4-Chlorobutanol induces unusual reversible and irreversible thermal unfolding of ribonuclease A: thermodynamic, kinetic, and conformational characterization.
The thermal denaturation of ribonuclease A has been studied by differential scanning calorimetry in the presence of 4-chlorobutan-1-ol. The thermal transitions were observed to be reversible at pH 5.5 in the presence of low concentration (up to 50 mM) of the alcohol, irreversible in the intermediate (50 mM < c < mM) and again reversible in the presence of 250 mM and higher concentrations of 4-chlorobutan-1-ol. In the presence of 50 mM 4-chlorobutan-1-ol, ribonuclease A is present in two conformational states unfolding at different temperatures. The reversible thermal transitions have been fitted to a two-state native-to-denatured mechanism. Irreversible thermal transitions have been analyzed according to two-state irreversible native-to-denatured kinetic model. Using the irreversible model, rate constant as a function of temperature and energy of activation of the irreversible process have been calculated. Circular dichroism and fluorescence spectroscopic results corroborate the DSC observations and indicate a protein conformation with poorly defined tertiary structure and high content of secondary structure in the presence of 50 mM 4-chlorobutan-1-ol at a temperature corresponding to the second transition. Similar results have been observed at pH 3.9.
Mehta
Ranjana
R
Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India.
Kundu
Agnita
A
Kishore
Nand
N
D000818:Animals; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D002724:Chlorobutanol / Q000494:pharmacology*; D002942:Circular Dichroism; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011487:Protein Conformation; D011489:Protein Denaturation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000187:drug effects*; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D012259:Ribonuclease, Pancreatic; D002724:Chlorobutanol