Chemical Dynamics of NO Hemoglobin Interactions

Montana State University, Bozeman MT

Investigators

Abstract

Singel MCB 0091228 The discovery by Furchgott of an endothelium-derived relaxing factor (EDRF) and its subsequent identification with NO raise an intriguing problem: in regulating vascular tension and blood flow, how does NO avoid being hoarded or destroyed by well-known reactions with hemoglobin (Hb) to form Hb-heme-iron(II) nitrosyl complexes or nitrate? The recent discovery, by Stamler and co-workers, of an S-nitrosylated derivative of hemoglobin (SNO-Hb) in vivo, and the ongoing elucidation of its biological activities, suggest a means to evade both such fates. Evidently, the previously unsuspected NO/Hb reaction channel - S-nitrosylation - can effectively compete with the Hb-Fe(II)-NO and nitrate forming reactions. Moreover, these studies suggest a dynamical cycle in which the NO-group is alternatively largely sequestered (Hb-Fe(II)-NO) or made available for delivery (Hb-SNO), in response to the cyclic variation in oxygen tension during the arterial/venous transit. The implicated iron-to-sulfur NO-group transfer chemistry appears to be linked to the cooperative oxygen delivery function of Hb. The broad aim of this project is to generate experimental results that test these ideas, and provide critical observations for further development of the model. A direct competition (competitive kinetics) method recently introduced by Gow et al. will be utilized to characterize the relative rates of the reactions that occur upon exposure of oxygenated Hb to NO and its congeners. The experiments are designed to detect the dependence of the relative kinetic behavior on the oxygen-saturation of Hb, and to probe the link between this behavior and the allosteric state of Hb. The reaction competition is assessed by detection of the heme-iron(II) nitrosyl-Hb, SNO-Hb, Fe(III)-metHb, and nitrate products through EPR (electron paramagnetic resonance) spectroscopy, UV/VIS spectroscopy, and chemiluminescence techniques. The chemical behavior will be characterized over a range of conditions -temperature, pH, CO2 tension, organic phosphate concentration - that: 1) focus on the standard physiological conditions relevant to testing the biological significance of the NO/Hb chemical pathways; but also 2) embrace non-physiological conditions that have assumed a standard role in studies of Hb allostery. Experiments designed to demonstrate that iron(II)-nitrosyl complexes in Hb can serve as a labile, accessible reservoir, rather than a sink of NO-groups (alpha-heme-iron(II) nitrosyls) will also be undertaken. EPR and chemiluminescence methods will be utilized to monitor the migration of the NO-group during variation of hemoglobin ligation state (oxy, deoxy, met). This reactive behavior will also be studied under conditions of physiological significance and under conditions known to affect the allosteric behavior of Hb.