scholarly journals Design and properties of a fluorescent indicator of intracellular free Na+ concentration

1988 ◽  
Vol 250 (1) ◽  
pp. 227-232 ◽  
Author(s):  
G A Smith ◽  
T R Hesketh ◽  
J C Metcalfe
Keyword(s):  
Aqueous Solubility ◽  
Ratio Method ◽  
Isosbestic Point ◽  
Buffer Solution ◽  
Acetoxymethyl Ester ◽  
Apparent Dissociation Constant ◽  
Fluorescent Indicator ◽  
Carboxylate Groups ◽  
Reporter Group ◽  
Benzenoid Ring

We have recently described a cryptand structure, FCryp-1, with appropriate properties for an indicator of intracellular free Na+ concentration using the 19F-n.m.r. chemical shift of the incorporated 5FBAPTA [1,2-bis-(2-amino-5-fluorophenoxy)ethane-NNN‘N’-tetra-acetic acid] reporter group to measure the free cytosolic Na+ concentration [(Na+]i) [Smith, Morris, Hesketh and Metcalfe (1986) Biochim. Biophys. Acta 889, 82-83]. FCryp-1 carries four carboxylate groups to confer aqueous solubility and the indicator is membrane-permeant when the carboxyls are esterified with acetoxymethyl ester groups. Here we describe the synthesis of FCryp-2 to provide a fluorescent indicator of [Na+]i. FCryp-2 retains the parent tribenzo (2:2:1) cryptand structure of FCryp-1, in which the benzenoid ring at C-21 in FCryp-1 is replaced by an indole derivative which acts as the fluorophor in FCryp-2. With excitation at 340 nm, FCryp-2 gives an emission maximum at 460 nm in the absence of Na+ which shifts to 395 nm when FCryp-2 is saturated with Na+, with an isosbestic point at 455 nm. The apparent dissociation constant of FCryp-2 in a buffer solution of 100 mM-KCl/20 mM-KH2PO4/K2HPO4, pH 7.0, at 37 degrees C is 6.0 mM and the free Na+ concentration can be measured either from the calibrated fluorescence intensity at 395 nm, which increases 25-fold when Na+ is bound to FCryp-2, or from the ratio of fluorescence intensities at 395 nm and 455 nm. The measurement of free [Na+] by either method is unaffected by K+, Ca2+ or Mg2+ in the normal intracellular concentration ranges. Free [Na+] measurements by the ratio method are unaffected by pH from 6.6 to 7.6.

scholarly journals ULTRAVIOLET SPECTROSCOPY AND ITS PHARMACEUTICAL APPLICATIONS- A BRIEF REVIEW

2018 ◽  
Vol 11 (2) ◽  
pp. 59 ◽  
Author(s):  
Dipali M Atole ◽  
Hrishikesh H Rajput
Keyword(s):  
Simultaneous Equation ◽  
Ratio Method ◽  
Isosbestic Point ◽  
Complex Matrix ◽  
Spectrophotometric Methods ◽  
Factor Method ◽  
Absorbance Ratio ◽  
Present Information ◽  
Different Types ◽  
Absorbance Difference

 Rapid and easy analytical methods are needed due to increasing number of multicomponent formulations, biotherapeutic products and samples of complex matrix in que. Number of Ultraviolet (UV) spectrophotometric methods used for these purpose. Different types of UV spectrometric methods developed on the basis of principle of additivity, absorbance difference, processing absorption spectra. The aim of this review is to present information on simultaneous equation method, difference spectrophotometry, derivative spectrophotometry, absorbance ratio spectra, derivative ratio spectra, successive ratio - derivative spectra, Q-absorbance ratio method, absorptivity factor method, dual wavelength method, absorption factor method, multivariate chemometric methods, and isosbestic point method. A brief summary on theories, mathematical background and some applications of these methods are presented here.

