Evaluating GSNO Catabolism in GSNOR KO Lungs

GSNOR KO lung tissue was evaluated to confirm diminished GSNO catabolism compared to WT. Lungs from six week old mice were homogenized in ice-cold RIPA lysis buffer containing protease inhibitors (sc-24948, Santa Cruz Biotechnology, Dallas, TX) and protein levels were quantified by Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL). Enzyme activity in lung homogenates was assessed by timed GSNO catabolism and evaluation by GSNO-dependent NADH consumption quantified by absorbance.^{27 (link)}GSNO-dependent NADH consumption: A known quantity of GSNO (100 μM) was loaded with co-reagents (200 μM NADH and 2 mM GSH) and equivalent protein quantities of frozen lung homogenates (100 μg) in PBS. At time 0, 10, 20, 30, and 40 minutes after incubation at 37°C, NADH absorbance (absorption at 340 nm) was measured (FLUOstar Omega spectrometer, BMG Labtech, Ortenberg, Germany) and NADH decrease was calculated and reported as % from time 0.^{27 (link)}