Refolding of chicken cystatin and human stefin A studied by fast protein liquid chromatography (FPLC).
Denaturant gradient gel-chromatography [Endo, S. et al. (1983) Anal. Biochem. 131, 108-120] has been used to follow renaturation of chicken cystatin and human stefin A. Slow gradient of GuHCl was applied to the size-exclusion Superose 12 column. The column was maintained at 5 degrees C by circulating water from a cryostate. When the denatured protein has been injected it started to refold on the column. By the simplified equation, according to [Hanai, R. et al. (1986) Biophys. Chem. 25, 27-36] approximate kinetics of folding was obtained. No intermediate species in folding could be detected from 6M to 2M GuHCl, only the close-to-denatured state and the close-to-native state.
Dept. Biochemistry, J. Stefan Institute, Ljubljana.
E
E
Zerovnik
J
J
Kos
B
B
Lenarcic
V
V
Turk
D000818:Animals; D002645:Chickens; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D055312:Cystatin A; D055313:Cystatin B; D015891:Cystatins / Q000032:analysis*; D006801:Humans; D007700:Kinetics; D011487:Protein Conformation; D011489:Protein Denaturation; D013696:Temperature
C524994:CSTB protein, human; D055312:Cystatin A; D015891:Cystatins; C100158:cystatin, egg-white; C026407:CSTA protein, human; D055313:Cystatin B