Abstract
The refolding of guanidine hydrochloride-denatured horse heart ferricytochrome c at pH 7.0 and 23 degrees C occurs in three kinetic phases as observed by stopped flow measurements using changes in Soret absorbance or in tryptophan fluorescence. The three kinetic phases have time constants of 10 +/- 5 ms, 240 +/- 30 ms, and 13 +/- 3 s accounting for 15 +/- 5%, 70 +/- 5%, and 15 +/- 5% of the total reaction, respectively. The intermediate kinetic phase can be selectively eliminated by conducting the refolding measurements at pH 5.0. Both the intermediate and slow kinetic phases can be eliminated by conducting the refolding measurements either at pH 7.0 or at pH 5.0 in the presence of an excess of an extrinsic ligand for an axial position of the heme iron. Similar results are obtained using tuna heart ferricytochrome c except that the fractional reaction in the fast and intermediate kinetic phases at pH 7.0 in the absence of extrinsic ligand are 29 +/- 2% and 58 +/- 2%, respectively. We suggest that both the intermediate and slow kinetic phases are generated by proline peptide isomerization occurring during and prior to the refolding procedure, respectively, and that their occurrence is dependent upon the conformation of the denatured protein and upon the ligation of methionine 80 in the folded product, respectively.
Highlights
EXPERIMENTALPROCEDURES have time constants of 10 f 5 ms, 240 k 30 ms, and 13 Materials-Horse heart, tuna heart(type XI), cow heart f 3 s accounting for 15 f 5%, 70 f 5%, and 15 k 5% of, and Saccharomyces cerevisiae cytochromes c the total reaction, respectively
We have recently reported (4) that the refolding of guanidine hydrochloride-denatured bullfrog heart ferricytochrome c, which contains an intramolecular disulfide bond, occurs in two kinetic phases having centisecond and second time constants,with 80%of the totalchange occurring in the centisecond phase
Measurements atp H 7.0-Addition of increasing amounts of guanidine hydrochloride to a solution of horse heart ferricytochrome c maintained at pH7.0 and 23 "C shifts the Soret maximum from 409 nm to 406 nm, eliminates the 695-nm absorbance bond, and increases the fluorescence of tryptophan 59 as shown in Fig. 1.These changes describe a common transition centeredat 2.7 M guanidine hydrochloride indicative of the conversion of the folded low spin native protein having methionine 80 as an axial ligand to adenatured low spin protein not having methionine as an axial ligand
Summary
Measurements atp H 7.0-Addition of increasing amounts of guanidine hydrochloride to a solution of horse heart ferricytochrome c maintained at pH7.0 and 23 "C shifts the Soret maximum from 409 nm to 406 nm, eliminates the 695-nm absorbance bond, and increases the fluorescence of tryptophan 59 as shown in Fig. 1.These changes describe a common transition centeredat 2.7 M guanidine hydrochloride indicative of the conversion of the folded low spin native protein having methionine 80 as an axial ligand to adenatured low spin protein not having methionine as an axial ligand. These changes describe a common transition centered at 2.3 f 0.1 M guanidine hydrochloride indicative of conversion of the folded low spin native protein to a denatured mixed spin form. Rapid dilution across the conformational transition at pH 3.0, 3 M guanidine hydrochloride diluted to 1 M, generates only two kinetic phases, the fast and slow phases having time constants of 20 f 5 ms and 16 f 2 s, respectively, with the fast phase accounting for 90 +- 2%of the totalreaction
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