To investigate the role and importance of the four methionines in recombinant human leptin, and the effect of methionine oxidation in leptin structural stability and biological activity.Methods
Oxidized leptin derivatives were prepared in the presence of H2O2 and t-butylhydroperoxide, separated by RP-HPLC, and characterized by peptide mapping and LC/MS. Their biophysical and biological properties were studied.Results
Six major species of oxidized leptins were detected: two mono-oxidized, one di-oxidized, two tri-oxidized, and one tetra-oxidized. Further oxidation at cystine disulfide was also detected. Kinetic analysis indicated that oxidation at Met1 and Met69 proceeded first and independently. In 48 mM t-butylhydroperoxide, the pseudo first-order rate constants, k1 and k69, were 1.5 × 10−3 and 2.3 × 10−4 min−1. No change in the secondary or tertiary structure was detected for Met1 mono-oxidized and Met1, Met69 di-oxidized leptins. The Met1 mono-oxidized leptin retained full potency as compared to native leptin. A slight decrease of thermostability and a significant loss of the in vitro bioactivity were observed for Met1, Met69 di-oxidized leptin. Both Met55 and Met137 were not oxidized in t-butylhydroperoxide but only in H2O2. They appeared to be much less accessible to oxidation and might interact with the hydrophobic core structure of the leptin molecule.Conclusions
The oxidation of leptin occurred in the order of Met1 > Met69 >> Met55 ≈ Met137, and the importance for maintaining leptin structural integrity was Met55 ≈ Met137 >> Met69≈ Met1. Met69, but not Met1, plays a critical role in the protein stability and activity.