Summary.
Oxyhaemoglobin (oxyHb) and methaemoglobin (metHb) react with S-nitrosocysteine (CysNO) to form nitroso derivatives. We test this reaction with a new method for evaluating transnitrosation reaction. The assay exploits an amperometric sensor developed in our laboratory. The results we obtain are in good agreement with those reported by others, although at much higher sensitivity, indicating the suitability of the method for examining high-mass nitroso compounds.
The S-nitrosylation of oxyHb at a CysNO/haem ratio of 1 : 1 is about 5% in 60 min. In the same experimental conditions, the nitrosylation of met-Hb reaches 25%. OxyHb and metHb derivatize by 50% in 60 min upon using a CysNO/haem ratio of 10 : 1.
The oxidation of haem iron occurs at ratios of haem/CysNO of 1 : 5 or higher.
We conclude that CysNO transfers NO+ both to metHb and oxyHb.
We propose that NO transfer in RBC may occur through transnitrosation reactions between high and low-mass nitrosothiols.
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References
Arnelle DR, Stamler JS (1995) NO+, NO and NO- donation by Snitrosothiols: implications for regulation of physiological functions by S-nitrosylation and acceleration of disulfide formation. Arch Biochem Biophys 318: 279–285
Askew SC, Barnett DJ, McAninly J, Williams DLH (1995) Catalysis by Cu2+ of nitri from S-nitrosothiols (RSNO). J Chem Soc Perkin Trans 2: 741–745
Gaston B, Sears S, Woods J, Hunt J, Ponaman M, McMahon T, Stamler JS (1998) Bronchodilator S-nitrosothiol deficienct in asthmatic respiratory failure. Lancet 351: 1317–1319
Gow AJ, Stamler JS (1998) Reaction between nitric oxide and haemoglobin under physiological conditions. Nature 391: 169–173
Gow AJ, Luchsinger BP, Pawloski JR, Singel DJ, Stamler JS (1999) The oxyhemoglobin reaction of nitric oxide. Proc Natl Acad Sci USA 96: 9027–9032
Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR (1982) Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal Biochem 126: 131–138
Ignarro RJ, Lippton H, Edwards JC, Baricos WH, Hyman AL, Kadowitz PJ, Gruetter CA (1981) Mechanism of vascular smooth muscle relaxation by organic nitrates, mitrites, nitroprusside and nitric oxide: evidence for the involvement of S-nitrosothiols as active intermediates. J Pharmacol Exp Ther 218: 739–749
Jia L, Bonaventura C, Bonaventura J, Stamler JS (1996) Snitrosohaemoglobin: a dynamic activity of blood involved in vascular control. Nature 380: 221–226
Kharitonov VG, Sundquist R, Sharma SV (1995) Kinetics of nitrosilation of thiols by nitric oxide in the presence of oxygen. J Biol Chem 270: 28158–28164
Kilbourn RJ, Joly G, Cashon B, De Angelo J, Bonaventura J (1994) Cell-free hemoglobin reverses the entotoxin-mediated hyperesponsivity of rat aortic rings to a-adrenergic agents. Biochem Biophys Res Commun 199: 155–162
McMahon TJ, Stone AE, Bonaventura J, Singel D, Stamler JS (2000) Functional coupling of oxygen-binding and vasoactivity in S-nitrosohaemoglobin. J Biol Chem 275: 16738–16745
Olsson I (1981) Methods Enzymol 76: 631–651
Palmerini CA, Arienti G, Mazzolla R, Palombari R (1998) A new assay for the determination of low-molecular weight nitrosothiols (nitrosoglutatione and nitrosocysteine), NO and nitrites by using a specific and sensitive solid-state amperometric sensor. Nitric Oxide 2: 375–380
Palmerini CA, Arienti G, Palombari R (2000) Determination of SNitrosohemoglobin using a solid-state amperometric sensor. Nitric Oxide 4: 546–549
Palmerini CA, Saccardi C, Arienti G and Palombari R (2002) Formation of nitrosothiols from gaseous nitric oxide at pH 7.4. J Biochem Mol Toxicol 16: 135–139
Pawloski JR, Hess DT, Stamler JS (2001) Export by red blood cells of nitric oxide bioactivity. Nature 409: 622–626
Perutz MF (1996) Blood. Taking the pressure off [news]. Nature 380: 205–206
Saville B (1958) A scheme for the colorimetric determination of micrograms amounts of thiols. Analyst 83: 670–672
Stamler JS, Jaraki O, Osborne J, Simon DI, Keaney JV, Vita J, Singel D, Valeri CR, Loscalzo J (1992) Nitric oxide circulates in mammalian plasma primarily as an S-nitroso adduct of serum albumin. Proc Natl Acad Sci USA 89: 7674–7677
Stamler JS, Jia L, Eu JP, McMahon TJ, Demchenko IT, Bonaventura J, Gernert K, Piantadosi CA (1997) Blood flow regulation by S-nitrosohaemoglobin in the physiological oxygen gradient. Science 276: 2034–2037
Williams DLH (1996) The mechanism of nitric oxide formation from S-nitrosothiols (thionitrites). Chem Commun: 1085– 1091
Williams DLH (1999) The chemistry of S-nitrosothiols. Acc Chem Res 32: 869–876
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Authors' address: Prof. Carlo A. Palmerini, Dipartimento di Scienze Biochimiche e Biotecnologie Molecolari, University of Perugia, Via del Giochetto, I-06127, Perugia, Italy, Fax: +39.075.585.7414; E-mail:arienti@unipg.it; crlpal@unipg.it
Abbreviations: Hb: haemoglobin; SNO-Hb: S-nitrosohaemoglobin; CysNO: S-nitrosocysteine; metHb: methaemoglobin; oxyHb: oxyhaemoglobin; SNO-metHb: S-nitrosomethaemoglobin; SNO-oxyHb: S-nitrosooxyhaemoglobin
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Palmerini, C., Palombari, R. & Arienti, G. Electrochemical assay of human haemoglobin S-nitrosylation by nitrosocysteine. Amino Acids 25, 59–62 (2003). https://doi.org/10.1007/s00726-002-0355-z
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DOI: https://doi.org/10.1007/s00726-002-0355-z