The effect of neighboring amino acid residues and solution environment on the oxidative stability of tyrosine in small peptides

Date of Completion

January 2003


Chemistry, Organic|Chemistry, Pharmaceutical|Health Sciences, Pharmacy




Tyrosine is susceptible to photo-oxidation and metal-catalyzed oxidation in aqueous solution. The effects of the physicochemical characteristics of the molecule and formulation variables on tyrosine oxidation were investigated in model dipetides, tripeptides and in glucagon using chromatographic, mass spectrometric and computation techniques in a kinetic study. ^ The ionization of the phenolic group of tyrosine and tyrosyl residue in model di- and tri-peptides (Gly-Tyr, N-Ac-Tyr, Glu-Tyr, Tyr-Arg, and Lys-Tyr-Lys) was influenced by the surrounding electrostatic environments. Positive neighboring residues lowered the tyrosyl pKa while the negative neighboring groups raised it. The tyrosyl peptides were oxidized by light under alkaline conditions by a zero order reaction. Hydroxyl by-products were observed in photo-oxidation when tyrosine was located immediately adjacent to positively charged groups. When the amino group tyrosine was linked to neutral and negatively charged residues, hydroxylation was retarded, and as a result, the rate of oxidation decreased. Tyrosine, Gly-Tyr, Glu-Tyr, Tyr-Arg, N-Ac-Tyr and Lys-Tyr-Lys could be oxidized by the hydrogen peroxide/metal ion system at neutral pH. Metal-catalyzed oxidation followed first order kinetics and yielded mainly monohydroxyl by-products. Adjacent negatively charged amino acids accelerated the reaction due to the affinity of the negative charges to metal-ions whereas positively charged amino acid residues disfavored the reaction. ^ The two tyrosyl residues (Tyr-10 and Tyr-13) in glucagon were oxidized at different rates. Tyr-10 that is adjacent to the negatively charged Asp was more susceptible to metal-catalyzed oxidation than Tyr-13 located next to the positively charged Lys. Conversely, the Asp residue slowed down the photo-oxidation of Tyr-10. ^ The strength of light exposure, oxygen headspace and the presence of the cationic surfactant, cetyltrimethylammonia chloride, had significant impact on the kinetics of tyrosyl photo-oxidation. In metal catalyzed oxidation, the bivalent Cu2+ had a stronger pro-oxidation effect than the trivalent Fe3+. An increase in metal-ion concentration or in ionic strength of the solutions further accelerated the reaction. EDTA and butylated hydroxyanisole were more effective anti-oxidants than the reducing agents, ascorbic acid and sodium metabisulfite, in retarding metal-catalyzed oxidation. ^