Abstract
The autocatalytic reaction between nitrite and the oxy form of globins involves free radicals. For myoglobin (Mb), an initial binding of nitrite to the iron-coordinated oxygen molecule was proposed; the resulting ferrous-peroxynitrate species was not detected, but its decay product, the high-valent ferryl form, was demonstrated in stopped-flow experiments. Reported here are the stopped flow spectra recorded upon mixing oxy Hb (native, as well as chemically-derivatized in the form of several candidates of blood substitutes) with a supraphysiological concentration of nitrite. The data may be fitted to a simple kinetic model involving a transient met-aqua form, in contrast to the ferryl detected in the case of Mb in a similar reaction sequence. These data are in line with a previous observation of a transient accumulation of ferryl Hb under auto-catalytic conditions at much lower concentrations of nitrite (Grubina, R. et al. J. Biol. Chem. 2007, 282, 12916). The simple model for fitting the stopped-flow data leaves a small part of the absorbance changes unaccounted for, unless a fourth species is invoked displaying features similar to the oxy and tentatively assigned as ferrous-peroxynitrate. Density functional theory (DFT) calculations support this latter assignment. The reaction allows for differentiating between the reactivities of various chemically modified hemoglobins, including candidates for blood substitutes. Polymerization of hemoglobin slows the nitrite-induced oxidation, in sharp contrast to oxidative-stress type reactions which are generally accelerated, not inhibited. Sheep hemoglobin is found to be distinctly more resistant to reaction with nitrite compared to bovine Hb, at large nitrite concentrations (stopped-flow experiments directly observing the oxy + nitrite reaction) as well as under auto-catalytic conditions. Copolymerization of Hb with bovine serum albumin (BSA) using glutaraldehyde leads to a distinct increase of the lag time compared to native Hb as well as to any other form of derivatization examined in the present study. The Hb-BSA copolymer also displays a slower initial reaction with nitrite under stopped-flow conditions, compared to native Hb.
Highlights
Under various stress conditions, the redox chemistry of hemoglobin is susceptible to toxic reactions due to the interaction with oxidative stress agents such as peroxide, yielding free radicals and highly-oxidizing states at the iron [1,2,3,4,5]
Having previously shown that ferryl is clearly detectable in the reaction on oxy Mb and nitrite under stopped-flow millisecond conditions at high nitrite concentrations where the initial oxy+nitrite reaction is directly observable [9], we report here on the distinctly different behavior of hemoglobin in the same reaction—revealing at the same time a potential application as a test for blood substitute candidates in terms of reactivity towards a component of nitrosative stress
The reaction between nitrite and oxy Hb is influenced by the concentration of reactants in complex manners, especially at lower nitrite concentrations—linked to the fact that several processes are thought to occur beyond the initial event of oxyHb being oxidized to met by nitrite, with NO2 as a likely product [12,13,14,15,16,17,19,20,40]
Summary
The redox chemistry of hemoglobin is susceptible to toxic reactions due to the interaction with oxidative stress agents such as peroxide, yielding free radicals and highly-oxidizing states at the iron (ferryl, Compound II) [1,2,3,4,5]. Having previously shown that ferryl is clearly detectable in the reaction on oxy Mb and nitrite under stopped-flow millisecond conditions at high nitrite concentrations where the initial oxy+nitrite reaction is directly observable [9], we report here on the distinctly different behavior of hemoglobin in the same reaction—revealing at the same time a potential application as a test for blood substitute candidates in terms of reactivity towards a component of nitrosative stress. Upon completion of this study, a recent report by Bulow and co-workers has provided additional evidence for the facts that the reaction of oxyHb with nitrite involves metHb and nitrate as final products, and ferryl Hb is an intermediate species of the reaction when working at a low-millimolar concentration level of nitrite, as initially observed under similar auto-catalytic conditions where, at ~10 s after mixing, a brief accumulation of ferryl was detected [31]
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