title: Effects of T- and R-state stabilization on deoxyhemoglobin-nitrite reactions and stimulation of nitric oxide signaling.
abstract: Recent data suggest that transitions between the relaxed (R) and tense (T) state of hemoglobin control the reduction of nitrite to nitric oxide (NO) by deoxyhemoglobin. This reaction may play a role in physiologic NO homeostasis and be a novel consideration for the development of the next generation of hemoglobin-based blood oxygen carriers (HBOCs, i.e. artificial blood substitutes). Herein we tested the effects of chemical stabilization of bovine hemoglobin in either the T- (THb) or R-state (RHb) on nitrite-reduction kinetics, NO-gas formation and ability to stimulate NO-dependent signaling. These studies were performed over a range of fractional saturations that is expected to mimic biological conditions. The initial rate for nitrite-reduction decreased in the following order RHb>bHb>THb, consistent with the hypothesis that the rate constant for nitrite reduction is faster with R-state Hb and slower with T-state Hb. Moreover, RHb produced more NO-gas and inhibited mitochondrial respiration more potently than both bHb and THb. Interestingly, at low oxygen fractional saturations, THb produced more NO and stimulated nitrite-dependent vasodilation more potently than bHb despite both derivatives having similar initial rates for nitrite reduction and a more negative reduction potential in THb versus bHb. These data suggest that cross-linking of bovine hemoglobin in the T-state conformation leads to a more effective coupling of nitrite reduction to NO-formation. Our results support the model of allosteric regulation of nitrite reduction by deoxyhemoglobin and show that cross-linking hemoglobins in distinct quaternary states can generate products with increased NO yields from nitrite reduction that could be harnessed to promote NO-signaling in vivo.
authors.affiliation: Department of Pathology, Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL, USA.
authors.forename: Nadiezhda
authors.initials: N
authors.lastname: Cantu-Medellin
authors.forename: Dario A
authors.initials: DA
authors.lastname: Vitturi
authors.forename: Cilina
authors.initials: C
authors.lastname: Rodriguez
authors.forename: Serena
authors.initials: S
authors.lastname: Murphy
authors.forename: Scott
authors.initials: S
authors.lastname: Dorman
authors.forename: Sruti
authors.initials: S
authors.lastname: Shiva
authors.forename: Yipin
authors.initials: Y
authors.lastname: Zhou
authors.forename: Yiping
authors.initials: Y
authors.lastname: Jia
authors.forename: Andre F
authors.initials: AF
authors.lastname: Palmer
authors.forename: Rakesh P
authors.initials: RP
authors.lastname: Patel
mesh_terms: D000494:Allosteric Regulation; D000704:Analysis of Variance; D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects; D002417:Cattle; D019069:Cell Respiration; D004563:Electrochemistry / Q000379:methods; D006454:Hemoglobins / Q000737:chemistry* / Q000378:metabolism; D006834:Hydrazines / Q000494:pharmacology; D066298:In Vitro Techniques; D008297:Male; D008928:Mitochondria / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D009572:Nitrite Reductases / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism* / Q000494:pharmacology; D010084:Oxidation-Reduction; D058105:Polymerization; D011487:Protein Conformation; D055550:Protein Stability; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D014664:Vasodilation
chemical_list: C105785:1-hexanamine-6-(2-hydroxy-1-methyl-2-nitrosohydrazine)-N-methyl; D006454:Hemoglobins; D006834:Hydrazines; D009573:Nitrites; D009569:Nitric Oxide; C100003:deoxyhemoglobin; D009572:Nitrite Reductases