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2.
Figure 6

Figure 6. Overview of human serum albumin (HSA).. From: Redox-Sensitivity and Site-Specificity of S- and N- Denitrosation in Proteins.

The protein secondary structure is shown schematically and the domains are colored-coded as follows: I, blue; II, green; III, yellow; IV, red. Cys-34 and Trp-214 are shown in a space-filling representation and colored by atom types (PDB ID: 1E78; [45]).

Frances L. Jourd'heuil, et al. PLoS One. 2010;5(12):e14400.
3.
Figure 3

Figure 3. Effect of superoxide dismutase (SOD) on the GSH-induced decomposition of NANT and NO production.. From: Redox-Sensitivity and Site-Specificity of S- and N- Denitrosation in Proteins.

(A), Time-course of NANT decomposition in the presence of GSH with or without SOD; representative of three experiments. (B), NO formation in solution upon incubation of NANT with GSH and SOD; representative of 4 experiments.

Frances L. Jourd'heuil, et al. PLoS One. 2010;5(12):e14400.
4.
Figure 1

Figure 1. Denitrosation of N-acetyl-nitroso-Trp (NANT) by glutathione (GSH).. From: Redox-Sensitivity and Site-Specificity of S- and N- Denitrosation in Proteins.

The decomposition of NANT (100 µM) was followed spectrophotometrically at 335 nm upon incubation with increasing concentrations of GSH in 100 mM phosphate buffer (pH 7.4) containing 100 µM DTPA. NANT decay followed apparent first order kinetics and kobs for NANT decomposition was plotted as a function of [GSH]. The values represent the mean ± SEM (n = 4).

Frances L. Jourd'heuil, et al. PLoS One. 2010;5(12):e14400.
5.
Figure 7

Figure 7. Site-directed nitrosation of human albumin.. From: Redox-Sensitivity and Site-Specificity of S- and N- Denitrosation in Proteins.

(A), The presence of mercury-sensitive and -resistant species in wild-type (WT), C34S and W214L recombinant HSA (3 mg/ml) treated with 20 µM DEA/NO for 30 min at 37°C was determined by reductive chemiluminescence. The effect of sodium azide (B) and GSH (C) on the amount of nitrosated Cys-34 and Trp-214 was determined after desalting of the samples. The values represent the mean ± SEM (n = 3).

Frances L. Jourd'heuil, et al. PLoS One. 2010;5(12):e14400.
6.
Figure 10

Figure 10. Transfer of NO between nitroso-N-acetyl-Trp and proteins.. From: Redox-Sensitivity and Site-Specificity of S- and N- Denitrosation in Proteins.

Cell lysates obtained from murine fibroblasts were incubated alone (Control), with 10 µM nitroso-N-acetyl-Trp (NOTrp), 10 µM sodium nitrite and N-acetyl-Trp (Trp + NO2), or 10 µM GSNO (GSNO) for 1 hr at 37°C. Nitrosated proteins were then determined using the biotin switch assay as described under Materials and Methods. Equal loading was verified using Coomassie blue staining. The immunoblot is representative of three experiments.

Frances L. Jourd'heuil, et al. PLoS One. 2010;5(12):e14400.
7.
Figure 2

Figure 2. The denitrosation of N-acetyl-nitroso-Trp (NANT) by glutathione (GSH) requires molecular oxygen.. From: Redox-Sensitivity and Site-Specificity of S- and N- Denitrosation in Proteins.

(A), Time-course of GSH-induced NANT decomposition in the presence or absence of molecular oxygen; representative of at least 4 experiments. (B), Representative chromatogram (detection at 335 nm) obtained from the reaction of 100 µM NANT with 1 mM GSH and O2. Chromatograms are representative of three independent experiments. (C), Time-course of O2 consumption in the presence of 100 µM NANT and 1 mM GSH; representative of 4 experiments. The solid lines represent the nonlinear regression fitting of data points (open symbols).

