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

作者：Zapata Adrian L; Kumar Murugaeson R; Pervitsky Dmitry; Farmer Patrick J^*

来源：Journal of Inorganic Biochemistry, 2013, 118: 171-178.

DOI：10.1016/j.jinorgbio.2012.10.005

• HNO
• Bimolecular rate constants
• Myoglobin
• Binding affinity

The reactions of several horse heart myoglobin species with nitrosyl hydride. HNO, derived from Angeli%26apos;s salt (AS) and Piloty%26apos;s acid (PA) have been followed by UV-visible, H-1 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 x 10(5) M-1 s(-1) at pH 7.0 and 1.1 x 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 x 10(5) M-1 s(-1) and 1.67 x 10(4) 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 x 10(5) 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 x 10(-5) s(-1), which gives the thermodynamic binding affinity of HNO to deoxymyoglobin as 4.2 x 10(9) 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.

• 出版日期2013-1