Abstract
Oxidations of hemoglobin in oxygen-saturated and deoxygenated media by 10 structurally variant alkyl nitrites have been examined in kinetic detail. Pronounced structural influences on rate constants, whose values span a range of 80 in oxidations of both oxy- and deoxyhemoglobin, have been observed. tert-Butyl nitrite provides the slowest oxidation rate that for deoxyhemoglobin terminates after only half of the available iron(II) heme units have been oxidized. Activation parameters have been determined for oxidations of oxyhemoglobin and deoxyhemoglobin by ethyl, isopropyl, and neopentyl nitrites from kinetic evaluation of these reactions as a function of temperature. The differences in free energies of activation (delta G) between hemoglobin R and T states range from 1.8 to 2.9 kcal/mol for the three alkyl nitrites examined. The composite data portray alkyl nitrite oxidations as inner sphere electron transfer processes whose kinetic characteristics reflect the ligand binding properties of hemoglobin. A sulfhydryl-induced alkyl nitrite oxidation of oxyhemoglobin that is most pronounced in oxygen-saturated media has been observed, and its cause has been traced to nitrosyl exchange of alkyl nitrites with the beta-93 cysteine sulfhydryl group of hemoglobin.
Highlights
Oxidations of hemoglobin in oxygen-saturated and transfer process is suggested if oxidation occurs on both the deoxygenated media by 10 structurally variant alkyl oxy and deoxy forms with the same kinetic dependence on nitriteshave been examinedinkineticdetail.Prohemoglobin and on the chemical oxidant and if iron ligands nounced structural influences on rate constants, whose that include dioxygen inhibitoxidation [11,12,13,14]
Sulfhydryl-induced Alkyl Nitrite Oxidation-Our prior evaluation of the kinetic characteristicsof ethyl nitrite oxidations of oxyhemoglobin identifiedan inverse firstorderkinetic dependence on the concentration of molecular oxygen that deviated from linearity when the oxygen concentration approached saturation [16]
If hemoglobin oxidation by alkyl nitrites occursby inner sphere electron transfer, asis suggested by structural influences on reactivity (Tables I and II),the observed rate enhancementfor oxidation of oxyhemoglobin could be a reflection of differential steric barriers in the R and T state conformations [34]
Summary
Oxidations of hemoglobin in oxygen-saturated and transfer process is suggested if oxidation occurs on both the deoxygenated media by 10 structurally variant alkyl oxy and deoxy forms with the same kinetic dependence on nitriteshave been examinedinkineticdetail.Prohemoglobin and on the chemical oxidant and if iron ligands nounced structural influences on rate constants, whose that include dioxygen inhibitoxidation [11,12,13,14]. Hydrolytic rate constants were determined at 25.0 “C in 0.05 M phosphate-bufferedsolution (pH 7.0) for each of the alkyl nitrites employed in kinetic investigations of hemoglobin oxidation. The compositions of nitrosylhemoglobin and methemoglobin from alkyl nitrite oxidations of deoxyhemoglobin were calculated from product absorbances observed at 542, 552, and 572 nm in each experiment, and the resulting composition determinations were averaged to obtain the reported values. Free cysteine added to trheeaction solution containing p-93 sulfhydryl-blocked HbO,did not increase the observed rate constant for hemoglobin oxidation even when a 10-fold molar excess of cysteine was employed. 0 kcal/mol on oxygen concentration (Fig. 1)for oxidation of p-93 sulfhydryl-blocked HbO, is identical to that for oxidation of unmodified hemoglobin, the use of iodoacetamide as theblocking reagent does not influence the kinetic character of oxyhemo-. Composite errors in AH$ and AS$ cancel in calculations of AG$
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