3-NITROTYROSINE IN THE PROTEINS OF HUMAN PLASMA DETERMINED BY AN ELISA METHOD

The modification of tyrosine residues in proteins to 3-nitrotyrosine b y peroxynitrite or other potential nitrating agents has been detected in biological systems that are subject to oxidative stress. A convenie nt semi-quantitative method has been developed to assay nitrated prote ins in biological fluids and homogenates using a competitive ELISA dev eloped in our laboratory. This assay selectivity detected 3-nitro-L-ty rosine residues in a variety of peroxynitrite-treated proteins (BSA, h uman serum albumin (HSA), alpha(1)-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 presenc e of nitrotyrosine in plasma proteins was detected by Western blot ana lysis and quantified by the ELISA. A concentration of 0.12+/-0.01 mu M nitro-BSA equivalents was measured in the proteins of normal plasma w hich was increased in peroxynitrite-treated plasma and was elevated in inflammatory conditions. HSA and low-density lipoprotein (LDL) isolat ed from plasma contained 0.085+/-0.04 and 0.03+/-0.006 nmol nitro-BSA equivalents/mg protein, respectively. Comparison of the level of nitra tion in peroxynitrite-treated HSA and LDL in the presence and absence of plasma indicates that nitration and presumably oxidation is inhibit ed by plasma antioxidants. The presence of nitrotyrosine in LDL is con sistent with previous reports implicating peroxynitrite in the oxidati ve modification of lipoproteins and the presence of a low concentratio n of oxidized LDL in the blood.