Nitrocytochrome c: synthesis, purification, and functional studies.
Posttranslational protein tyrosine oxidation, to yield 3-nitrotyrosine, is a biologically relevant protein modification related with acute and chronic inflammation and degenerative processes. It is usually associated with a decrease or loss in protein function. However, in some proteins, tyrosine nitration results in an increase or gain in protein function. Nitration of cytochrome c by biological oxidants in vitro can be achieved via different mechanisms, which include reactions with peroxynitrite, nitrite plus hydrogen peroxide, and nitric oxide plus hydrogen peroxide, and result in a loss in its electron transport capacity and in a higher peroxidatic activity. This chapter describes the methodology for studying chemical and biological properties of nitrocytochrome c. In particular, we report methods to synthesize tyrosine-nitrated cytochrome c, purify cytochrome c mononitrated species, map the sites of tyrosine nitration, and investigate the functional consequences of nitrated cytochrome c on mitochondrial electron transport properties, peroxidatic activity, and apoptosome assembly.
Souza
José M
JM
Department of Biochemistry, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay.
Castro
Laura
L
Cassina
Adriana María
AM
Batthyány
Carlos
C
Radi
Rafael
R
D000818:Animals; D045304:Cytochromes c / Q000138:chemical synthesis / Q000302:isolation & purification / Q000502:physiology; D006801:Humans; D009566:Nitrates / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology
D009566:Nitrates; D045304:Cytochromes c