A practicable method to prepare nitrated proteins with peroxynitrite and low concentration of sodium hydroxide.

Peroxynitrite is an ion acting as a powerful oxidant and nucleophile, which plays a key role in the inflammation and aging process by nitrating tyrosine or tryptophan residues of the proteins. Nitration of a target protein is considered to be a proper method to study the behavioral change of the proteins being nitrated. The commonly used methods for peroxynitrite preparation in vitro usually contain high concentration of sodium hydroxide, which easily induces hydrolysis of target proteins. Accordingly, the method for peroxynitrite preparation was optimized in vitro by changing the sequence of hydrochloric acid and sodium hydroxide added. After different amount of hydrochloric acid added to the system following sodium nitrite, peroxynitrite can be yielded in a concentration up to 60 mM with sodium hydroxide as low as 17 mM. More importantly, biological activity of the target protein was well maintained after protein nitration since low sodium hydroxide was used.