: the first, next to the distal side of the haem, whose occupancy might alter the electrostatic potential surrounding the bound N(-)(3); the second, involving residues of helices B and G which are far from the haem iron atom, thus implying a long range effect on the bound N(-)(3). Based on the evidence that no significant conformational changes are found in the three-dimensional structures of N(-)(3)-free and N(-)(3)-bound myoglobin and on previous results on N(-)(3) binding to ferric myoglobin mutants in CD3 positions, we favour the first hypothesis, suggesting that the functional heterotropic modulation of monomeric myoglobin is mainly depending on a decrease of the positive charge density induced by the binding of anions to the haem distal side.

Allosteric Regulation↗
PubMed2002

A concise route to triazolobenzodiazepine derivatives via a one-pot alkyne-azide cycloaddition reaction.

A new and efficient one-pot synthesis of [1,2,3]triazolo[1,5-a][1,4] benzodiazepin-6(4H)-ones is described starting from readily available anthranilic acids. A small array of the title compounds were assembled via a four-step sequence involving diazotisation, azide addition followed by amide bond formation employing polymer supported carbodiimide and subsequent 1,3-dipolar cycloaddition reaction.

Alkynes↗
PubMed2000

Spectroscopic properties and ligand field analysis of cis-diazido(1,4,8,11-tetraazacyclotetradecane)chromium(III) azide.

The emission and excitation spectra of cis-[Cr(cyclam)(N3)2](N3) (cyclam = 1,4,8,11-tetraazacyclotetradecane) taken at 77 K are reported. The infrared and visible spectra at room temperature are also measured. The vibrational intervals due to the electronic ground state are extracted from the far-infrared and emission spectra. The ten electronic bands due to spin-allowed and spin-forbidden transitions are assigned. Using the observed transitions, a ligand field analysis has been performed to determine the bonding property of azido group in the chromium(III) complex. It is found that azide ligand has weak sigma- and pi-donor properties toward chromium(III) ion. The zero-phonon line in the excitation spectrum splits into two components by 249 cm(-1), and the large 2Eg splitting can be reproduced by the ligand field theory.

Absorption↗
PubMed2001

PM3, AM1, MNDO and MINDO3 semi-empirical IR spectra simulations for compounds of interest for Titan's chemistry: diazomethane, methyl azide, methyl isocyanide, diacetylene and triacetylene.

Four semi-empirical methods (PM3, AM1, MNDO and MINDO3) have been tested to find the best auxiliary tool for the gas chromatography/Fourier transform IR spectroscopy/mass spectrometry (GC/FTIR/MS) identification of five compounds of interest for Titan's atmospheric chemistry as test compounds: diacetylene, triacetylene, diazomethane, methyl azide, methyl isocyanide. Of the four methods, MINDO3 can be considered as the most appropriate method to facilitate the identification of such and similar compounds, since (1) the simulated IR spectra best match the experimental spectra for four compounds of five studied; and (2) MINDO3 provides the best linearity between the calculated and experimental frequencies (correlation coefficient of 0.995; a scaling factor of 0.84 can be applied to afford better correspondence between the calculated and experimental wavenumbers). None of the semi-empirical methods tested is able to predict (even approximately) infrared band intensities, and therefore a spectral intensity pattern.

Acetylene↗
PubMedDate not supplied

Heterobifunctional cross-linking agents incorporating perfluorinated aryl azides.

New heterobifunctional cross-linking reagents were developed that possess a photoactive tetrafluorinated phenyl azide as the photoactive terminus and a chemically reactive succinimidyl ester as the electrophilic terminus. These reagents, succinimidyl N-(4-azido-2,3,5,6-tetrafluorobenzoyl)tyrosinate (9) and succinimidyl 2-(4-azido-2,3,5,6-tetrafluorophenyl)thiazole-4-carboxylate (15), were designed to possess either an 125I or 35S radiolabel, respectively. In a biochemical study, the latter reagent was coupled to Lys-75 of calmodulin (CaM), and the radioiodinated monoadduct was photochemically cross-linked, in a calcium-dependent manner, to the porcine erythrocyte plasma membrane Ca2+, Mg2(+)-ATPase. Densitometry scans of the gel indicated a reproducible 22% cross-linking of the CaM with one of the Ca2+,Mg2(+)-ATPase bands. Since the purification of the Ca2+,Mg2(+)-ATPase results in micelles having Ca2+,Mg2(+)-ATPase with its CaM binding site oriented both to the inside and outside of the micelle, the amount of Ca2+,Mg2(+)-ATPase available for cross-linking was reduced by approximately half, suggesting that the actual cross-linking efficiency was on the order of 40%.