Micromolar Ca2+ from sparks activates Ca2+-sensitive K+ channels in rat cerebral artery smooth muscle

2001 ◽  
Vol 281 (6) ◽  
pp. C1769-C1775 ◽  
Author(s):  
Guillermo J. Pérez ◽  
Adrian D. Bonev ◽  
Mark T. Nelson
Keyword(s):  
Smooth Muscle ◽  
Laser Scanning ◽  
Cerebral Arteries ◽  
Bk Channels ◽  
Bk Channel ◽  
Hill Coefficient ◽  
Channel Activity ◽  
Acetoxymethyl Ester ◽  
Open Probability ◽  
Apparent Dissociation Constant

The goal of the present study was to test the hypothesis that local Ca2+ release events (Ca2+ sparks) deliver high local Ca2+concentration to activate nearby Ca2+-sensitive K+ (BK) channels in the cell membrane of arterial smooth muscle cells. Ca2+ sparks and BK channels were examined in isolated myocytes from rat cerebral arteries with laser scanning confocal microscopy and patch-clamp techniques. BK channels had an apparent dissociation constant for Ca2+ of 19 μM and a Hill coefficient of 2.9 at −40 mV. At near-physiological intracellular Ca2+ concentration ([Ca2+]i; 100 nM) and membrane potential (−40 mV), the open probability of a single BK channel was low (1.2 × 10−6). A Ca2+spark increased BK channel activity to 18. Assuming that 1–100% of the BK channels are activated by a single Ca2+ spark, BK channel activity increases 6 × 105-fold to 6 × 103-fold, which corresponds to ∼30 μM to 4 μM spark Ca2+ concentration. 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid acetoxymethyl ester caused the disappearance of all Ca2+sparks while leaving the transient BK currents unchanged. Our results support the idea that Ca2+ spark sites are in close proximity to the BK channels and that local [Ca2+]i reaches micromolar levels to activate BK channels.

scholarly journals Study of Complexes of Tannic Acid with Fe(III) and Fe(II)

2019 ◽  
Vol 2019 ◽  
pp. 1-6 ◽  
Author(s):  
Zhaofeng Fu ◽  
Rui Chen
Keyword(s):  
Tannic Acid ◽  
Ph Value ◽  
Ratio Method ◽  
Iron Ions ◽  
Test Paper ◽  
Buffer Solution ◽  
Carbonate Buffer ◽  
Color Changes ◽  
Different Color ◽  
Complex Ratio

UV-Vis absorption spectra of tannic acid were gained at pH 1.0∼9.0. Due to the pH value dependence of complex, the stoichiometry of tannic acid with iron ions was tested in buffer solution by the mole ratio method. The result suggests that the complex ratio of tannic acid to Fe(III) is 1 : 1 and to Fe(II) 3 : 1 in the carbonate buffer solution, and the complex ratio of iron-tannic complexes is 1 : 1 at pH 2.2. Due to the different color changes of tannic acid with iron ions in the coordination reactions, a tannic acid test paper was designed. The concentrations of Fe(III) more than 5.000 × 10−6 mol/L and the concentrations of Fe(II) more than 1.000 × 10−5 mol/L in aqueous solution can be detected by this test paper.

scholarly journals Ca2+-dependent and phospholipid-independent binding of annexin 2 and annexin 5

2002 ◽  
Vol 367 (3) ◽  
pp. 895-900 ◽  
Author(s):  
Nicole D. BROOKS ◽  
Jean E. GRUNDY ◽  
Nadine LAVIGNE ◽  
Mélanie C. DERRY ◽  
Christina M. RESTALL ◽  
...  
Keyword(s):  
Binding Sites ◽  
Dependent Manner ◽  
Homologous Proteins ◽  
Anionic Phospholipid ◽  
Apparent Dissociation Constant ◽  
Excess Amount ◽  
Annexin 2 ◽  
Covalently Linked ◽  
Reporter Group ◽  
Dissociation Constant Kd

