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 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.
PURPOSE: To investigate the role and importance of the four methionines in recombinant humanleptin, 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: F Zhang; M B Basinski; J M Beals; S L Briggs; L M Churgay; D K Clawson; R D DiMarchi; T C Furman; J E Hale; H M Hsiung; B E Schoner; D P Smith; X Y Zhang; J P Wery; R W Schevitz Journal: Nature Date: 1997-05-08 Impact factor: 49.962
Authors: J L Halaas; K S Gajiwala; M Maffei; S L Cohen; B T Chait; D Rabinowitz; R L Lallone; S K Burley; J M Friedman Journal: Science Date: 1995-07-28 Impact factor: 47.728
Authors: Jin Yin; Jhih-Wei Chu; Margaret Speed Ricci; David N Brems; Daniel I C Wang; Bernhardt L Trout Journal: Pharm Res Date: 2005-01 Impact factor: 4.200
Authors: Jin Yin; Jhih-Wei Chu; Margaret Speed Ricci; David N Brems; Daniel I C Wang; Bernhardt L Trout Journal: Pharm Res Date: 2004-12 Impact factor: 4.200
Authors: Jared S Bee; Michele Davis; Erwin Freund; John F Carpenter; Theodore W Randolph Journal: Biotechnol Bioeng Date: 2010-01-01 Impact factor: 4.530