HNO-myoglobin reactivity examined by SVD and global analysis.


Analyses of reactions involving HNO are inherently difficult as it must be generated in situ, and undergoes several concurrent and competitive reactions; thus its concentration flux is difficult to characterize. Global kinetic modeling, using singular value decomposition software, allows for investigation of complex time course spectra. Using this method, the determined rate for trapping of HNO by metmyoglobin, which produces NO-myoglobin, is found to be 2.7 x 10⁵ M^-1s^-1 at pH 7.0 and 1.1 x 10⁵ M^-1s^-1 at pH 9.4. The reaction of deoxymyoglobin with HNO generates the adduct HNO-myoglobin directly. It is followed by a secondary reaction of the adduct with HNO yielding NOmyoglobin; the determined bimolecular rate constants for these reactions are 3.48 x 10⁵ M^-1s^-1 and 1.67 x 104 M^-1s^-1 respectively, both of which are independent of pH. The derived spectrum for HNO-myoglobin is characterized by a Soret absorbance maximum at 423 nm with an extinction coefficient of 1.66 x 10⁵ M^-1cm^-1. The rate constant for unimolecular loss of HNO from HNO-myoglobin was determined by competitive trapping with CO at 8.9 x 10^-5 s^-1, which produced a thermodynamic binding affinity of HNO to deoxymyoglobin of 3.9 x 10^10 M^-1. Also in this report, sequential absorbance spectra are used to analyze reactions of HNO with a mixture of met- and deoxy-Mb, (which have highly overlapping spectra) and also to model the flux of HNO over the course of the reaction. The determined trapping rate constants, 2.76 x 10⁵ M^-1s^-1 for met-Mb and 3.74 x 10⁵ M^-1s^-1 for deoxy-Mb, are qualitatively similar, thus little kinetic preference is predicted in physiological reactivity of HNO. Lastly, the study of a pH and O₂ dependent RNS is generated in situ by the reaction of NO-Mb with O₂, which introduces the possibility of heme modifications and N-bound ferrous RNS adducts. These results suggest that the formation of HNO-ferrous adducts and their intermediates represent an important consideration in the biological action of HNO-releasing drugs.



SVD- singular value decomposition. Mb- myoglobin. cGMP- cyclic guanosine monophosphate. cAMP- cyclic adenosine monophosphate. AS- Angeli’s Salt or trioxodinitrate. PA- Piloty’s Acid or benzenesulfohydroxamic acid. MSHA- N-methane sulfonylhydroxylamine. MLA- marquardt-levenberg algorithm. RKI- runge-kutta integration.