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. Author manuscript; available in PMC: 2017 Oct 1.
Published in final edited form as: Free Radic Biol Med. 2016 Sep 5;99:418–425. doi: 10.1016/j.freeradbiomed.2016.09.005

Fig. 2. Nitrosyl yield from unliganded hemes.

Fig. 2

A Representative EPR spectra of HbNO yields from reactions of 100 μM deoxyHb (dHb) or 100 μM deoxygenated metHb and equimolar SSNO− mixture preincubated for 10 and 30 minutes. B Representative EPR spectra of MbNO yields from 100 μM of metMb and equimolar SSNO− and 100 μM of metMb with MAHMANOnoate yielding 100 μM NO. C Average HbNO yield from a 100 μM SSNO− mixture preincubated for 10 or 30 minutes, degassed, and reacted with 100 μM metHb in anaerobic buffer and 100 μM deoxyHb (n=3). Average nitrosyl yields from metHb and SSNO− at 10 and 30 minutes were 17.8 ± 1.9 μM and 14.2 ± 1.0 μM, p < 0.05. Average nitrosyl yields from deoxyHb and SSNO− at 10 and 30 minutes were 8.3 ± 4.5 μM and 5.9 ± 2.3 μM, p=0.27. Nitrosyl yields from deoxyHb + SSNO− at 10 and 30 minutes are significantly less than yields from metHb + SSNO− at the same preincubation times. *= p-value < 0.050 and **= p-value = 0.005. D Nitrosyl yield from 100 μM SSNO− mixture at 10 or 30 minutes, degassed, and reacted with 100 μM metHb (n=6) and 100 μM metMb (n=4) for 10 minutes (*= p-value < 0.005). MAHMANOnoate (yield 100 μM NO) reacted with 100 μM metHb and metMb for 10 minutes in deoxygenated buffer for 10 minutes showed significantly different nitrosyl yields. E Representative EPR spectra of HbNO yield from the reaction of 100 μM SSNO− pre-incubated for 10 minutes and 100 μM metHb in the absence (solid blue) and presence of (solid black) O2. Using the SSNO− incubated for 30 minutes, HbNO yield is shown in the absence (black dot) and presence (blue dash) of O2. F Representative EPR spectra of HbNO yield from 100 μM metHb and MAHMANOnoate (100 μM NO yield) reacted under aerobic and anaerobic conditions for 10 minutes. G Average HbNO yield from 10 minute SSNO− mixture (100 μM) and metHb (100 μM) in aerobic and anaerobic conditions(n=6, n=3, respectively). Average HbNO yield from 30 minute SSNO− mixture (100 μM) and metHb (100 μM) in aerobic and anaerobic conditions (n=6, n=3, respectively). Also shown is MAHMANOnoate (100 μM NO yield) and 100 μM metHb reacted for 10 minutes in aerobic and anaerobic conditions (n=3). At 10 and 30 minutes, nitrosyl yields were significantly different between atmospheric conditions. Likewise, nitrosyl yields were significantly different between atmospheric treatments of metHb and MAHMANOnoate (†= p-value = 0.05, *= p-value < 0.01, **= p-value = 0.001,). H Spontaneous release of NO from 1.0 mM SSNO− mixture stock. Injection was 5 μL into PBS pH 7.4.