title: In vitro methionine oxidation of recombinant human leptin.
abstract: OBJECTIVE
  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 x 10(-3) and 2.3 x 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 approximately Met137, and the importance for maintaining leptin structural integrity was Met55 approximately Met137 >> Met69 approximately Met1. Met69, but not Met1, plays a critical role in the protein stability and activity.
authors.affiliation: Department of Protein Chemistry, Amgen Inc., Thousand Oaks, California 91320-1789, USA. jliu@amgen.com
authors.forename: J L
authors.initials: JL
authors.lastname: Liu
authors.forename: K V
authors.initials: KV
authors.lastname: Lu
authors.forename: T
authors.initials: T
authors.lastname: Eris
authors.forename: V
authors.initials: V
authors.lastname: Katta
authors.forename: K R
authors.initials: KR
authors.lastname: Westcott
authors.forename: L O
authors.initials: LO
authors.lastname: Narhi
authors.forename: H S
authors.initials: HS
authors.lastname: Lu
mesh_terms: D000273:Adipose Tissue / Q000378:metabolism*; D003902:Detergents / Q000737:chemistry; D010450:Endopeptidases / Q000737:chemistry; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D020738:Leptin; D008666:Metalloendopeptidases; D008715:Methionine / Q000737:chemistry*; D009765:Obesity / Q000235:genetics; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D010545:Peroxides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins / Q000737:chemistry; D020122:tert-Butylhydroperoxide
chemical_list: D003902:Detergents; D020738:Leptin; D010545:Peroxides; D011506:Proteins; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D020122:tert-Butylhydroperoxide; D008715:Methionine; D006861:Hydrogen Peroxide; D010450:Endopeptidases; D008666:Metalloendopeptidases; C058921:endoproteinase Asp-N