A singular value decomposition approach for kinetic analysis of reactions of HNO with myoglobin

The reactions of several horse heart myoglobin species with nitrosyl hydride, HNO, derived from Angeli's salt (AS) and Piloty's acid (PA) have been followed by UV–visible, 1H NMR and EPR spectroscopies. Spectral analysis of myoglobin-derived speciation during the reactions was obtained by using singular value decomposition methods combined with a global analysis to obtain the rate constants of complex sequential reactions. The analysis also provided spectra for the derived absorbers, which allowed self-consistent calibration to the spectra of known myoglobin species. Using this method, the determined rate for trapping of HNO by metmyoglobin, which produces NO-myoglobin, is found to be 2.7 × 105 M− 1 s− 1 at pH 7.0 and 1.1 × 105 M− 1 s− 1 at pH 9.4. The reaction of deoxymyoglobin with HNO generates the adduct HNO-myoglobin directly, but is followed by a secondary reaction of that product with HNO yielding NO-myoglobin; the determined bimolecular rate constants for these reactions are 3.7 × 105 M− 1 s− 1 and 1.67 × 104 M− 1 s− 1 respectively, and 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 × 105 M− 1 cm− 1. The rate constant for unimolecular loss of HNO from HNO-myoglobin was determined by competitive trapping with CO at 8.9 × 10− 5 s− 1, which gives the thermodynamic binding affinity of HNO to deoxymyoglobin as 4.2 × 109 M− 1. These results suggest that the formation of HNO-ferrous heme protein adducts represents an important consideration in the biological action of HNO-releasing drugs.

Graphical abstractThe reactions of myoglobin species with HNO have been analyzed using SVD methods to obtain rate constants of complex sequential reactions. Bimolecular rates for reactions of HNO with metmyoglobin, deoxymyoglobin and HNO-myoglobin are described, as well as unimolecular loss of HNO from HNO-myoglobin, determined by competitive trapping with CO.Figure optionsDownload full-size imageDownload as PowerPoint slideHighlights► SVD global fitting obtains self-consistent analysis of complex sequential reactions. ► Bimolecular rate constants for the reactions of HNO with metMb, deoxyMb, and HNO-Mb ► Reaction of HNO with HNO-Mb has implications for pharmacology of HNO donors. ► First demonstration of HNO dissociation from HNO-Mb ► First determination of HNO/deoxyMb binding affinity