Stabilization and activation of alpha-chymotrypsin in water-organic solvent systems by complex formation with oligoamines.
Formation of enzyme-oligoamine complexes was suggested as an approach to obtain biocatalysts with enhanced resistance towards inactivation in water-organic media. Complex formation results in broadening (by 20-40% v/v ethanol) of the range of cosolvent concentrations where the enzyme retains its catalytic activity (stabilization effect). At moderate cosolvent concentrations (20-40% v/v) complex formation activates the enzyme (by 3-6 times). The magnitude of activation and stabilization effects increases with the number of possible electrostatic contacts between the protein surface and the molecules of oligoamines (OA). Circular dichroism spectra in the far-UV region show that complex formation stabilizes protein conformation and prevents aggregation in water-organic solvent mixtures. Two populations of the complexes with different thermodynamic stabilities were found in alpha-chymotrypsin (CT)-OA systems depending on the CT/OA ratio. The average dissociation constants and stoichiometries of both low- and high-affinity populations of the complexes were estimated. It appears that it is the low-affinity sites on the CT surface that are responsible for the activation effect.
Chemistry Department, M.V. Lomonosov Moscow State University, 119899 Moscow, Russia. helena_koudriachova@hotmail.com
Elena V
EV
Kudryashova
Tatiana M
TM
Artemova
Alexei A
AA
Vinogradov
Alexander K
AK
Gladilin
Vadim V
VV
Mozhaev
Andrey V
AV
Levashov
D002384:Catalysis; D002918:Chymotrypsin / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D004789:Enzyme Activation; D004795:Enzyme Stability; D000431:Ethanol; D007700:Kinetics; D011073:Polyamines / Q000737:chemistry*; D011487:Protein Conformation; D012997:Solvents / Q000737:chemistry*; D055672:Static Electricity; D013816:Thermodynamics; D014867:Water
D011073:Polyamines; D012997:Solvents; D014867:Water; D000431:Ethanol; D002918:Chymotrypsin; C117608:alpha-chymotrypsin