Abstract
The autoxidation of hemoglobin as a function of oxygen pressure and the effect of added nitroblue tetrazolium have been studied. It has been shown that the enhanced autoxidation at intermediate oxygen pressures can only be partially explained by the outer-sphere reaction of oxygen with deoxygenated chains. An additional enhancement associated with the properties of partially oxygenated hemoglobins has been attributed to the mobility on the distal side of the heme which facilitates the nucleophilic displacement of bound oxygen by the distal histidine. Nitroblue tetrazolium, in addition to reacting with the superoxide formed during autoxidation, is shown to oxidize directly both deoxygenated and oxygenated hemoglobin in two steps, proceeding through the one-electron reduced intermediate, a tetrazolinyl radical. Even though the oxygenated chains are oxidized by nitroblue tetrazolium, much less overall reduction of nitroblue tetrazolium is observed at high oxygen pressures. This phenomenon is attributed to the reoxidation by oxygen of both the tetrazolinyl radical and the formazan. The reaction with nitroblue tetrazolium is also found to contribute to enhanced oxidation at intermediate oxygen pressures. This behavior is explained by the two-electron reaction with nitroblue tetrazolium and the oxygen dependence of the various processes involved in the reaction of nitroblue tetrazolium.
Highlights
Can only be partially explained by the outer-sphere re- Levy et al, 1988)that the enhanced autoxidatiofnhemoglobin action of oxygenwithdeoxygenatedchains
Evenate oxygen pressures (Levy et al, 1988;Brooks, 1935;George though the oxygenated chains are oxidized by nitrobluaend Stratmann, 1952;Wallace et al, 1982)was confirmed,and tetrazolium,much less overallreductionofnitroblue kinetic modeling was used to determine the mechanism(s) retetrazolium is observed at high oxygen pressures
The oxygen (Wallace et al1.,982;Brantley et al, 1993;Dickerson et reaction with nitroblue tetrazoliiusmalso found to con-al., 1993)(Reaction 1) and the nucleophilic displacement (Waltribute to enhanced oxidation at intermediate oxygen lace et al, 1974a, 197413; Satoh and Shikama, 1981)of oxygen pressures. This behavior is explained by the two-elec- from partially oxygenated hemoglobin (Reaction 2)were both tron reaction with nitroblue tetrazolium and the ofxoyugnednto contribute to the enhanced autoxidation
Summary
Nitroblue tetrazoliumi,n addition to reacting with cal productionand potential damage to erythrocytes and other tissues under hypoxic conditions.A method such as the reduction of NBT would makiet possible to quantitate the oxidative stress produced underhypoxic conditions In this pathpeer we have studied quantitatively the autoxidationof hemosuperoxide formed during autoxidatioins,shown to oxi- globin as a function of oxygen pressure and investigated the dize directly both deoxygenated and oxygenated hemeoff-ect of adding NBT (Fig.). The abbreviations used are: NBT, nitroblue tetrazolium; PBS, phos- a gas line and evacuation of gas using a vacuum pump were continued phate-buffered saline; NBTr, reduced NBT PO,, oxygen pressure.
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