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Extra info for Methods of Biochemical Analysis: v. 30 (Methods of Biochemical Analysis Series: No)
A typical time-resolved fluorescence of +OH* is shown in Figure 2 1. The initial rapid decay represents the dissociation of the protons in the liposomes. Each dissociativeevent increases the population of [+O-* + H + ] at the expense of [+OH*] (the square brackets represent an event taking place is a single liposome). Thus, with time the recombination reaction that occurs as the [+O-* + H+] vesicles become more probable, until the velocities of the forward and backward reactions (each taking place in different vesicles) become equal.
Insertion of Equations (16) and (17) into Equation (19) leads, in the range pK* < pH C pKo, to a solution y1 = k23 + k,,n,. As the decay times of the free hydroxypyrene in its undissociated state and in its ionized state are the same and equal to that of bound +O-* (see Table I), we can approximate kf,nl-(bund)= k’f,nr(bund). ) Using the approximation of k,,, and the calculated values of k23 and k2, the rate of recombination (k21*H+) can be obtained. 7~ (20) the rate of dissociation in the cavity k I 2 is obtained.
The rate constant of the reaction is thus controlled by two radii, the Debye radius (RD), where electrostatic interaction dominates, and the radius of the reaction sphere, out of which the proton cannot escape. The rate constant of this reaction is given by Equation (21) where ED is the sum of the diffusion coefficient of the proton and the proton emitter. 3 x 1 0 - ~ cm2/sec. 0 X lo9 sec-’, that is, the proton will recombine with its conjugated base within 1 nsec. This time scale is short enough to be followed by the time resolved fluorescence of +OH*.