Rate of nitric oxide scavenging by hemoglobin bound to haptoglobin |
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Authors: | Ivan Azarov Xiaojun He Anne Jeffers Swati Basu Burak Ucer Roy R Hantgan Andrew Levy Daniel B Kim-Shapiro |
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Institution: | aDepartment of Physics, Wake Forest University, Winston-Salem, NC 27109, USA;bDepartment of Biochemistry, Wake Forest University, School of Medicine, Winston-Salem, NC 27157, USA;cRappaport Faculty of Medicine, Technion Institute of Technology, Haifa, Israel |
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Abstract: | Cell-free hemoglobin, released from the red cell, may play a major role in regulating the bioavailability of nitric oxide. The abundant serum protein haptoglobin, rapidly binds to free hemoglobin forming a stable complex accelerating its clearance. The haptoglobin gene is polymorphic with two classes of alleles denoted 1 and 2. We have previously demonstrated that the haptoglobin 1 protein–hemoglobin complex is cleared twice as fast as the haptoglobin 2 protein–hemoglobin complex. In this report, we explored whether haptoglobin binding to hemoglobin reduces the rate of nitric oxide scavenging using time-resolved absorption spectroscopy. We found that both the haptoglobin 1 and haptoglobin 2 protein complexes react with nitric oxide at the same rate as unbound cell-free hemoglobin. To confirm these results we developed a novel assay where free hemoglobin and hemoglobin bound to haptoglobin competed in the reaction with NO. The relative rate of the NO reaction was then determined by examining the amount of reacted species using analytical ultracentrifugation. Since complexation of hemoglobin with haptoglobin does not reduce NO scavenging, we propose that the haptoglobin genotype may influence nitric oxide bioavailability by determining the clearance rate of the haptoglobin–hemoglobin complex. We provide computer simulations showing that a twofold difference in the rate of uptake of the haptoglobin–hemoglobin complex by macrophages significantly affects nitric oxide bioavailability thereby providing a plausible explanation for why there is more vasospasm after subarachnoid hemorrhage in individuals and transgenic mice homozygous for the Hp 2 allele. |
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Keywords: | Nitric oxide Hemoglobin Haptoglobin Kinetics Time-resolved absorption spectroscopy Analytical centrifugation |
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