Thermal stability of lactate dehydrogenase and alcohol dehydrogenase incorporated into highly concentrated gels
Kulis, I.Iu.
Biokhimiia 44(3): 417-423
1979
ISSN/ISBN: 0320-9725 PMID: 465589 Document Number: 139164
The rate constants for inactivation of pig muscle lactate dehydrogenase and horse liver alcohol dehydrogenase in solution at 65.degree. C (pH 7.5) are 0.72 and 0.013 min-1, respectively. The enzyme incorporation into acrylamide gels results in immobilized enzymes whose residual activity is 18-25% of the original one. In 6.7% gels the rate of thermal inactivation for lactate dehydrogenase is decreased nearly 10-fold; the inactivation rate for alcohol dehydrogenase is increased 4.6-fold as compared to the soluble enzymes. In 14 and 40% gels the inactivation constants for lactate dehydrogenase are 6.3 .cntdot. 10-3 and 5.9 .times. 10-4 min-1, respectively. In 60% gels the thermal inactivation of lactate dehydrogenase is decelerated 3600-fold as compared to the native enzyme. The enthalpy and entropy for the inactivation of the native enzyme are 62.8 kcal/mol and 116.9 cal/(mol .cntdot. .degree.C) for the native enzyme and those of gel-incorporated (6.7%) enzyme are 38.7 kcal/mol and 42 cal/(mol .cntdot. .degree.C), respectively. The thermal stability of alcohol dehydrogenase in 60% gels is increased 12-fold. To prevent gel swelling, methacrylic acid and allylamine were added to the matrix followed by treatment with dicyclohexylcarbodiimide. The enzyme activity of the modified gels is 2.7-3% of that for the 6.7% gels. The stability of lactate dehydrogenase in such gels is significantly increased. A mechanism of stabilization of the subunit enzymes in highly concentrated gels is discussed.