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Cited by 259Nitric Oxide and Endothelin EffectsHemoglobin structure and functionRedox biology and oxidative stress

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S-Nitrosation of Serum Albumin by Dinitrosyl-Iron Complex

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Boese, Matthias, Mordvintcev, Peter I., Vanin, Anatoly F., Busse, Rudi, Mülsch, Alexander·1995·Journal of Biological Chemistry
DOI 10.1074/jbc.270.49.29244
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Abstract

The objective of this study was to identify a potential mechanism for S-nitrosation of proteins. Therefore, we assessed S-nitrosation of bovine serum albumin by dinitrosyl-iron-di-L-cysteine complex [(NO)2Fe(L-cysteine)2], a compound similar to naturally occurring iron-nitrosyls. Within 5-10 min, (NO)2Fe(L-cysteine)2 generated paramagnetic albumin-bound dinitrosyl-iron complex and S-nitrosoalbumin in a ratio of 4:1. Although S-nitroso-L-cysteine was concomitantly formed in low amounts, its concentration was not sufficient to account for formation of S-nitrosoalbumin via a trans-S-nitrosation reaction. Low oxygen tension did not affect S-nitrosation by the dinitrosyl-iron complex thus excluding the involvement of oxygenated NOx-species in the nitrosation reaction. Blockade of albumin histidine residues by pyrocarbonate, which prevented formation of dinitrosyl-iron-albumin complex, did not inhibit S-nitrosation of albumin. Thus, S-nitrosation of albumin by (NO)2Fe(L-cysteine)2 can proceed by direct attack of a nitrosyl moiety on the protein thiolate, without previous binding of the iron. We conclude that protein-bound dinitrosyl-iron complexes detected in high concentrations in certain tissues provide a reservoir of S-nitrosating species, e.g. low molecular dinitrosyl iron complexes.

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Figures

Image 1
CC-BY · From the PDF
FIG. 1 Representative EPR spectra of dinitrosyl-iron complexes. ESR spectra were recorded at 77 K, modulation amplitude 5 Gauss ( a-d ), and at 295 K, 0.5 Gauss ( e ) as described under 'Experimental Procedures.' The position of the g factors is indicated by arrows . Note that spectra a-d were recorded at 4-fold higher magnetic field sweep than spectrum e , as indicated by the calibration bars (4 and 1 millitesla). a , DNIC-L-cysteine (25 m M); b , desalted DNIC-BSA (30 m M); c , BSA-DNIC (30 m M) from N -ethylmaleimide-treated BSA; d , DNIC (40 m M) in Griess reagent; e , DNIC-L-cysteine (200 m M) recorded at 295 K. For assignment of structures see 'Results.'
FIG. 1 Representative EPR spectra of dinitrosyl-iron complexes. ESR spectra were recorded at 77 K, modulation amplitude 5 Gauss ( a-d ), and at 295 K, 0.5 Gauss ( e ) as described under 'Experimental Procedures.' The position of the g factors is indicated by arrows . Note that spectra a-d were recorded at 4-fold higher magnetic field sweep than spectrum e , as indicated by the calibration bars (4 and 1 millitesla). a , DNIC-L-cysteine (25 m M); b , desalted DNIC-BSA (30 m M); c , BSA-DNIC (30 m M) from N -ethylmaleimide-treated BSA; d , DNIC (40 m M) in Griess reagent; e , DNIC-L-cysteine (200 m M) recorded at 295 K. For assignment of structures see 'Results.'CC-BY · From the PDF
Image 3
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Image 4
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FIG. 3 Time course of S -nitrosation of BSA by DNIC. BSA (1 mM; 0.4 mM free thiol) was incubated in 100 mM potassium phosphate (pH 7.4) with 0.75 mM DNIC. At the indicated times a sample was withdrawn to determine the concentration of S -nitrosothiol by the Saville/Griess reaction. Means 1 S.E. of five experiments.
FIG. 3 Time course of S -nitrosation of BSA by DNIC. BSA (1 mM; 0.4 mM free thiol) was incubated in 100 mM potassium phosphate (pH 7.4) with 0.75 mM DNIC. At the indicated times a sample was withdrawn to determine the concentration of S -nitrosothiol by the Saville/Griess reaction. Means 1 S.E. of five experiments.CC-BY · From the PDF
FIG. 4 Excess free L-cysteine is required for S -nitrosation of BSA by DNIC. BSA (1 mM; 0.4 mM free thiol) was incubated for 5 min at 4 °C with DNIC (0.75 mM) and desalted at 4 °C by a Sephadex G-25 column. The time course of S -nitrosothiol formation by DNIC-BSA was then assessed at 37 °C in the absence ( E ) and presence ( ● ) of 20 mM L-cysteine. Means 1 S.E. of three experiments.
FIG. 4 Excess free L-cysteine is required for S -nitrosation of BSA by DNIC. BSA (1 mM; 0.4 mM free thiol) was incubated for 5 min at 4 °C with DNIC (0.75 mM) and desalted at 4 °C by a Sephadex G-25 column. The time course of S -nitrosothiol formation by DNIC-BSA was then assessed at 37 °C in the absence ( E ) and presence ( ● ) of 20 mM L-cysteine. Means 1 S.E. of three experiments.CC-BY · From the PDF
FIG. 5 Separation of S -nitroso-BSA and low mass S -nitrosothiols derived from DNIC by gel chromatography. BSA (0.4 mM free thiol) was incubated with DNIC (0.4 mM) for 5 min at 37 °C. Subsequently S -nitroso-BSA was separated from S -nitroso-L-cysteine by chromatography on Sephadex G-25. Individual fractions (#) of the column eluate were tested for S -nitrosothiol ( E ) and protein ( ● ). Shown is a representative experiment out of three.
FIG. 5 Separation of S -nitroso-BSA and low mass S -nitrosothiols derived from DNIC by gel chromatography. BSA (0.4 mM free thiol) was incubated with DNIC (0.4 mM) for 5 min at 37 °C. Subsequently S -nitroso-BSA was separated from S -nitroso-L-cysteine by chromatography on Sephadex G-25. Individual fractions (#) of the column eluate were tested for S -nitrosothiol ( E ) and protein ( ● ). Shown is a representative experiment out of three.CC-BY · From the PDF
FIG. 6 TransS -nitrosation of BSA by S -nitroso-L-cysteine. BSA (0.5 mM free thiol) was incubated for 5 min with an equal amount of S -nitroso-L-cysteine. The protein was separated from the low molecular compounds by gel chromatography on Sephadex G-25 and S -nitrosothiol ( E ) and protein ( ● ) was measured in individual fractions. This is a representative experiment out of three.
FIG. 6 TransS -nitrosation of BSA by S -nitroso-L-cysteine. BSA (0.5 mM free thiol) was incubated for 5 min with an equal amount of S -nitroso-L-cysteine. The protein was separated from the low molecular compounds by gel chromatography on Sephadex G-25 and S -nitrosothiol ( E ) and protein ( ● ) was measured in individual fractions. This is a representative experiment out of three.CC-BY · From the PDF
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Citations

