EGTA

EGTA ethyleneglycol-bis(P-ami noethyl ether) -N,N -tetraacetic acid 5.4 10.9... 

The titration with EDTA, using solochrome black as indicator, will yield the calcium content of the sample (if no magnesium is present) or the total calcium and magnesium content if both metals are present. To determine the individual elements, calcium may be evaluated by titration using a suitable indicator, e.g., Patton and Reeder s indicator or calcon — see Sections 10.48 and 10.60, or by titration with EGTA using zincon as indicator — see Section 10.61. The difference between the two titrations is a measure of the magnesium content. 

DETERMINATION OF CALCIOM IN THE PRESENCE OF MAGNESIUM USING EGTA AS TITRANT 10.61... 

Procedure. Prepare an EGTA solution (0.05M) by dissolving 19.01 g in 100 mL sodium hydroxide solution (1M) and diluting to 1 L in a graduated flask with de-ionised water. Prepare the indicator by dissolving 0.065 g zincon in 2 mL sodium hydroxide solution (0.1M) and diluting to 100 mL with de-ionised water, and a buffer solution (pH 10) by dissolving 25 g sodium tetraborate, 3.5 g ammonium chloride, and 5.7 g sodium hydroxide in 1 L of de-ionised water. 

The EGTA solution may be standardised by titration of a standard (0.05M) calcium solution, prepared by dissolving 5.00 g calcium carbonate in dilute hydrochloric acid contained in a 1 L graduated flask, and then after neutralising with sodium hydroxide solution diluting to the mark with de-ionised water, use zincon indicator in the presence of Zn-EGTA solution (see below). 

To determine the calcium in the calcium-magnesium mixture, pipette 25 mL of the solution into a 250 mL conical flask, add 25 mL of the buffer solution and check that the resulting solution has a pH of 9.5-10.0. Add 2mL of the Zn-EGTA solution and 2-3 drops of the indicator solution. Titrate slowly with the standard EGTA solution until the blue colour changes to orange-red. 

The calcium content may then be determined by titration wth EDTA using either Patton and Reeder s indicator or calcon (Section 10.60), or alternatively by titration with EGTA (see Section 10.61). 

Calcium, D. of - continued in limestone or dolomite, (fl) 813 in presence of barium, (ti) 333 with CDTA, (ti) 333 with lead by EDTA, (ti) 333 with magnesium by EDTA, 328 by EGTA, (ti) 331 by flame emission, (aa) 804 Calcium oxalate, thermal analysis 498 Calcon 318 Calculators 133 Calibration of apparatus, 87 of burettes, 88 of graduated flasks, 88 of pipettes, 88 of weights, 74... 

Ethylenedioxybis ethyliminodi( acetic acid)] see EGTA Evolution methods 444 External indicators 375 Extinction see Absorbance Extinction coefficient 649 molar, 649 specific, 649... 

The interaction between aequorin and a chelator must be carefully considered when estimating Ca2+ concentrations with aequorin in a calcium buffer containing EDTA or EGTA. This is particularly crucial when using a common calcium buffer system that contains a constant total concentration of a chelator in the buffer solutions of various Ca2+ concentrations in such a buffer system, a buffer of lower Ca2+ concentration contains a higher concentration of the free form of the chelator, resulting in an increased inhibition. 

Fig. 4.1.8 Influence of various calcium chelators on the relationship between Ca2 " concentration and the luminescence intensity of aequorin, at 23-25°C (panel A) in low-ionic strength buffers (I < 0.005) and (panel B) with 150 mM KC1 added. Buffer solutions (3 ml) of various Ca2+ concentrations, pH 7.05, made with or without a calcium buffer was added to 2 pi of 10 pM aequorin solution containing 10 pM EDTA. The calcium buffer was composed of the free form of a chelator (1 or 2mM) and various concentrations of the Ca2+-chelator (1 1) complex to set the Ca2+ concentrations (the concentration of free chelator was constant at all Ca2+ concentrations). The curves shown are obtained with 1 mM MOPS (A), 1 mM gly-cylglycine ( + ), 1 mM citrate (o), 1 mM EDTA plus 2mM MOPS ( ), 1 mM EGTA plus 2 mM MOPS ( ), 2 mM NTA plus 2 mM MOPS (V), and 2 mM ADA plus 2 mM MOPS (A). In the chelator-free buffers, MOPS and glycylglycine, Ca2+ concentrations were set by the concentration of calcium acetate. Reproduced with permission, from Shimomura and Shimomura, 1984. the Biochemical Society.