Annexins are a family of homologous proteins that associate with anionic phospholipid (aPL) in the presence of Ca2+. Evidence that the function of one annexin type may be regulated by another was recently reported in studies investigating cytomegalovirus—aPL interactions, where the fusogenic function of annexin 2 (A2) was attenuated by annexin 5 (A5). This observation suggested that A2 may bind directly to A5. In the present study, we demonstrated this interaction. The A2—A5 complex was first detected utilizing (covalently linked) fluorescein-labelled A5 (F-A5) as a reporter group. The interaction required concentrations of Ca2+ in the millimolar range, had an apparent dissociation constant [Kd(app)] of 1nM at 2mM Ca2+ and was independent of aPL. A2 bound comparably with F-A5 pre-equilibrated with an amount of aPL that could bind just the F-A5 or to an excess amount of aPL providing sufficient binding sites for all of F-A5 and A2. A2—A5 complex formation was corroborated in an experiment, where [125I]A2 associated in a Ca2+-dependent manner with A5 coated on to polystyrene. Surface plasmon resonance was used as a third independent method to demonstrate the binding of A2 and A5 and, furthermore, supported the conclusion that the monomeric and tetrameric forms of A2 bind equivalently to A5. Together these results demonstrate an A2—A5 interaction and provide an explanation as to how A5 inhibits the previously reported A2-dependent enhancement of virus—aPL fusion.

scholarly journals Validated Analytical Spectrofluorimetric Method for Quantitation of Diphenhydramine HCl in Commercial Dosage Forms

2018 ◽  
Vol 1 (4) ◽  
pp. 12-26
Author(s):  
Syed Najmul Hejaz Azmi ◽  
Aisha Al-Mahroqi ◽  
Khoula Al-Mamari ◽  
Shaima Al-Shukaili
Keyword(s):  
Dynamic Range ◽  
Fluorescence Emission ◽  
Reference Method ◽  
Pharmaceutical Formulations ◽  
Ion Pair ◽  
Ratio Method ◽  
Eosin Y ◽  
Stoichiometric Ratio ◽  
Buffer Solution

Diphenhydramine HClis a weakly fluorescent drug having tertiary amine group forming ion pair complex with eosin Y in dichloromethane at pH 5 in disodium hydrogen phosphate-citric acid buffer solution. The complex formation was the basis for the development of new analytical method for determination of active diphenhydramine in pharmaceutical formulations. The stoichiometric ratio between diphenhydramine and eosin Y was studied by mole ratio method and found to be 2:1. The ion-pair complex showed maximum fluorescence emission intensity at 554 nm with excitation at 259 nm. The linear dynamic range was obtained in the concentration range of 2-22 µg mL-1 with a linear equation of FI = 0.361 + 13.675 C. The apparent Gibb’s free energy (ΔGº) was calculated and found to be -80.783 KJ mol-1, confirmed the feasibility of the reaction. The proposed method was successfully applied to the determination of diphenhydramine HCl in pharmaceutical formulations and in good agreement with the reference method.

scholarly journals Measurement of intrasynaptosomal free calcium by using the fluorescent indicator quin-2

1984 ◽  
Vol 219 (1) ◽  
pp. 149-158 ◽  
Author(s):  
R H Ashley ◽  
M J Brammer ◽  
R Marchbanks
Keyword(s):  
Cerebral Cortex ◽  
Plasma Membrane ◽  
Guinea Pig ◽  
Calcium Ionophore ◽  
Ionized Calcium ◽  
Free Calcium ◽  
Acetoxymethyl Ester ◽  
Fluorescent Indicator ◽  
Calcium Indicator ◽  
Hydrolysis Of

The recently synthesized calcium indicator quin −2 was incorporated into synaptosomes from guinea-pig cerebral cortex following uptake and internal hydrolysis of quin −2 tetra-acetoxymethyl ester. Incubation in physiological media containing 1 mM- or 2 mM-CaCl2 led to equilibrium cytosolic ionized calcium concentrations of 85 +/- 10 nM and 205 +/- 5 nM respectively (mean +/- S.E.M. from eight and eighteen preparations respectively). Cytosolic Ca2+ was elevated following increases in external Ca2+ concentration, plasma membrane depolarization, mitochondrial inhibition, calcium ionophore addition or replacement of external sodium by lithium. Preliminary experiments were performed to assess changes in cytosolic Ca2+ accompanying the release of the neurotransmitter acetylcholine.

