Salts and glycine increase reversibility and decrease aggregation during thermal unfolding of ribonuclease-A.
Ribonuclease-A (RNase-A) has been a model for studying protein folding and unfolding. However, we show here that its unfolding at neutral pH is complicated by aggregation. Circular dichroism thermal scans showed that reversibility of RNase-A after heating is only about 63%. In accordance with this observation, native-polyacrylamide gel electrophoresis of the sample heated at 75 degrees C showed formation of soluble oligomers. Ammonium sulfate at 0.4 M caused about a 3 degrees C higher melting temperature and nearly complete reversibility, while glycine and NaCl at 0.4 M significantly increased reversibility and decreased aggregation without affecting melting temperature. These results demonstrate that aggregation makes thermal unfolding of RNase-A at least partially irreversible and salts and glycine increase reversibility and decrease aggregation.
Kita
Yoshiko
Y
Alliance Protein Laboratories, Thousand Oaks, CA 91360, USA.
Arakawa
Tsutomu
T
D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005998:Glycine / Q000494:pharmacology*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D013816:Thermodynamics
D012259:Ribonuclease, Pancreatic; D005998:Glycine