Oxidation of human insulin-like growth factor I in formulation studies, II. Effects of oxygen, visible light, and phosphate on methionine oxidation in aqueous solution and evaluation of possible mechanisms.

PURPOSE

The oxidation of methionine in human Insulin-like Growth Factor I (hIGF-I) in aqueous solution was studied with respect to oxygen, visible light and sodium phosphate.

METHODS

Aqueous solutions of hIGF-I were prepared with different amounts of phosphate and dissolved oxygen. The solutions were stored either in darkness or exposed to artificial visible light. The oxidized hIGF-I was quantified by RP-HPLC. A two level full factorial experimental design, with two levels of each of the three factors studied, was used.

RESULTS

Oxidation was found to be positively correlated with light oxygen content and, interestingly, phosphate. The increasing effect of phosphate on the oxidation appears not to originate from metal contaminants. The influence of both oxygen and phosphate increased with time. The pH dependence of oxidation indicated the formation of a phosphorylated sulfonium ion as an oxidation intermediate. A significant interaction effect between phosphate and visible light suggested participation of radicals.

CONCLUSIONS

Factorial experiments provide a valuable tool when studying complex mechanisms with interacting factors. The oxidation of methionine in hIGF-I is significantly affected by light but also by the presence of phosphate buffer.