Abstract
Non-enzymatic nitrite induced collagen cross-linking results in changes reminiscent of age-related damage and parallels the well-known model system, non-enzymatic glycation. We have recently observed that nitrite modification of basement membrane proteins can induce deleterious effects on overlying retinal pigment epithelial cells in studies relevant to age-related macular degeneration. The present work was undertaken in order to confirm 3-nitro-tyrosine (3-NT) as a product of the reaction and to identify the site specificity of nitration in collagen IV, a major component of basement membranes. Human collagen type IV was modified via incubation with 200 mM NaNO(2) (pH=7.38) for one week at 37(o)C. The modified protein was prepared in 2 different ways, including acid hydrolysis and trypsin digestion for site specificity determination. The samples were analyzed by LC/MS using a C(12) RP column. Site specificity was determined from tandem MS/MS data utilizing TurboSEQUEST software and the Swiss-Prot sequence database. 3-NT was detected in protein digests and acid hydrolysates of nitrite modified collagen IV. Positive identification with standard 3-NT was confirmed by identical R(t), lambda(max)=279 nm and 355 nm, and m/z=227. Analyses of tryptic digests identified four sites of tyrosine nitration, alpha1(IV)Y348, alpha1(IV)Y534, alpha2(IV)Y327, and alpha2(IV)Y1081. These sites are located in the triple-helical region of the protein and provide clues regarding potential sites for nitrite modification in collagen type IV.
Highlights
IntroductionReactions involving nitrite ion are becoming increasingly recognized as a means through which oxides of nitrogen (including nitric oxide and nitrogen dioxide) exert negative effects on human tissues
Reactions involving nitrite ion are becoming increasingly recognized as a means through which oxides of nitrogen exert negative effects on human tissues
The results indicate that tyrosine nitration by reaction with nitrite in collagen IV can form 3-nitrotyrosine (3-NT)
Summary
Reactions involving nitrite ion are becoming increasingly recognized as a means through which oxides of nitrogen (including nitric oxide and nitrogen dioxide) exert negative effects on human tissues. Nitrite can act as a reactive substrate for an array of chemical reactions and include interactions with H+ (as nitrous acid), H2O2 (through heme peroxidase or free metals), and UV light. Nitrite can mediate nitrosation reactions through the formation of nitrosonium ion (NO+) and/or dinitrogen trioxide (N2O3) in acidic electrophilic additions. Oxo-metal complex formation by heme peroxidase/H2O2 or free Fe/H2O2 in Fenton-type reactions, can cause nitration through single electron transfer to nitrite forming nitrogen dioxide (NO2 ) (Thomas et al, 2002; Radi, 2004). Based on UV-Vis absorption, the authors reported that nitrite can modify tyrosine residues in type I collagen and form 3-NT (Paik et al, 2001)
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