Determinationofnitration degrees for thebirchpollen allergen Bet v 1

Nitration of tyrosine residues in the major birch pollen allergen Bet v 1 may alter the allergenic potential of the protein. The kinetics and mechanism of the nitration reaction, however, have not yet been well characterized. To facilitatefurtherinvestigations,anefficientmethodtoquantify the nitration degree (ND) of small samples of Bet v 1 is required. Here, we present a suitable method of high- performance liquid chromatography coupled to a diode array detector (HPLC-DAD) that can be photometrically calibrated using the amino acids tyrosine (Tyr) and nitrotyrosine (NTyr) without the need for nitrated protein standards. The new method is efficient and in agreement with alternative methods based on hydrolysis and amino acid analysis of tetranitromethane (TNM)-nitrated Bet v 1 standards as well as samples from nitration experiments with peroxynitrite. The results confirm the applicability of the new method for the investigation of the reaction kinetics and mechanism of pro- tein nitration. Keywords Proteintyrosinenitration .Betv1.0101 . HPLC-DAD .Nitrationdegree