Demetalation reactions of four Chl a analogs were quantitatively analyzed by monitoring absorbance changes at their Soret peak positions. The apparent demetalation rate constant of demetalation is denoted as k′ in this article, and the k′ is followed by an equation, k′ = k[H+]n. Figure 3 depicts kinetic plots of the Soret absorbance of four Chl a analogs as well as Chl a in acetone/water (3/1, vol/vol) at the proton concentration of 1.2 × 10−3 m. Their demetalation reactions were regarded as pseudo-first-order reactions because of linear time-dependency of the logarithm of Soret absorbance in their reactions. This is consistent with the reaction conditions in which the proton concentration was much higher than the concentration of the Chl a analogs. Such pseudo-first-order demetalation kinetics was also observed at the lower proton concentrations of 2.5 × 10−4 and 1.2 × 10−4 m. Therefore, apparent reaction rate constants of the demetalation, k′s, could be estimated by fitting the time courses of the Soret absorbance of the Chl a analogs to the following kinetic equation:
where A0, A and A∞ are their Soret absorbance at the onset of the measurement, at time t, and at the complete demetalation, respectively. The k′-values of the four Chl a analogs are summarized in Fig. 4, and are compared with those of Chl a. The k′s in Fig. 4 are the averages of more than three independent measurements; their standard deviations were less than 6, 11, 12, 5 and 4% of the averages of the k′s for DV-Chl a, mesoChl a, pyroChl a, PChl a and Chl a, respectively. The logarithm of apparent demetalation rate constants, log k′, of all the Chl a analogs linearly increased with that of proton concentrations, log [H+]. The slopes of log k′ plots against log [H+] in Fig. 4 were determined to be 1.9, 2.1, 2.0, 2.2 and 1.9 for DV-Chl a, mesoChl a, pyroChl a, PChl a and Chl a, respectively. The relationships between log k′ and log [H+] allow us to estimate the number of protons that participate in the rate-limiting step of the demetalation reactions, as the k′s are followed by the equation, k′ = k[H+]n, where n denotes the proton number involved in the rate-limiting step [26-28]. The present results indicate the participation of two protons in the rate-limiting step of removal of the central magnesium from Chl a analogs under the acidic conditions. This is consistent with previous works, which mentioned that the proton number in the rate-limiting steps of demetalation of Chl a/a′, BChl a, Zn-Chl a and Zn-BChl a was ca 2 [26-28]. These suggest that the rate-limiting step of (B)Chl demetalation is the attack of two protons on two nitrogen atoms in the cyclic tetrapyrroles under acidic conditions.
Figure 3. Kinetic plots for demetalation of DV-Chl a (open circle), mesoChl a (open square), pyroChl a (open triangle), PChl a (open diamond) and Chl a (closed square) in acetone/water (3/1, vol/vol) at the proton concentration of 1.2 × 10−3 m. Absorbance changes were monitored at 440, 427, 433, 435 and 431 nm for DV-Chl a, mesoChl a, pyroChl a, PChl a and Chl a respectively. A0, A and A∞ are Soret absorbance of Chl a analogs at the onset of measurements, at time t, and at the complete demetalation, respectively.
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Figure 4. Demetalation rate constants, k′s, of DV-Chl a (open circle), mesoChl a (open square), pyroChl a (open triangle), PChl a (open diamond) and Chl a (closed square) in acetone/water (3/1, vol/vol) dependent on the examined proton concentrations [H+].
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