Abstract
The redox properties and thiol reactivity of quinones play critical roles in their therapeutic and toxicological properties. The present study was undertaken to investigate the binding activity of ubiquinone 0 (UQ(0)) to human oxyhemoglobin (HbO(2)) using electron spin resonance (ESR). Addition of UQ(0) to HbO(2) resulted in the immediate detection of a five-line ESR spectrum characteristic of the semiquinone radical of UQ(0) (UQ(0)). With time the HbO(2) adduct with UQ(0), which was characterized by a broad immobilized ESR spectrum, was gradually formed. Matrix-assisted laser desorption/ionization time-of-flight mass spectra analysis showed that UQ(0) bound to the beta-chain of HbO(2). Superoxide dismutase dose-dependently suppressed the intensity of the broad spectrum and accelerated its formation. However, N-ethylmaleimide, a thiol-blocking agent, completely eliminated its formation. The nonspecific protease mixture pronase also prevented its formation and resulted in the gradual appearance of a 4-line spectrum from the 5-line spectrum of UQ(0). The structure of the species responsible for the 4-line spectrum was confirmed and identified by the reaction of UQ(0) with reduced glutathione. In human red blood cells, UQ(0) rapidly bound to glutathione but more slowly to HbO(2). These results suggest that UQ(0) reacted with both ferrous heme and the reactive beta-93 cysteinyl residue of HbO(2) to generate its corresponding semiquinone radical. Subsequently UQ(0) bound to the beta-93 cysteinyl residue of HbO(2) to form a covalent-binding adduct responsible for the broad spectrum.
Highlights
Electron Spin Resonance Investigation of Semiquinone Radicals Formed from the Reaction of Ubiquinone 0 with Human Oxyhemoglobin*
In human red blood cells, ubiquinone 0 (UQ0) rapidly bound to glutathione but more slowly to HbO2. These results suggest that UQ0 reacted with both ferrous heme and the reactive -93 cysteinyl residue of HbO2 to generate its corresponding semiquinone radical
Interaction of Ubiquinone 0 with Human Oxyhemoglobin—When 0.5 mM UQ0 was mixed with 1.2 mM human HbO2 in borate buffer, a 5-line electron spin resonance (ESR) spectrum appeared immediately (Fig. 1A)
Summary
Electron Spin Resonance Investigation of Semiquinone Radicals Formed from the Reaction of Ubiquinone 0 with Human Oxyhemoglobin*. The present study was undertaken to investigate the binding activity of ubiquinone 0 (UQ0) to human oxyhemoglobin (HbO2) using electron spin resonance (ESR). In human red blood cells, UQ0 rapidly bound to glutathione but more slowly to HbO2 These results suggest that UQ0 reacted with both ferrous heme and the reactive -93 cysteinyl residue of HbO2 to generate its corresponding semiquinone radical. UQ0 bound to the -93 cysteinyl residue of HbO2 to form a covalent-binding adduct responsible for the broad spectrum. Adriamycin possesses antitumor activity and is important therapeutically [14] Because quinones and their corresponding semiquinone species have strong electrophilic character, they can readily react with available nucleophiles, especially sulfhydryl groups, and thereby form covalent adducts
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