Frances L. Jourd'heuil, et al. PLoS One. 2010;5(12):e14400.
8.
Figure 4

Figure 4. Formation of nitrite (NO2−) and nitrate (NO3−, panel A), glutathione disulfide (GSSG, panel B), and S-nitrosoglutathione (GSNO, panel C) during NANT decomposition.. From: Redox-Sensitivity and Site-Specificity of S- and N- Denitrosation in Proteins.

N-acetyl nitroso Trp (100 µM) was incubated with GSH 1 mM for 30 min in the presence or absence of O2 and SOD at 37°C and product formation was determined by anion pairing HPLC. The values represent the mean ± SEM (n = 5–9); ND  =  not detectable.

Frances L. Jourd'heuil, et al. PLoS One. 2010;5(12):e14400.
9.
Figure 8

Figure 8. Stability of nitrosated human serum albumin in the presence of GSH and ascorbate.. From: Redox-Sensitivity and Site-Specificity of S- and N- Denitrosation in Proteins.

HSA (3 mg/ml) was incubated with 20 µM DEA/NO in 100 mM phosphate buffer (pH 7.4) containing 100 µM DTPA for 30 min at 37°C. Samples were then incubated for an additional 30 min either alone or in the presence of GSH (1 mM) and ascorbate (Asc; 300 µM) in 100 mM phosphate buffer (pH 7.4) containing 100 µM DTPA, after which the samples were desalted. Nitrosation at Cys-34 and Trp-214 was then determined using reductive chemiluminescence. The values represent the mean ± SEM (n = 3).

Frances L. Jourd'heuil, et al. PLoS One. 2010;5(12):e14400.
10.
Figure 5

Figure 5. Validation of nitrosamine determination by tri-iodide-based chemiluminescence assay.. From: Redox-Sensitivity and Site-Specificity of S- and N- Denitrosation in Proteins.

(A), Flow chart for the determination of nitroso species using the tri-iodide chemiluminescence assay. Details may be found under Materials and Methods. (B), N-acetyl-nitroso-Trp (NANT, 10 µM) was preincubated with HgCl2 or NEM in the presence of acidified sulfanilamide. The samples were then quantified by gas phase chemiluminescence as described under Materials and Methods. The values represent the mean ± SEM (n = 3). (C), Stock solutions of GSNO and NANT of known concentrations were diluted and mixed together in 100 mM phosphate buffer containing DTPA (100 µM) and nitrite (40 µM) to obtain a final concentration of 5 µM for each compound. Concentrations were immediately determined using the same tri-iodide based chemiluminescence assay (n = 3, mean ± SEM).

Frances L. Jourd'heuil, et al. PLoS One. 2010;5(12):e14400.
11.
Figure 9

Figure 9. Stability of nitrosated fragment of helix IIa-h2 of HSA containing Trp-214.. From: Redox-Sensitivity and Site-Specificity of S- and N- Denitrosation in Proteins.

A, A peptide (#2) corresponding to the fragment of helix IIa-h2 of HSA was nitrosated using acidified nitrite. The peptide was then diluted to obtain a final concentration of nitroso Trp of 10 µM (based on an ε of 6100 M−1.cm−1 at 335 nm) and the stability of the nitrosated peptide was determined by reductive chemiluminescence. There was no evidence for an NO-dependent signal in the control peptide (#1) that did not contain the Trp residue but that was exposed to acidified nitrite. Peptide #2 (10 µM) was also incubated in the presence of light for 30 min or with HgCl2 (final concentration 4.9 mM) for 15 min. The values represent the mean ± SEM (n = 3). B, The nitrosated peptide (10 µM) was incubated for 30 min either alone or in the presence of GSH (1 mM) and ascorbate (Asc; 300 µM) in 100 mM phosphate buffer (pH 7.4) containing 100 µM DTPA. Nitrosation of Trp was then determined using reductive chemiluminescence upon pretreatment of paired samples with or without HgCl2 to elimate the contribution of RSNOs and as described under Materials and Methods. The values represent the mean ± SEM (n = 3); *p<0.05 vs. peptide alone.

Frances L. Jourd'heuil, et al. PLoS One. 2010;5(12):e14400.

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