N-acetyl-L-cysteine (NAC) improves antioxidant potentials of RBCs to provide protection against
oxidative stress induced hemolysis. The antioxidant mechanism of NAC to reduce oxidative stress in RBC,
studied through inactivation of pro-oxidant MetHb. NAC causes irreversible inactivation of the MetHb in an
H2O2 dependent manner, and the inactivation follows the pseudo- first- order kinetics. The kinetic constants
are ki = 8.5μM, kinact = 0.706 min-1 and t1/2 = 0.9 min. Spectroscopic studies indicate that MetHb accepts NAC
as a substrate and oxidizes through a single electron transfer mechanism to the NACox. The single e- oxidation
product of NAC has been identified as the 5, 5’- dimethyl-1- pyrroline N- oxide (DMPO) adduct of the sulfur
centered radical (aN = 15.2 G and aH=16. 78 G). Binding studies indicate that NACox interacts at the heme
moiety and NAC oxidation through MetHb is essential for NAC binding. Heme-NAC adduct dissociated from
MetHb and identified (m/z 1011.19) as 2:1 ratio of NAC/heme in the adduct. TEMPO and PBN treatment
reduces NAC binding to MetHb and protects against inactivation confirms the role of thiyl radical in the
inactivation process. Furthermore, scavenging thiyl radicals by TEMPO abolish the protective effect of NAC in
hemolysis. Current work highlights antioxidant mechanism of NAC through NAC thiyl radical generation, and
MetHb inactivation to exhibit protection in RBC against oxidative stress induced hemolysis.
Keywords: Antioxidant, free radicals, hemolysis, malaria, NAC, RBC.
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