Ca2+ binding to bovine lactoferrin enhances protein stability and influences the release of bacterial lipopolysaccharide

2002 ◽  
Vol 80 (1) ◽  
pp. 41-48 ◽  
Author(s):  
Paola Rossi ◽  
Francesco Giansanti ◽  
Alberto Boffi ◽  
Maria Ajello ◽  
Piera Valenti ◽  
...  
Keyword(s):  
Protein Stability ◽  
Bacterial Lipopolysaccharide ◽  
Bovine Lactoferrin ◽  
Bacterial Cells ◽  
Weakly Bound ◽  
Apparent Dissociation Constant ◽  
Outer Membranes ◽  
Carboxylate Groups ◽  
Ft Ir ◽  
Phosphate Buffered Saline

Bovine lactoferrin (bLf) is known to damage the outer membrane of Gram-negative bacteria by binding to bacterial lipopolysaccharide (LPS). We report that LPS is released from bacterial outer membranes also when apo- or metal-saturated Lf is separated from bacterial cells by a dialysis membrane. This process occurs in phosphate-buffered saline with no added Ca2+ and Mg2+ and is hindered by addition of these cations. The effect of bLf is similar to that induced by EDTA and has been ascribed to chelation of Ca2+. In fact, it may be envisaged that Ca2+-binding sites on LPS have different affinities and that bLf can remove those ions that are more weakly bound. Ca2+ binding does not alter Lf iron-binding properties significantly or its UV and CD spectral features but brings about changes in the FT-IR bands due to carboxylate residues. Ca2+ binding is characterized by an apparent dissociation constant of 6 µM and a stoichiometry of 1.55 Ca2+ per Lf molecule; it enhances bLf stability towards chemical and thermal denaturation. The increase in stability takes place in both the apo- and iron-saturated forms but not in the desialilated protein, indicating that the carboxylate groups of the sialic acid residues present on two of the glycan chains are involved in Ca2+ binding.Key words: lactoferrin, calcium, antibacterial activity, lipopolysaccharides, protein stability, denaturation.

SIMULTANEOUS SPECTROPHOTOMETRIC DETERMINATION OF DAPAGLIFLOZIN AND SAXAGLIPTIN IN FIXED DOSE COMBINATION (FDC) BY Q-ABSORBANCE RATIO METHOD

INDIAN DRUGS ◽  
2021 ◽  
Vol 57 (11) ◽  
pp. 63-75
Author(s):  
Gan Ee How ◽  
◽  
Venkata Subrahmanya Lokesh Bontha
Keyword(s):  
Uv Spectra ◽  
Fixed Dose Combination ◽  
Fixed Dose ◽  
Ratio Method ◽  
Isosbestic Point ◽  
Good Precision ◽  
Absorbance Ratio ◽  
Ich Guidelines ◽  
Dose Combination

A simple Q-absorbance ratio method have been developed for the determination of dapagliflozin (DAPA) and saxagliptin (SAXA) in fixed dose combination (FDC) using UV-Visible spectrophotometer. In this method, the UV spectra of DAPA and SAXA were overlaid to obtain wavelength at isosbestic point (λiso) of 217.6 nm and at absorption maximum (λmax) of DAPA at 224.2 nm, which are involved in the formation of Q-absorbance equation. Validation of method was done according to ICH guidelines. DAPA and SAXA obeyed Beers law in the concentration range of 2-25 µg/mL and 5-25 µg/mL, respectively. Good accuracy of method was determined by recovery studies and found to be in the range of 103.1-104.6% for DAPA and 97.7-102.4% for SAXA. This method has shown good precision (%RSD < 2.0). Statistical analysis like one-way ANOVA and student t-test were conducted and the reported method was accurate. This method was found to be simple, cheap, eco-friendly accurate and precise and can be used for routine analysis of DAPA and SAXA in FDC for testing regularly in manufacturing units.

