The fluorescent indicators Fura-2 and PBFI are widely used for the determination of intracellular concentrations of Ca²⁺ and K⁺, respectively. To investigate the complex forming reaction between Fura-2 and Ca²⁺, and between PBFI and K⁺ in the ground and excited states, steady-state and time-resolved measurements were performed. The fluorescence decay surfaces were analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution: (1) for Fura-2: k₀₁ equals 1.2 X 10⁹ s^-1, k₂₁ equals 1.0 X 10¹¹ M^-1x^-1, k₀₂ equals 5.5 X 10⁸s^-1, k₁₂ equals 2.2 X 10⁷s^-1 (2) for PBFI: k₀₁ equals 1.1 X 10⁹s^-1, k₂₁ equals 2.7 X 10⁸M^-1s^-1, k₀₂ equals 1.8 X 10⁹s^-1, k₁₂ equals 1.4 X 10⁹s^-1 k₀₁ and k₀₂ denote the deactivation rate constants of the free and bound forms of the indicator, respectively k₂₁ represents the bimolecular rate constant of binding of the cation by the indicator whereas k₁₂ is the rate constant of dissociation of the cation:indicator complex. For both probes the effect of the excited-state reaction can be neglected in the determination of K_d and/or the ion concentration.

Date Published: 17 August 1994
PDF: 11 pages
Proc. SPIE 2137, Time-Resolved Laser Spectroscopy in Biochemistry IV, (17 August 1994); doi: 10.1117/12.182705

Published in SPIE Proceedings Vol. 2137:
Time-Resolved Laser Spectroscopy in Biochemistry IV
Joseph R. Lakowicz, Editor(s)