Fig. 4.1.14 Relationship between Ca2+ concentration and the initial light intensity of various recombinant semisynthetic aequorins and w-aequorin J (a semisynthetic natural aequorin made from isoform J). The curve number corresponds to the number of semisynthetic aequorin used in Table 4.1.4. A sample aequorin (3 (Ag) was in 3 ml of calcium-buffer solution containing 1 mM total EGTA, 100 mM KC1,1 mM Mg2+ and 1 mM MOPS (pH 7.0), at 23-24°C. From Shimomura etal., 1993a, with permission from Elsevier.

Fig. 7.4.3 Relationships between the total light emission of polynoidin and the concentrations of the photoprotein (A), H2O2 (B), Fe2+ (C), and EGTA (D). The basic buffer solution used was 50 mM phosphate buffer, pH 6.6, containing 3 mM H2O2, 0.1 mM FeSC>4 and 10 mM EGTA the concentrations of H2O2, FeSC>4 and EGTA were varied in B, C and D, respectively. FeSC>4 was added last to initiate light emission. From Nicolas et al., 1982, with permission from the American Society for Photobiology.

EGTA enhanced the luminescence up to 10 times at the concentration of 10 mM (panel D). DETAP AC (diethylenetriaminepentaacetic acid) was nearly as effective as EGTA, but EDTA (10 mM) diminished the light emission. 

The frozen shells were ground in a cold mortar with 50 mM sodium acetate buffer, pH 5.8, containing 10 mM EGTA and 0.2 M NaCl, then the mixture was centrifuged. The pellets were re-extracted with the same buffer, and centrifuged. All supernatants were combined, and the photoprotein was precipitated with ammonium sulfate. The photoprotein in the precipitate was purified by four steps of column chromatography at near 0°C. Due to the instability of the photoprotein, efforts were made to reduce the time required for purification. 

Step 1. Gel filtration on Sephadex G-100, in 10 mM sodium phosphate buffer, pH 6.5, containing 5mM EGTA and 0.2 M NaCl, which was the basic buffer used throughout the purification process. 

Elimination of a cofactor needed for luminescence. The chelators EDTA and EGTA efficiently quench the luminescence of Ca2+-activated systems such as aequorin, obelin and mnemiopsin. The luminescence systems that require ferrous ions, such as extracts of the polychaete Chaetopterus, can be inhibited by 8-hydroxyquinoline and... 

A skinned fiber is a muscle fiber, the sarcolemma of which has been mechanically removed or which is made freely permeable to small molecules, such as Ca2+, Mg2+, EGTA, ATP, soluble enzymes and others by a chemical agent (saponin, (3-escin or Staphylococcus a-toxin). The organization of the sarcoplasmic reticulum (SR) and myofibrils is kqrt as they are in the living muscle. 

Earlandite structure, 6,849 Edge-coalesced icosahedra eleven-coordinate compounds, 1, 99 repulsion energy coefficients, 1,33,34 Edta — see Acetic acid, ethylenediaminetetra-Effective atomic number concept, 1,16 Effective bond length ratios non-bonding electron pairs, 1,37 Effective d-orbital set, 1,222 Egta — see Acetic acid,... 

Microtubules can be reconstituted in vitro at 37 °C from a solution that contains a physiological mixture of brain tubulin, MAPs, small amounts of guanosine 5 -triphosphate (GTP), magnesium ions, and the calcium-chelating agent EGTA [ethylene glycol-bis(2-aminoethyl ether) N, N -tetraacetic acid]. Tubulin assembly is inhibited by low temperature and by the presence of calcium ions. 

Figure 11 depicts two characteristics of the Indo-l-detected calcium response. The intracellular Ca rise is primarily from intracellular stores because adding EGTA to chelate extracellular Ca does not inhibit the response (Figure 11, upper panel). 

Figure 11. Effects of EGTA or treatment with islet activating protein (lAP or pertussis toxin) on the 6-HCH-induced Ca response detected in Indo-l-labeled cells. Cells were treated for 2 hours at 37 C with (lAP) or without (Control) 10 Jg/mL lAP, then labeled with Indo-1. Cells were washed and resuspended at 2 x 10 cells/mL buffer and stimulated with 6-HCH (solid trace). In some cases (dashed traces), stimulation was preceded by the addition of 5-mAf EGTA 10 s before stimulation. Other experimental conditions are as in Figure 9.

Figure 7. Sensitivity of the FMLP-induced calcium signal to removal of extracellular calcium. Indo-l-loaded neutrophils were stimulated with 10 M FMLP in a medium of normal osmolality (320 mosmol/kg) and indo-1 fluorescence was recorded as described in Figure 6. Trace 1 Cells in a medium with normal calcium (1.5 mN). Trace 2 EGTA added to chelate extracellular calcium before stimulation extracellular calcium (1.5 milf) readded 70 s after stimulation. Trace 3 Cells in a medium with normal calcium EGTA added 70 s after stimulation to chelate extracellular calcium.

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