Cites

Show all 40 references

Research works printed but not linked: 10

  1. Becker, 1995, Eur. J. Biochem.
  2. Mülsch, 1994, Drug Res., 44, 3a
  3. Vanin, 1975, Studia Biophys., 49, 13
  4. Burbaev, 1971, Zhurnal Strukt. Chimii (USSR), 12, 252
  5. Vanin, 1975, Biofizika (USSR), 20, 1068
  6. Bottomley, 1978, Accts. Chem. Res., 11, 158, 10.1021/ar50124a006 · DOI
  7. Zhang, 1983, Cancer Res., 43, 339
  8. Lu, 1982, Z. Gastoenterol., 20, 361
  9. Croisy, 1984, IARC Sci. Publ., 57, 327
  10. Croisy, 1980, IARC Sci. Publ., 31, 83

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Metadata

Authors
Boese, M., Mordvintcev, P. I., Vanin, A. F., Busse, R., & Mülsch, A.
Affiliations
Goethe University Frankfurt (DE)
Corresponding author
Mülsch, Alexander
ORCID
Vanin, Anatoly F.
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
1995-12
Volume
270
Issue
49
Pages
29244-29249
ISSN
0021-9258 (ISSN-L)
DOI
10.1074/jbc.270.49.29244
OpenAlex
W1977448081
Cited by
259
References
40
Citations by year
2026: 1 · 2025: 3 · 2024: 5 · 2023: 5 · 2022: 7 · 2021: 13 · 2020: 5 · 2019: 9 · 2018: 6 · 2017: 5 · 2016: 7 · 2015: 4 · 2014: 9 · 2013: 10 · 2012: 8
FWCI
4.99
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Topics
  • Health Sciences › Medicine › Physiology (medical) › Nitric Oxide and Endothelin Effects
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Hemoglobin structure and function
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
Keywords
S-nitrosylation, bovine serum albumin, dinitrosyl iron complexes, S-nitroso-L-cysteine, histidine residues, oxygen partial pressure, nitric oxide
MeSH terms
Animals, Cattle, Cysteine, Oxygen, Serum Albumin, Bovine

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