Flavonoids as reductants of ferryl hemoglobin

The ferryl derivatives of hemoglobin are products of the reactions of oxyand methemoglobin with hydrogen peroxide. Ferryl hemoglobins, either with or without a radical site on the protein moiety, are oxidizing species. Plant polyphenols, flavonoids, have been shown to act as antioxidants in vivo and in vitro. Reactions of metand oxyhemoglobin with hydrogen peroxide in the presence of catechin, quercetin and rutin were studied. These flavonoids accelerated reduction of ferryl hemoglobin to methemoglobin. The rate constants of the reactions of ferryl hemoglobin with catechin, quercetin and rutin were in the order of 102 M–1 s–1, i.e. similar to the rate constants of ferryl hemoglobin with intracellular reducing compounds like urate or ascorbate. The beneficial effect of flavonoids against oxidative damage of hemoglobin caused by hydroperoxides, reported in the literature, is probably, at least in part, connected with the ability of flavonoids to scavenge ferryl hemoglobin.

Flavonoids are plant polyphenols and are present as common components in human diet.Their ability to act as antioxidants has been extensively L. Gebicka and E. Banasiak studied (Heim et al., 2002, and references therein).On the other hand, their prooxidant activity has also been reported (Cotelle, 2001, and references therein).It has recently been shown that the flavonoid (-)epigallocatechin gallate, one of the main catechins isolated from green tea, efficiently reduces ferrylHb, but also enhances the autooxidation of human hemoglobin (Jia & Alayash, 2008).
In the present study we investigated the kinetics of the reduction of ferrylHb by three flavonoids: quercetin, rutin and catechin.

MATERIALS AND METHODS
Hemoglobin from bovine blood was obtained from Sigma.Methemoglobin was prepared by oxidation of Hb with a 2-fold excess of potassium ferricyanide and passage through a Sephadex G-25 column using 10 mM phosphate buffer (pH 7.0) as an eluent.Oxyhemoglobin was obtained by reduction of Hb with a 10-fold excess of sodium dithionite.The solution was then bubbled with O 2 and purified on a Sephadex G-25 column.Concentrations of metHb and oxyHb were determined spectrophotometrically using ε 405 = 1.79 × 10 5 M -1 cm -1 and ε 500 = 1.00 × 10 4 M -1 cm -1 for metHb, ε 415 = 1.25 × 10 5 M -1 cm -1 and ε 577 = 1.38 × 10 4 M -1 cm -1 for oxyHb, expressed per heme (Antonini & Brunori, 1971).FerrylHb was prepared by incubation 1.5 × 10 -5 M MetHb with an equimolar amount of H 2 O 2 in relation to the heme group in 10 mM phosphate buffer at room temperature for 5 min (Sztiller et al., 2006).
Quercetin, rutin and catechin were obtained from Sigma.Stock solutions of quercetin and rutin were prepared in DMSO.Stock solution of catechin was prepared in water.
Absorbance spectra of oxy-, met-and ferryl hemoglobin were recorded in the range of 350-600 nm.Absorbance maxima of selected haemoglobin species are shown in Table 1.All spectrophotometric measurements were carried out at ambient temperature (23±1 o C) using a Hewlett-Packard 8452A diodearray spectrophotometer.Water from MilliQ Plus was used throughout.

