Hemoglobin: a mechanism for the generation of hydroxyl radicals.

Oxyhemoglobin (HbO2) reduces Fe(III)NTA aerobically to become methemoglobin (metHb) and Fe(II)NTA. These conditions are favorable for the generation via Fenton chemistry of the hydroxyl radical that was measured by HPLC using salicylate as a probe. The levels of hydroxyl radicals generated are a function of both the percent metHb formed and the chemical nature of the buffer. The rates of formation of both metHb and hydroxyl radicals were dependent upon the concentration of Fe(III)NTA. Of the buffers tested, HEPES was the most effective scavenger of hydroxyl radicals while the other buffers scavenged in the order: HEPES > Tris > MPOS > > NaCL approximately unbuffered. The addition of catalase to remove H2O2 or bathophenanthroline to chelate Fe(II) inhibited virtually all hydroxyl radical formation. Carbonyl formation from free radical oxidation of amino acids was found to be 0.1 mol/mol of hemoglobin. These experiments demonstrate the ability of hemoglobin to participate directly in the generation of hydroxyl radicals mediated by redox metals, and provide insight into potential oxidative damage from metals released into the blood during some pathologic disorders including iron overload.

Intrinsic stoichiometric equilibrium constants for the binding of zinc(II) and copper(II) to the high affinity site of serum albumin.

Intrinsic stoichiometric equilibrium constants were determined for zinc(II) and copper(II) binding to bovine and human serum albumin. Data were obtained from equilibrium dialysis experiments. Metals were presented to apoprotein as metal chelates in order to avoid metal hydrolysis and to minimize nonspecific metal-protein interactions. Scatchard analysis of the binding data indicated that the high affinity class for both zinc and copper was comprised of one site. Results of binding experiments done at several pH values suggested that while both histidyl and carboxyl groups appear to be involved in copper binding, histidyl residues alone were sufficient for zinc binding. These amino acid residues were used in combination to model several binding sites used in the formulation of equilibria expressions from which stoichiometric constants were calculated. The log10K for bovine serum albumin were calculated to be 7.28 for Zn(II) and 11.12 for Cu(II). Those for human serum albumin were determined to be 7.53 and 11.18 for Zn(II) and Cu(II), respectively. These constants were used in equilibria to simulate speciation of metal-albumin and metal-chelator and to illustrate relative binding affinities. This comparison of binding strengths was possible only through the calculation of an intrinsic stoichiometric binding constant.

Site-directed mutagenesis of histidine residues involved in Cu(II) binding and reduction by sperm whale myoglobin.

Sperm whale myoglobin (Mb) reduces Cu(II) through a site-specific mechanism involving complexation by one or more surface histidine residues. Three mutants of Mb, derived from recombinant wild-type Mb, were designed in which surface histidine residues exhibiting strong Cu(II) binding were replaced with amino acids with comparatively poor metal binding characteristics. The kinetics of Cu(II)(Gly)2 reduction by native Mb, recombinant wild-type Mb, and the mutants were compared. Recombinant wild-type Mb reduced Cu(II) at a rate similar to that of native Mb. Two single mutations (His-48----Ala and His-116----Asp) decreased the rate by 31% and 7%, respectively, relative to wild-type Mb and decreased the rate by 38% and 16%, respectively, relative to native Mb. A double mutation (His-113----Ala, His-116----Asp) decreased the rate only slightly more than the single mutation at His-116. Previous NMR studies showed that His-113 exhibits the strongest Cu(II) binding of all surface histidines, but the present experiments suggest that it plays little or no role in the reduction of Cu(II) by Mb. His-48, located 12.7 A from the Fe(II)-heme, participates in one-third of the redox activity of the protein. His-116 appears to play a minor role in the overall redox activity of Mb, but its involvement shows that Mb has the ability to reduce Cu(II) through a histidine residue located more than 20 A from the Fe(II)-heme. These experiments demonstrate that electron transport from the Fe(II)-heme to site-specifically bound Cu(II) can be mediated through multiple pathways in sperm whale Mb.