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
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C. The regeneration reaction
was 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 t
wo-disulfide-containing species, most likely those already containing t
wo 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.