Animals↗
PubMedDate not supplied

Synthesis and binding of new polyfluorinated aryl azides to alpha-chymotrypsin. New reagents for photoaffinity labeling.

The preparation of several new, water-soluble, polyfluorinated aryl azide reagents for use in photoaffinity labeling studies is described. The ability of some of these reagents and their nonfluorinated controls to noncovalently bind to alpha-chrymotrypsin is reported. It is found that polyfluorination does not interfere with ligand binding.

Affinity Labels↗
PubMedDate not supplied

N-hydroxysuccinimide ester functionalized perfluorophenyl azides as novel photoactive heterobifunctional cross-linking reagents. The covalent immobilization of biomolecules to polymer surfaces.

The synthesis of N-hydroxysuccinimide (NHS) functionalized perfluorophenyl azides (PFPAs) 2 and 3 is described together with a general method for the covalent modification of polymer surfaces using heterobifunctional, photoactivable cross-linking reagents 1 and 3. The NHS-active ester group becomes covalently attached to the polymer surface via an efficient CH bond insertion reaction of the photogenerated, highly reactive nitrene intermediate derived from the PFPA. The NHS ester is capable of further reaction with a variety of primary amine-containing reagents including biomolecules by way of amide formation. The method is illustrated as follows. Photolysis of polystyrene (PS) and poly-(3-octylthiophene) (P3OT) thin films spin-coated with NHS PFPA ester 1 or 3 gave films 8 or 9, respectively. Each film was then exposed to an aqueous solution of horseradish peroxidase (HRP), giving films 10 or 11, respectively. The amounts of HRP immobilized on PS and P3OT were calculated from enzyme activity assays to be 0.5 +/- 0.1 ng/mm2 for 10a, 1.0 +/- 0.2 ng/mm2 for 11a, 0.2 +/- 0.1 ng/mm2 for 10b, 0.3 +/- 0.1 ng/mm2 for 11b. Using this surface functionalization methodology, biotin-streptavidin-biotin-HRP was constructed on the PS film. The storage stability of HRP thus immobilized through the extended linker, biotin-streptavidin-biotin, was enhanced as compared to that of HRP directly immobilized on the PS surface.

Azides↗
PubMed1993

Evaluation of nitrogen nuclear hyperfine and quadrupole coupling parameters for the proximal imidazole in myoglobin-azide, -cyanide, and -mercaptoethanol complexes by electron spin echo envelope modulation spectroscopy.

Electron spin echo envelope modulation (ESEEM) spectroscopy and computer simulation of spectra has been used to evaluate the nitrogen nuclear hyperfine and quadrupole coupling parameters for the proximal imidazole nitrogen directly coordinated to iron in three low-spin heme complexes, myoglobin-azide, -cyanide, and -mercaptoethanol (MbN3, MbCN, and MbRS). The variability in the weak electron-nuclear coupling parameters reveals the electronic flexibility within the heme group that depends on properties of the exogenous ligands. For example, the isotropic component of the nitrogen nuclear hyperfine coupling ranges from 4.4 MHz for MbN3 to 2.2 MHz for both MbCN and MbRS. The weaker coupling in MbCN and MbRS is taken as evidence for delocalization of unpaired electron spin from iron into the exogenous anionic ligands. The value of e2Qq, the nuclear quadrupole coupling constant for the axial imidazole nitrogen in MbCN and MbRS, was 2.5 MHz but was significantly larger, 3.2 MHz, in MbN3. This large value is considered evidence for a weakened sigma bond between the proximal imidazole and ferric iron in this form, and for a feature contributing to the origin of the high spin-low spin equilibrium exhibited by MbN3 [Beetlestone, J., & George, P. (1964) Biochemistry 5, 707-714]. The ESEEM results have allowed a correlation to be made between the orientation of the g tensor axes, the orientation of the p-pi orbital of the proximal imidazole nitrogen, and sigma- and pi-bonding features of the axial ligands. Furthermore, the proximal imidazole is suggested to act as a pi-acceptor in low-spin heme complexes in order to support strong sigma electron donation from the lone pair orbital to iron. An evaluation of the nitrogen nuclear hyperfine coupling parameters for the porphyrin pyrrole sites in MbRS reveals a large inequivalence in isotropic components consistent with an orientation of rhombic axes (and g tensor axes) that eclipses the Fe-Npyrrole vector directions.

