A. Spector et al., Investigation of the mechanism of action of microperoxidase-11, (MP11), a potential anti-cataract agent, with hydrogen peroxide and ascorbate, EXP EYE RES, 71(2), 2000, pp. 183-194
The interaction of hydrogen peroxide, ascorbate and microperoxidase-ll (MP1
1), a ferriheme undecapeptide derived from cytochrome c, has been investiga
ted using spectrophotometry, oxymetry, electron paramagnetic resonance (EPR
), and mass spectroscopy techniques. It is shown that in 50 mM phosphate pH
7.0-7.4 in the absence of other reactants H2O2 induces a concentration-dep
endent decrease in absorption at the Soret band (399 nm) of the microperoxi
dase, with concomitant H2O2 decomposition and oxygen evolution. The reactio
n causes irreversible heme degradation, concomitant with loss of enzymatic
activity. Ascorbate effectively protects MP11 from degradation and inhibits
oxygen evolution. At ascorbate concentrations greater than that of H2O2, m
icroperoxidase degradation is almost completely prevented. Mass spectrometr
y showed that H2O2 oxidizes the microperoxidase to a mono-oxygenated produc
t, which did not form if ascorbate was included in the reaction system, The
re appears to be a 1:1 relationship between H2O2 degradation and ascorbate
oxidation. EPR experiments revealed that an ascorbate radical was formed du
ring the reaction, These reactions may be described by a scheme where a put
ative 'compound I' of the microperoxidase is reduced by ascorbate back to t
he original redox state (ferric) of the peroxidase in two one-electron step
s, concomitantly with oxidation of the ascorbate to an ascorbate radical or
in one two-electron transfer step forming dehydroascorbate. In the absence
of ascorbate, the 'compound I' reacts further with the peroxide causing mi
croperoxidase degradation and partial oxygen evolution. These observations
are relevant to the interaction of ferrihemes with H2O2 and ascorbic acid a
nd may he pertinent for the potential application of MP11 as an anti-catara
ct agent, (C) 2000 Academic Press.