3-Nitrotyrosine in the proteins of human plasma determined by an ELISA method

Abstract

The modification of tyrosine residues in proteins to 3-nitrotyrosine by peroxynitrite or other potential nitrating agents has been detected in biological systems that are subject to oxidative stress. A convenient semi-quantitative method has been developed to assay nitrated proteins in biological fluids and homogenates using a competitive ELISA developed in our laboratory. This assay selectivity detected 3-nitro-l-tyrosine residues in a variety of peroxynitrite-treated proteins (BSA, human serum albumin (HSA), alpha1-antiprotease inhibitor, pepsinogen and fibrinogen) and also in a nitrated peptide, but had a low affinity for free 3-nitro-L-tyrosine and 3-chloro-L-tyrosine. The IC50 values for the inhibition of antibody binding by different nitrated proteins were in the range 5-100 nM, suggesting that the antibody discriminated between nitrotyrosine residues in different environments. The presence of nitrotyrosine in plasma proteins was detected by Western blot analysis and quantified by the ELISA. A concentration of 0. 12+/-0.01 microM nitro-BSA equivalents was measured in the proteins of normal plasma which was increased in peroxynitrite-treated plasma and was elevated in inflammatory conditions. HSA and low-density lipoprotein (LDL) isolated from plasma contained 0.085+/-0.04 and 0. 03+/-0.006 nmol nitro-BSA equivalents/mg protein, respectively. Comparison of the level of nitration in peroxynitrite-treated HSA and LDL in the presence and absence of plasma indicates that nitration and presumably oxidation is inhibited by plasma antioxidants. The presence of nitrotyrosine in LDL is consistent with previous reports implicating peroxynitrite in the oxidative modification of lipoproteins and the presence of a low concentration of oxidized LDL in the blood.

Nitrated proteins in bronchoalveolar lavage fluid of patients at risk of ventilator-associated bronchopneumonia

“…The nitration of tyrosine residues in proteins and peptides in vivo is presented as a footprint of reactive nitrogen species production [10] and more specifically as the marker of ONOO -production [17]. The antibodies used in the present study for NTP measurement are presented as specific to nitrotyrosine and NTPs [17]. This was confirmed by addition of increased exogenous concentrations of nitrotyrosine that were quantitatively recognized by the antibodies.…”

“…High levels of nitrotyrosine or NTPs have already been found in tissues and biological fluids from patients with acute lung injury and other diseases involving oxidative damage [10±12, 19]. The nitration of tyrosine residues in proteins and peptides in vivo is presented as a footprint of reactive nitrogen species production [10] and more specifically as the marker of ONOO -production [17]. The antibodies used in the present study for NTP measurement are presented as specific to nitrotyrosine and NTPs [17].…”

“…mL -1 was taken as the lower limit of detection. The ELISA is specific for free nitrotyrosine and nitrotyrosine in proteins in a sequence-independent manner [17].…”

Evaluation of the antioxidant response in the plasma of healthy or hypertensive subjects after short-term exercise

“…Many molecular species are attacked by peroxynitrite and, among them, tyrosine and cysteine residues of proteins are the major targets. 27,45 The concentration of N-Tyr represents a footprint of the peroxynitrite formation, and its measure is widely used to evaluate the level of nitrosative stress. 13 Peroxynitrite is produced in the plasma of hypertensives and normotensives during the short-term physical effort, as increased N-Tyr levels in circulating proteins were found.…”

“…27 Major modifications were using TBS (130 mmol l À1 NaCl, 20 mmol l À1 Tris-HCl, pH 7.3) instead of PBS as buffer, sheep anti-N-Tyr IgG and DAS-HRP as primary and secondary antibodies, respectively. The extent of tyrosine nitration, in standard solutions of nitrated BSA, was determined by measuring the absorbance at 430 nm, using the molar extinction coefficient of 4500 mol l À1 cm À1 at pH 9.6 28 : 9.3 residues of N-Tyr per BSA molecule were detected.…”