Animals↗
PubMed1992

Introduction of reactive cysteine residues in the epsilon subunit of Escherichia coli F1 ATPase, modification of these sites with tetrafluorophenyl azide-maleimides, and examination of changes in the binding of the epsilon subunit when different nucleotides are in catalytic sites.

Cysteine residues have been exchanged for serine residues at positions 10 and 108 in the epsilon subunit of the Escherichia coli F1 ATPase by site-directed mutagenesis to create two mutants, epsilon-S10C and epsilon-S108C. These two mutants and wild-type enzyme were reacted with [14C]N-ethylmaleimide (NEM) to examine the solvent accessibility of Cys residues and with novel photoactivated cross-linkers, tetrafluorophenyl azide-maleimides (TFPAM's), to examine near-neighbor relationships of subunits. In native wild-type F1 ATPase, NEM reacted with alpha subunits at a maximal level of 1 mol/mol of enzyme (1 mol/3 alpha subunits) and with the delta subunit at 1 mol/mol of enzyme; other subunits were not labeled by the reagent. In the mutants epsilon-S10C and epsilon-S108C, Cys10 and Cys108, respectively, were also labeled by NEM, indicating that these are surface residues. Reaction of wild-type enzyme with TFPAM's gave cross-linking of the delta subunit to both alpha and beta subunits. Reaction of the mutants with TFPAM's also cross-linked delta to alpha and beta and in addition formed covalent links between Cys10 of the epsilon subunit and the gamma subunit and between Cys108 of the epsilon subunit and the alpha subunit. The yield of cross-linking between sites on epsilon and other subunits depended on the nucleotide conditions used; this was not the case for delta-alpha or delta-beta cross-linked products. In the presence of ATP+EDTA the yield of cross-linking between epsilon-Cys10 and gamma was high (close to 50%) while the yield of epsilon-Cys108 and alpha was low (around 10%).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1984

Photoaffinity labeling of the alpha 1-adrenergic receptor using an 125I-labeled aryl azide analogue of prazosin.

alpha 1-Adrenergic receptor probes, which can be radioiodinated to yield high specific activity radioligands, have been synthesized and characterized. 2-[4-(4-Amino-benzoyl)piperazin-1-yl]-4-amino-6,7-dimethoxyquin azoline (CP63,155), an arylamine analogue of the selective alpha 1-adrenergic antagonist prazosin, and its iodinated derivative, 2-[4-(4-amino-3-[125I]iodobenzoyl)piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline [( 125I]CP63,789), bind reversibly and with high affinity (KD = 1 nM and 0.6 nM, respectively) to rat hepatic membrane alpha 1-adrenergic receptors. Conversion of [125I]CP63,789 to the aryl azide yields a photolabile derivative, 2-[4-(4-azido-3-[125I]iodobenzoyl)piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline [( 125I]CP65,526), which prior to photolysis binds competitively and with high affinity (KD = 0.3 nM). Binding of [125I]CP63,789 and [125I]CP65,526 (prior to photolysis) is rapid and saturable. Both ligands identify similar alpha 1-adrenergic receptor binding site concentrations as the parent probe, [3H]prazosin. Specific binding by these iodinated ligands is stereoselective and inhibited by a variety of adrenergic agents with a specificity typical of the alpha 1-adrenergic receptor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography of [125I]CP65,526-labeled rat hepatic membranes reveal major protein species with molecular weights of 77K, 68K and 59K. Each protein binds adrenergic ligands with stereoselectivity and with a specificity typical of the alpha 1-adrenergic receptor. Inclusion of multiple protease inhibitors during membrane preparation prior to SDS-PAGE does not alter the labeling of these peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