Abstract
We have studied the heme oxidation kinetics of purified human hemoglobin (Hb) in the presence of lipid vesicles of dipalmitoyl phosphatidylcholine and bovine brain phosphatidylserine that exhibited minimal lipid peroxidation. We showed that the lipid vesicles enhanced Hb oxidation and that small unilamellar vesicles (SUVs) exerted a larger effect than large unilamellar vesicles (LUVs). We have determined pseudo first-order rate constants for the initial disappearance of oxygenated ferrous Hb (k0) and for the initial formation of several ferric Hb species (methemoglobin, hemichrome, and choleglobin) in the presence of SUVs and LUVs. k0 and other rate constants depended linearly on lipid-to-hemoglobin molar ratio (lipid/Hb), with k0SUV (h-1) = k0auto (h-1) + 3.7 x 10(-3) x lipid/Hb, and k0LUV (h-1) = k0auto (h-1) + 0.2 x 10(-3) x lipid/hb, where k0auto is the rate constant for Hb autoxidation in the absence of vesicles. Thus, in the absence of lipid peroxidation products, lipid vesicles themselves promote Hb oxidation by enhancing the rate of Hb oxidation. The enhanced oxidation was inhibited by catalase, but not by butylated hydroxytoluene. The rate constants were independent of Hb concentration, in the range of about 3.1 to 100 microM. We suggest that the lipid surface properties, including surface curvature, surface energy, and hydrophobicity, promote hemoglobin oxidation.
Highlights
We have studied the hemeoxidation kinetics of pu- absence of membrane components, whereas i n vivo Hb oxirified human hemoglobin (Hb)in the presence of lipid dation occurs in an environment enclosed by cellmembranes
Effects of BPS LUVs on Hb Oxidation Rate Constants-We found that the extruded LUVs enhanced Hb oxidation to a lesser extent than sonicated SUVs, at similar lipid/Hb
When the process of vesicle fusion induced by Hb wasslower than that of oxyHb oxidation, reliable kinetic information for the initial oxidation process of Hb in the presence of SUVs, or LUVs, could be obtained optically in the 500-700-nm region
Summary
We have studied the hemeoxidation kinetics of pu- absence of membrane components, whereas i n vivo Hb oxirified human hemoglobin (Hb)in the presence of lipid dation occurs in an environment enclosed by cellmembranes. These SUV-enhanced spectral changes were not observedwhen oxyHb was replaced with oxygenated hemolysate or HbCO (Fig. IC). The excellent linear fit suggests that reliable rate constants for the initial disappearance of oxyHb in the presence of lipid vesicles can be obtained optically. Reproducibility of these data was very good. We prepared samples with various Hb concentrations, ranging from 0.20 (3.1 pM) to 7 mgfml (109.3 yM) and lipid concentrations ranging from 0.25 to 9 mg/ml to maintain lipid/Hb of 100 or 200.We found that at either lipid/Hb value, the values of ktUVremained similar as the Hb concentrations increased from 12.4 to 437 I.~Min heme (Fig. 4).Since
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