RESULTS AND DISCUSSION
We found that quercetin, rutin and catechin added to oxyHb at a molar ratio [flavonoid]/[heme] in the range of 1-15 did not significantly change the rate of autooxidation of oxyhemoglobin during the first 0.5 h.Our results differ from those published by Jia and Alayash (2008) who have observed an enhanced rate of autooxidation of cross-linked Hb in the presence of epigallocatechin gallate (EGCG).
They explained the effect of EGCG by the formation of H 2 O 2 during EGCG autooxidation and its subsequent reaction with oxyhemoglobin.Under our experimental conditions the rate of flavonoid autooxidation was probably significantly lower than the rate of autooxidation of oxyhemoglobin.It has been shown that the rate of H 2 O 2 formation during autooxidation of epigallocatechins is relatively high in comparison to that measured during autooxidation of catechin (Mochizuki et al., 2002;Munoz-Munoz et al., 2008) The absorbance spectra obtained after mixing oxyHb or metHb with H 2 O 2 were characteristic for ferryl derivative of hemoglobin (ferrylHb with and without protein radical are spectroscopically indistinguishable).The values of the rate constant of the reaction of bovine metHb with H 2 O 2 found in the literature vary from 13 M -1 s -1 (Nagababu et al., 2002) to 3.7 × 10 3 M -1 s -1 (Patel et al., 1996).We measured the rate of this reaction under pseudo-first order conditions by following the absorbance changes at 406 nm (near a maximum of MetHb) and found the second-order rate constant equal to 98 M -1 s -1 at pH 7.0.The reaction product, ferrylHb with a globin-based radical, also reacts with H 2 O 2 to restore metHb (Nagababu & Rifkind, 2000), but this reaction is much slower.After mixing 3 × 10 -5 M MetHb with 1.5 × 10 -4 M H 2 O 2 an absorbance spectrum characteristic for ferrylHb appeared after several minutes (Fig. 1).This spectrum was stable during the next several minutes and then slowly disappeared.When metHb was mixed with H 2 O 2 in the presence of catechin, quercetin or rutin, a rapid decrease of the absorbance maximum of metHb, without any shift and then an increase of the absorbance maximum was observed (Fig. 2).Such absorbance changes were not observed when H 2 O 2 was absent in the reaction mixture.It means that the investigated flavonoids reduce ferryl derivatives of hemoglobin in reactions characteristic for heme peroxidases: (2) where compound I is a ferryl derivative (Fe(IV)=O) with a radical site on porphyrin or protein, compound II is a ferryl derivative without a radical site, and SH is a substrate to be oxidized.In the case of hemoglobin, ferrylHb with and without a globinbased radical are analogs of peroxidase compounds I and II, respectively.The lack of an absorbance maximum (or shoulder) characteristic for ferryl derivatives (at 414 nm) means that the rates of disappearance of ferryl derivatives in the reactions with flavonoids are comparable or higher than the rates of their formation.It is worth noting that under our experimental conditions the flavonoids did not react directly with H 2 O 2 (we did not detect absorbance spectra of oxidized catechin, quercetin or rutin).After mixing 2.2 × 10 -5 M oxyHb with 1.5 × 10 -4 M H 2 O 2 a decrease of the absorbance maximum at 414 nm was observed during 15 min of the reaction (Fig. 3).When the investigated flavonoids were also added to this mixture, the decrease of absorbance at 414 nm was followed by a build-up of a spectrum with a maximum at 406 nm, characteristic for metHb (Fig. 4).Under such reaction conditions, fer-rylHb which was formed in the reaction of oxyHb with H 2 O 2 underwent reduction to metHb by the flavonoid.
In order to determine the rate constants of the reactions of the investigated flavonoids with ferryl-Hb, we preformed ferrylHb, mixed it with the flavonoids and observed the increase of absorbance at 406 nm, characteristic for metHb (Fig. 5).We assumed that the preformed ferryl species was an anolog of peroxidase compound II (ferrylHb with a protein radical is unstable with t 1/2 = 50 s (McArthur & Davies, 1993).We were unable to study this reaction under pseudo-first order conditions as catechin, quercetin and rutin, and their oxidation products absorb in the same wavelength range as hemoglobin.Thus, second-order rate constants were estimated from the initial reaction rates between ferrylHb and the flavonoids, measured for several flavonoid concentrations and taking ∆ε 406 (metHb -ferrylHb) = 0.64 × 10 5 M -1 cm -1 determined by us.The rate of autoreduction of ferrylHb was included into the calculation.It    Absorbance spectra taken every 1 min after mixing 2.2 × 10 -5 M oxyHb, 1.5 × 10 -4 M H 2 O 2 and 3.1 × 10 -4 M catechin, l = 1 mm.
L. Gebicka and E. Banasiak was at least one order of magnitude lower than the rate of the reduction of ferrylHb by the investigated flavonoids.The estimated rate constants of the reactions of ferrylHb with quercetin, rutin and catechin are collected in Table 2.They are of the order of 10 2 M -1 s -1 , i.e are similar to the rate constants of the reactions of ferrylHb with such reducing compounds like ascorbate, urate, nitrite and epigallocatechin gallate (Table 3).
A beneficial effect of flavonoids against oxidative damage of hemoglobin and red blood cells caused by hydroperoxides has been reported (Cesquini et al., 2003;Pereira et al., 2003).This effect seems to be, at least in part, connected with the ability of flavonoids to scavenge ferrylHb which is formed in the reaction of oxyHb with hydrogen peroxide or organic hydroperoxides.FerrylHb reacts with oxyHb to form metHb (comproportionation reaction (Giulivi & Davies, 1990).Thus, if we take into account the following reaction schemes: a) in the absence of flavonoids (or other reducing agents): b) in the presence of flavonoids (or other reducing agents): we can see that the presence of flavonoids under conditions where ferrylHb is formed may prevent half of the oxyHb molecules from conversion to metHb (Grinberg et al., 1994).Absorbance spectra taken every 30 s after mixing 2.5 × 10 -6 M ferrylHb with 1 × 10 -5 M rutin, l = 1 cm.Inset: Kinetic traces measured at 406 nm.