REACTIVE INORGANIC OXY-SPECIES OF C, N, S, AND P - Oxidation of Amino Acids, Peptides, and Proteins: Kinetics and Mechanism - page 215

100

90

80

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20 60 80 100

0 40

[CO 3 •- ] ( µ M)

Figure 5.7. Relative activity (black squares) and relative absorbance at 405 nm (black
circles) of HRP versus [CO •− ] ; [HRP] = 2.3 µM, pH = 8.4, dose rate = 1.1 gy/min
(adapted from gebicka et al. [57] with the permission of Springer Americas).

The rate constants of the reactions of horseradish peroxidase (HRP) and
cytochrome c are similar at pH 8.0-8.4 and at pH 10.0 (Table 5.2) [55, 57]. The
rate constant for catalase has been determined only at pH 8.4 and was on the
order of 10 9 /M/s [57]. The pulse radiolysis experiments demonstrated CO •− did
not react directly with the heme iron of HRP, cytochrome c , and catalase. The
loss of activity was 30% and 20% for HRP and catalase, respectively, after
reactions with 100 µM of CO •− . A decrease in the absorbance of the Soret
region of HRP (and catalase) with an increase in the cumulative concentration
of CO •− was observed (Fig. 5.7) [57]. However, the decrease was lower than
the decrease of the enzyme activity. This suggests destruction of the heme
center was the main, but not exclusive, cause for the loss of enzyme activity.
Significantly, the changes in absorbances were not observed in the Soret region
of HRP, cytochrome c , and catalase after pulse radiolysis experiments. This
indicates the production of globin radicals decayed completely via dimerization/
disproportionation processes under the studied conditions of the experiments
(Fig. 5.7).
The reactivities of CO •− with native and denatured bovine serum albumin
(BSA) were similar (Table 5.2) and may likely be a result of the reactions with
all of the solvent-exposed reactive residues, including TrpH and TyrOH (Eq.
5.15). The characteristic absorption maximum of the Trp • radical in the visible
region was observed (Fig. 5.8) [59]. The decay of Trp • occurred as a result of
electron transfer from TyrOH of either the same (intramolecular, Eq. 5.16a)
or different albumin molecule (intermolecular, Eq. 5.16b), giving TyrO • . The
decay rate of the absorption band at ∼510 nm for Trp • was equal to the forma-
tion rate of the absorption band at 410 nm for TyrO • :