Affordable Access

Publisher Website

α-Chymotrypsin stability in aqueous-acetonitrile mixtures: is the native enzyme thermodynamically or kinetically stable under low water conditions?

Journal of Molecular Catalysis B Enzymatic
Publication Date
DOI: 10.1016/s1381-1177(98)00105-2
  • α-Chymotrypsin
  • Organic Solvent
  • Aqueous-Acetonitrile Mixtures
  • Enzyme Denaturation/Renaturation
  • Kinetic Stability
  • Biology
  • Physics


Abstract Like many proteins, α-chymotrypsin is denatured in 50% volume aqueous-acetonitrile mixtures. However, it also shows high catalytic activity in 70% or more acetonitrile. Good activity in two different aqueous organic composition ranges has been described for several other enzymes. The stability of the native protein under low water conditions is generally believed to be a kinetic phenomenon, though there are also arguments for thermodynamic stability. We have distinguished between these possibilities by studying the effects of changing medium composition at different times. In preliminary experiments, we found catalytic activity could be recovered by adding neat acetonitrile to chymotrypsin in a 50% mixture, suggesting that the enzyme could renature under these conditions. However, in the 50% mixture, the true initial activity at 30°C is not zero, as the literature suggests. Instead, there is an initial burst of product formation over a few minutes, after which the enzyme becomes inactivated. By pre-incubating a 50% aqueous-acetonitrile mixture at 30°C prior to enzyme addition, the product burst could be eliminated. Activity could not then be recovered by slow addition of acetonitrile to the denatured enzyme. In contrast, it was possible to renature by dilution with aqueous buffer so that regeneration of catalytic activity was achieved. Thus, the good practical performance at high acetonitrile concentrations almost certainly results from a high kinetic barrier towards denaturation. The kinetics of enzyme denaturation in 50% and 70% acetonitrile were also investigated both at 30 and 20°C. Loss of catalytic activity was faster at higher temperature and at lower acetonitrile concentrations.

There are no comments yet on this publication. Be the first to share your thoughts.