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. Author manuscript; available in PMC: 2017 Dec 1.
Published in final edited form as: Free Radic Biol Med. 2016 Sep 25;101:20–31. doi: 10.1016/j.freeradbiomed.2016.09.020

Figure 2.

Figure 2

Spectroscopic analysis of RSSH oxidation. (a) 5 mM MCPD and 10 mM TEMPOL in 0.1 M phosphate buffer containing 50 μM DTPA at pH 7.4 over a 30 minute period. The loss in absorbance at 426 nm corresponds to the reduction of TEMPOL to TEMPOL-OH. Inset: Reaction of 5 mM NAP and 10 mM TEMPOL. (b) Reduction of 10 mM K3Fe(CN)6 by 5 mM MCPD in 0.1 M phosphate buffer at pH 7.4 over a 30 minute period. The loss in absorbance at 420 nm corresponds to the reduction of the Fe(III) center to Fe(II). (c) Reduction of 10 mM TEMPOL in the presence of 5 mM NaSH and 5 mM GSSG in 0.1 M phosphate buffer containing 50 μM DTPA at pH 7.4 over a 30 minute period. The loss in absorbance at 426 nm corresponds to the reduction of TEMPOL to TEMPOL-OH (presumably via oxidation of GSSH). (d) UV-vis spectra for various redox states of myoglobin. Dashed line: The UV-vis spectrum of 5 μM MbFeIII alone (λ = 408, 502 and 530 nm) in 100 mM phosphate buffer (pH 7.4). Solid line: The UV-Vis spectrum that results after 5 μM MbFeIII is reduced by 50 μM MCPD for 30 minutes in 100 mM phosphate buffer (pH 7.4). The increase in absorbance at λ = 542 and 580 nm corresponds to the reduction of the MbFeIII center to MbFeII and subsequent binding of O2 to give MbFeIIO2. Inset: UV-vis spectra of both 5μM MbFeIII and 5 μM MbFeIII treated with 50 μM NAP for 30 minutes in 100 mM phosphate buffer (pH 7.4). No spectral changes are observed after the addition of NAP to the solution of MbFeIII in this case. (e) EPR spectra for the reduction of TEMPO by MCP-SSH. Dashed line: EPR spectrum of 5 mM TEMPO in methanol. Solid line: Resulting spectrum after a 30 minute incubation of 5 mM TEMPO with 5 mM MCPD in the presence of 10 mM tetrabutylammonium hydroxide in methanol. The loss in signal intensity corresponds to the reduction of TEMPO to TEMPO-OH by MCP-SSH.