Kinetic Studies of the Regeneration of Recombinant Hirudin Variant 1 with Oxidized and Reduced Dithiothreitol
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- 作者：Theodore W. Thannhauser ; David M. Rothwarf ; and Harold A. Scheraga
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：February 25, 1997
- 年：1997
- 卷：36
- 期：8
- 页码：2154 - 2165
- 全文大小：326K
- 年卷期：v.36,no.8(February 25, 1997)
- ISSN：1520-4995
文摘
The regeneration of native recombinant hirudin variant 1 (rHV1)from the reduced unfoldedform to the fully oxidized native state has been carried out withmixtures of oxidized and reduceddithiothreitol at pH 8.3 and 12 
C. The regeneration reactionwas quenched at various times by theaddition of 2-aminoethyl methanethiosulfonate to block unreactedsulfhydryl groups. The quenched protein-folding intermediates were fractionated by both capillaryelectrophoresis and a combination of anionexchange and reverse phase HPLC and characterized by mass spectrometry,amino acid analysis, anddisulfide analysis. These intermediates (before quenching) werefound to interconvert rapidly so as toachieve a steady-state distribution early in the regeneration process.The experimental data were fitted toa steady-state kinetic scheme. The analysis reveals that therate-determining step in the regeneration ofrHV1 with oxidized and reduced dithiothreitol involves the oxidation ofone or more two-disulfide-containing species, most likely those already containing two nativedisulfide bonds. This regenerationmechanism is different from one that has been proposed by Chatrenet andChang [(1993) J. Biol. Chem.268, 20988]. The differences are discussed, and possibleexplanations for the differences are presented.
C. The regeneration reactionwas quenched at various times by theaddition of 2-aminoethyl methanethiosulfonate to block unreactedsulfhydryl groups. The quenched protein-folding intermediates were fractionated by both capillaryelectrophoresis and a combination of anionexchange and reverse phase HPLC and characterized by mass spectrometry,amino acid analysis, anddisulfide analysis. These intermediates (before quenching) werefound to interconvert rapidly so as toachieve a steady-state distribution early in the regeneration process.The experimental data were fitted toa steady-state kinetic scheme. The analysis reveals that therate-determining step in the regeneration ofrHV1 with oxidized and reduced dithiothreitol involves the oxidation ofone or more two-disulfide-containing species, most likely those already containing two nativedisulfide bonds. This regenerationmechanism is different from one that has been proposed by Chatrenet andChang [(1993) J. Biol. Chem.268, 20988]. The differences are discussed, and possibleexplanations for the differences are presented.