F. Auchere et al., Purification and structure of the major product obtained by reaction of NADPH and NMNH with the myeloperoxidase/hydrogen peroxide/chloride system, EUR J BIOCH, 268(10), 2001, pp. 2889-2895
The first spectrophotometric study of the reaction of the myeloperoxidase/H
2O2/Cl- system with NADPH and NMNH showed that the reaction products were n
ot the corresponding oxidized nucleotides and that modifications would take
place on the nicotinamide part of the molecule [Auchere, F. & Capeillere-B
landin, C. (1999) Biochem. J. 343, 603-613]. In this report, in order to ob
tain more precise information on the structural modifications and mechanism
of the reaction, we focus on the purification and isolation of products de
rived from NADPH and NMNH by RP-HPLC. Electrospray ionization mass spectra
indicated that the relative height of the peaks reflected that of the natur
al isotopic abundance of Cl-35 and Cl-37, providing evidence that the produ
cts derived from NADPH and NMNH were monochlorinated. Moreover, calculated
masses revealed the 1 : 1 addition of HOCl to the molecule. Various 1D and
2D NMR experiments provided data for the assignments of the chemical shifts
of protons and carbons and the coupling constants of the protons of the ch
lorinated nucleotides. Further NOESY experiments allowed the characterizati
on of the spatial structure of the chlorinated product and showed that tran
s HOCl addition occurred at the C5=C6 carbon double bond of the nicotinamid
e ring, leading to a chlorohydrin.