A fluorescent indicator for measuring cytosolic free magnesium

1989 ◽  
Vol 256 (3) ◽  
pp. C540-C548 ◽  
Author(s):  
B. Raju ◽  
E. Murphy ◽  
L. A. Levy ◽  
R. D. Hall ◽  
R. E. London
Keyword(s):  
Dissociation Constant ◽  
Rat Hepatocytes ◽  
Ratio Method ◽  
Isolated Rat Hepatocytes ◽  
Fluorescent Indicator ◽  
Fura 2 ◽  
Calcium Indicators ◽  
Intracellular Probe ◽  
Free Magnesium ◽  
Isolated Rat

The previously developed chelator O-aminophenol-N,N,O-triacetic acid (APTRA) (L. A. Levy, E. Murphy, B. Raju, and R. E. London. Biochemistry 27: 4041-4048, 1988) has been modified to yield a fluorescent analogue which can be utilized as an intracellular probe for ionized Mg2+. The fluorescent analogue, FURAPTRA, with a magnesium dissociation constant of 1.5 mM, is structurally analogous to the calcium chelator fura-2 and exhibits a similar excitation shift on magnesium complexation. Hence, data on the intracellular Mg2+ concentration can be obtained using an analogous ratio method. The acetoxymethyl form of the chelator is readily loaded into cells and has been used to determine a cytosolic free Mg2+ concentration of 0.59 mM for isolated rat hepatocytes. As a consequence of the relatively high levels of cytosolic Mg2+, the problem of ion buffering is much less severe than for the analogous calcium indicators.

scholarly journals Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator.

1982 ◽  
Vol 94 (2) ◽  
pp. 325-334 ◽  
Author(s):  
R Y Tsien ◽  
T Pozzan ◽  
T J Rink
Keyword(s):  
Cell Types ◽  
Atp Depletion ◽  
Free Calcium ◽  
Fluorescence Signal ◽  
Resting Cells ◽  
Tetracarboxylic Acid ◽  
Acetoxymethyl Ester ◽  
Intact Mouse ◽  
Fluorescent Indicator ◽  
The Face

A new, fluorescent, highly selective Ca2+ indicator , "quin2", has been trapped inside intact mouse and pig lymphocytes, to measure and manipulate cytoplasmic free Ca2+ concentrations, [Ca2+]i. Quin2 is a tetracarboxylic acid which binds Ca2+ with 1:1 stoichiometry and an effective dissociation constant of 115 nM in a cationic background mimicking cytoplasm. Its fluorescence signal (excitation 339 nm, emission 492 nm) increases about fivefold going from Ca-free to CA-saturated forms. Cells are loaded with quin2 by incubation with its acetoxymethyl ester, which readily permeates the membrane and is hydrolyzed in the cytoplasm, thus trapping the impermeant quin2 there. The intracellular quin2 appears to be free in cytoplasm, not bound to membranes and not sequestered inside organelles. The fluorescence signal from resting cells indicates a [Ca2+]i of near 120 nM. The millimolar loadings of quin2 needed for accurately calibrated signals do not seem to perturb steady-state [Ca2+]i, but do somewhat slow or blunt [Ca2+]i transients. Loadings of up to 2mM are without serious toxic effects, though above this level some lowering of cellular ATP is observed. [Ca2+]i was well stabilized in the face of large changes in external Ca2+. Alterations of Na+ gradients, membrane potential, or intracellular pH had little effect. Mitochondrial poisons produced a small increase in [Ca2+]i, probably due mostly to the effects of severe ATP depletion on the plasma membrane. Thus intracellulary trapped chelators like quin2 offer a method to measure or buffer [Ca2+]i in hitherto intractable cell types.

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