In vitro methionine oxidation of recombinant human leptin

被引:44
作者
Liu, JL [1 ]
Lu, KV [1 ]
Eris, T [1 ]
Katta, V [1 ]
Westcott, KR [1 ]
Narhi, LO [1 ]
Lu, HS [1 ]
机构
[1] Amgen Inc, Dept Prot Chem, Thousand Oaks, CA 91320 USA
关键词
recombinant human leptin; leptin oxidation; oxidation kinetics; protein characterization; protein structure;
D O I
10.1023/A:1011998331254
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
Purpose, 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 Met(1) and Met(69) proceeded first and independently. In 48 mM t-butylhydroperoxide, the pseudo first-order rate constants, k(1) and k(69), were 1.5 x 10(-3) and 2.3 x 10(-4) min(-1). No change in the secondary or tertiary structure was detected for Met(1) mono-oxidized and Met(1), Met(69) di-oxidized leptins. The Met(1) 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 Met(1), Met(69) di-oxidized leptin. Both Met(55) and Met(137) 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 Met(1) > Met(69) >> Met(55) approximate to Met(137), and the importance for maintaining leptin structural integrity was Met(55) approximate to Met(137) >> Met(69) approximate to Met(1). Met(69), but not Met(1), plays a critical role in the protein stability and activity.
引用
收藏
页码:632 / 640
页数:9
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