PHOTOPHYSICS OF THE FLUORESCENT K+ INDICATOR PBFI

The fluorescent indicator PBFI is widely used for the determination of intracellular concentrations of K+. To investigate the binding reaction of K+ to PBFI in the ground and excited states, steady-state and time-resolved measurements were performed. The fluorescence decay surface was analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution at pH 7.2: k(01) = 1.1 x 10(9) s(-1), k(21) = 2.7 x 10(8) M(-1)s(-1), k(02) = 1.8 x 10(9) s(-1), and k(12) = 1.4 x 10(9) s(-1). k(01) and k(02) denote the respective deactivation rate constants of the K+ free and bound forms of PBFI in the excited state, k(21) represents the second-order rate constant of binding of K+ to the indicator in the excited state whereas k(12) is the first-order rate constant of dissociation of the excited K+-PBFI complex, From the estimated values of k(12) and k(21), the dissociation constant K-d* in the excited state was calculated. It was found that pK(d)* (-0.7) is smaller than pK(d) (2.2), The effect of the excited-state reaction can be neglected in the determination of K-d and/or the K+ concentration. Therefore, intracellular K+ concentrations can be accurately determined from fluorimetric measurements by using PBFI as K+ indicator.