Application of the method of thermal denaturation for investigation of alpha-chymotrypsin adducts with poly.
The thermostability of conjugates, non-covalent complexes and mixtures of alpha-chymotrypsin (alpha-ChT) with poly(alkylene oxides)--poly(ethylene glycol) (PEG) with molecular mass of 1.9 kD and diblock copolymers of ethylene and propylene oxides (proxanols)--has been investigated. It was shown that the addition of PEG in concentration up to 2 wt. % to the solution of alpha-ChT did not affect the rate of the enzyme thermoinactivation. Meanwhile the addition of proxanol in the same concentration resulted in twofold decrease in the rate constant for the slow inactivation step, k2. Even more pronounced decrease in the thermoinactivation rate was observed for alpha-ChT--proxanol complexes obtained by heating or under the action of high pressure. The general tendency in the behavior of complexes of both types was the decrease in the k2 constant as the temperature or pressure used for complex preparation increased. The highest stabilizing effect was observed for complex obtained by heating up to 52 degreesC and containing maximal number of polymer chains (molar ratio proxanol/alpha-ChT was 10). For this complex fourfold decrease in the k2 value was observed. Covalent attachment of PEG or proxanol to enzyme gives maximal stabilizing effect with up to tenfold decrease in the k2 value. The investigation of the thermal denaturation kinetics of alpha-ChT and its adducts with poly(alkylene oxides) by means of fluorescence spectroscopy has shown that the presence of polymer chains practically does not affect the rate of protein denaturation registered by the decrease in the intensity of protein fluorescence. The polymer chains, probably, diminish the rate of melting of the active site-containing region of the protein molecule. At the same time, the overall denaturation rate is independent of the presence of polymer chains in the vicinity of the protein globule.
Efremova
N V
NV
Bakh Institute of Biochemistry, Russian Academy of Sciences, Leninskii pr. 33, Moscow, 117071 Russia.
Sorokina
E M
EM
Kurganov
B I
BI
Topchieva
I N
IN
D000818:Animals; D002417:Cattle; D002918:Chymotrypsin / Q000737:chemistry*; D007700:Kinetics; D011092:Polyethylene Glycols / Q000737:chemistry*; D011489:Protein Denaturation; D013696:Temperature
D011092:Polyethylene Glycols; D002918:Chymotrypsin