Refolding of Cytochrome C Using Stopped-Flow

Download PDF August 24, 2022


CD spectra provide information regarding the secondary and tertiary structure of proteins. While the far-UV region elucidates structural information of the peptide backbone chain, the near-UV region of the spectrum highlights changes involving the aromatic amino acid residues. Therefore, coupling a CD spectrometer with a stopped-flow system is considered one of the best methods for analyzing the unfolding and refolding of proteins. This system now provides not only structural information pertaining to the protein in question, but also supplies this data on a sub millisecond time scale. The user can now obtain a more detailed picture of the time scale for when each protein unfolds and refolds.

This application notes demonstrates the use of the J-1500 CD spectrometer and SFS-492 Stopped-Flow system to monitor the refolding process of cytochrome c.

J-1500 Circular Dichroism Spectrophotometer
J-1500 CD Spectrometer


Measurement conditions
222 nm
289 nm
Data acquisition interval5 msec10 msec
Spectral bandwidth4 nm2 nm
Response time4 msec8 msec
Syringe 12 mg/mL cyt c: 4.3 M GuHCl10 mg/mL cyt c: 4.3 M GuHCl
Syringe 1 loading volume30 µL
Syringe 20.1 M acetic acid buffer (pH 6.3)
Syringe 2 loading volume270 µL
Accumulations36 times24 times
Flow rate1.5 mL/sec

An aqueous solution of cytochrome c denatured by guanidine hydrochloride (GuHCl) was diluted with 0.1 M acetic acid buffer solution (1:9). The refolding process was observed at 222 and 289 nm to monitor the secondary structure changes and aromatic side chain residue environment, respectively.


J-1500, circular dichroism, stopped-flow, SFS-492, protein folding, biochemistry, refolding


The change in the CD value at 222 nm reflects the fast refolding of the secondary structure within 200 msec (Figure 1). The change at 289 nm reflecting the aromatic side chains residue environment was slower than the change at 222 nm (Figure 2). This slower change appears in the latter step of the refolding process, which indicates the brief existence of an intermediate state with a refolded secondary structure and unfolded aromatic side chain residues.

Figure 1. Refolding measurement of cytochrome c monitored at 222 nm
Figure 2. Refolding measurement of cytochrome c monitored at 289 nm


This application note monitors the existence of an intermediate state by probing the refolding of denatured cytochrome c using a JASCO CD spectrometer and the SFS-492 Stopped-Flow system.


1. Elove, G. A., Chaffotte, A. F., Roder, H., and M. E. Goldberg, Biochemistry (1992), 31, 6876.
2. Chaffotte, A. F., Guillou, Y., and M. E. Goldberg, Biochemistry (1992), 31, 9694.

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About the Author

Leah Pandiscia received her PhD from Drexel University where she studied Biophysical Chemistry. She is a Spectroscopy Applications Scientist.