Abstract
The effect on stability of differences in amino acid sequences has been studied in three proteins from a homologous group. Cow and horse ferricytochromes c, which differ by 3 residues, were used for the investigations together with a less closely related member, Candida krusei. C. krusei cytochrome c differs from horse and cow by 6 additional N-terminal amino acids. Further, it differs from horse by 43 of the 103 residues common to both of theirs sequences. The effects of these difference on their relative stabilities has been studied by denaturation both in guanidine hydrochloride solution and in acid. Both techniques were followed by measurements with optical rotation at 300 nm. At pH 6.5 and 25°C. the midpoints of the Gu HCl denaturation curves are 1.89 M Gu HCl for C. krusei, 2.41 for horse, and 2.62 for cow. An analysis of these data indicates that cow is a minimum of 700 cal./mol more stable than horse and 2800 cal./mol more stable than C. krusei in the presence of 2-3 M Gu HCl. The acid denaturations of horse and cow showed that they had midpoint pH values of 2.60 and 2.44, respectively. Near pH 2.5 the difference in stability from an analysis between these curves is 627 cal./mol with cow the more stable. At the midpoint condition in acid horse and C. krusei had similar stabilities; however, under physiological conditions C. krusei might be less stable than horse by a small margin. In this latter respect, cow will be more stable than horse by at least 700 cal./mol. Most factors which contribute to the difference in stability suggest that horse should be more stable than cow ferricytochrome c. It was found that the midpoints of the acid denaturation curves occur at lower pH values when the reaction is monitored using absorbance at 395 nm. This is a clear demonstration that eh mechanism of denaturation for ferricytochrome c is more complex than a simple two-state process. The optical properties of the Gu HCl denatured proteins suggest that they have similar, randomly-coiled conformations. In contrast, acid-denatured cow ferricytochrome c appears to be less unfolded than the other two proteins.
Knapp, John Andrew (1974). The guanidine hydrochloride and acid denaturation of the cytochrome c from horse, cow, and Candida krusei. Doctoral dissertation, Texas A&M University. Texas A&M University. Libraries. Available electronically from
https : / /hdl .handle .net /1969 .1 /DISSERTATIONS -175782.