Spectrophotometric determination of reaction stoichiometry and equilibrium constants of metallochromic indicators

Dorogi, Peter L.; Neumann, Eberhard

doi:10.1016/0301-4622(81)80010-5

Cited by 9 publications

(6 citation statements)

References 15 publications

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“…Due to the high association constant of the AIII-Ca complex, initially the free Ca concentration in the vesicles was < 10-8 M corresponding to an electrochemical gradient of about 100 inV. The exact stoichiometry of AIII-Ca complexes is currently under dispute; 2: 1, 1 : 1 and even 1:2 complexes are being discussed [3,4,6]. Nevertheless, assuming a 1"1 AIII/Ca complex for the time being, interesing quantitative considerations can be made based on the experimental data.…”

Section: Discussionmentioning

confidence: 99%

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Calcium channels of the excitable ciliary membrane fromParamecium: An initial biochemical characterization

et al. 1983

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Summary.A stopped-flow spectrophotometric technique was used to study the kinetics of Ca flux into ciliary membrane vesicles from Paramecium tetraurelia wild-type and several 'pawn' mutants with defective Ca conductances. 15 mM Arsenazo III was used as metallochromic indicator and as intravesicular Ca trap. The absolute amount of Ca-permeable vesicles was significantly reduced in preparations from the 'pawn' mutants compared to wild-type. However, influx kinetics were identical for vesicles from wild-type and 'pawn' mutant Paramecia when the fraction of Ca-permeable vesicles was taken into account. Ca influx was rapid with a time constant of about 1.5 sec and an initial saturation rate of arsenazo III of about 50%/single vesicle x sec-1. Ca influx rates were half-maximal at approximately 20 gM Ca. Comparisons of Ba toxicity tested with a behavioral assay, Ca inward conductances under voltage-clamp conditions and Ca influx kinetics between wild-type and the 'pawn' mutants pwA (d4-94), leaky pwB (d4-96) and the double mutant pwA/pwB indicated that Ca transport in all types of ciliary membrane vesicles occurred through similar Ca gates.Key Words calcium 9 ion channel 9 Paramecium . mutants 9 cilia -membrane -stopped-flow spectroscopy

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Section: Discussionmentioning

confidence: 99%

“…The concentration of AIII in the membrane vesicles was 15 raM, the dissociation constant of the AIII-Ca complex is about 5 to 10 gM [4,6], thus > 99% of Ca entering the vesicle lumen was complexed by the dye. Consequently, the changes in Ca saturation of AIII quantitatively reflected the Ca influx.…”

Section: Discussionmentioning

confidence: 99%

Calcium channels of the excitable ciliary membrane fromParamecium: An initial biochemical characterization

et al. 1983

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“…(8). The dyes were dissolved in piperazine-ZV,l\r'-bis(2-ethanesulfonic acid) (Pipes buffer) at pH 7.0; buffer molarity was 0.03 M for ArsI and 0.05 M for TMX titrations.…”

Section: P~ri~c~~ion Of I~ica~orsmentioning

confidence: 99%

“…Several theoretical and graphical techniques are now in the literature and were developed to evaluate spectrophotometric titration curves in terms of Ca-dye-binding stoichiometries and equilibrium binding constants (2,(6)(7)(8). Determination of these quantities is especially difficult for dyes with high Ca affinity because a large fraction of the calcium may be bound to dye, giving nonlinear double-reciprocal plots (2).…”

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Arsenazo I and tetramethylmurexide as optical calcium indicators

Dorogi

Santarius

Neumann

1982

Analytical Biochemistry

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Calcium-binding stoichiometry, dissociation equilibrium constants at zero ionic strength @Lo), and molar extinction difference coefficients (At,) at the wavelength )r of the metallochromic indicators arsenazo I (ArsI) and tetramethylmurexide (TMX) were reevaluated with a computerized method based on mass conservation and thermodynamic consistency checks. This new method is shown to provide a more critical assessment of the assumed calcium-dye complexing model than is afforded by the commonly used reciprocal-plot method. The analyses of spectrophotometric Ca titrations confirm that both dyes form only 1: 1 complexes in aqueous solution. For TMX, R" = 1.3 X lOA3 hp and At&,, = 1.5 X I@ M-' cm-'; for Arsl, R" = 5.8 X 10el M and A6562 = 1.8 X 104 M-' cm-' at pH '7.0 and T = 293OK. The discriminatory power of the analytical method is demonstrated by comparison of these results with those found for a different dye, arsenazo III, which complexes Ca in l:l, 1:2, and 2:l forms.

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Kinetic calibration of the calcium indicator arsenazo III. I. Stopped-flow spectroscopy

Clemen

Rabl

Neumann

1988

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Spectrophotometric determination of reaction stoichiometry and equilibrium constants of metallochromic indicators

Cited by 9 publications

References 15 publications

Calcium channels of the excitable ciliary membrane fromParamecium: An initial biochemical characterization

Calcium channels of the excitable ciliary membrane fromParamecium: An initial biochemical characterization

Arsenazo I and tetramethylmurexide as optical calcium indicators

Kinetic calibration of the calcium indicator arsenazo III. I. Stopped-flow spectroscopy

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