Time-resolved response of fluorescent alkali ion indicators and detection of short-lived intermediates upon binding to molecular cavities.
Stopped-flow kinetic studies have been performed to determine the kinetic parameters of K(+) binding to the fluorescent cryptand F222 and of Na(+)binding to F221 at pH 8.O. The results clearly indicate that a comparatively stable intermediate is formed before the rate-limiting binding step occurs with a rate constant around 30 s(-1) under the chosen experimental conditions. The conversion of the intermediate to the final cation complex is assigned to the final penetration of the already bound, but still partially solvated cation into the ligand's cavity. The main fluorescence intensity change found upon cation binding is attributed to the second reaction step, and not to the fast, initial binding reaction. The comparatively slow overall binding reaction is interpreted on the bases of a special solvate substitution mechanism which, in principle, can also account for the 1500 times slower binding of Ca 2(+) to F221. With regard to time-resolved analytical Na(+) and K(+) determinations, the response times under the chosen conditions are around 20 ms. Differentiation between Na(+) and Ca(2+), for example, is possible with F221 on the basis of completely different response times.
Max-Planck-Institute of Biophysics, Kennedy-Allee 70, D-60596, Frankfurt, Germany.
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Doludda
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Kastenholz
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Lewitzki
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Grell