KINETIC-STUDY OF HEAVY-METAL SALT EFFECTS ON THE ACTIVITY OF L-LACTATE DEHYDROGENASE IN SOLUTION OF IMMOBILIZED ON AN OXYGEN-ELECTRODE

A sensitive and convenient biosensor for detection of heavy metal salt s has been developed. The method is based on the effects of heavy meta l salts on the catalytic activity of L-lactate dehydrogenase (LDH) in solution or coimmobilized with L-lactate oxidase (LOD) on an oxygen el ectrode. At metal concentrations below 100 mu M, the kinetic behavior, with the LDH substrate NADH, showed a competitive inhibition with hig h affinity during the first 10 s. With increased incubation time, irre versible first order inactivation with respect to enzyme concentration was observed. This irreversible inactivation of LDH in solution was d ose dependant. The efficiencies obtained for the different heavy metal salts were: HgCl2 > AgNO3 > Pb(COOCH3)(2) > CuSO4 > ZnCl2. HgCl2 and AgNO3 were effective in the nanomolar range while the other metal salt s acted at the micromolar level. LDH is protected by saturating amount s of substrate NADH against the effects of the heavy metal salts studi ed. The pKs for LDH catalytic activity and inactivation by heavy metal salts were similar. The results suggest binding of the heavy metal sa lts to the enzyme active site. Except for lead acetate, all heavy meta l detection was in the range of European norms. For AgNO3, CuSO4 and H gCl2, the sensor limit of detection reached the European norm values w hereas with ZnCl2 it was well below. The immobilization of LDH conside rably decreased the amount of enzyme consumed by permitting repetitive assays. The efficiency of inactivation by the heavy metal salts was r educed in comparison with LDH in solution. Restoration of activity of the inactivated immobilized enzyme was obtained with DTT, EDTA, KCN an d NADH treatment. This opens up possibilities for detection of toxic c ompounds using simple procedures suitable for assays in a variety of m onitoring conditions in environmental and food pollution control. (C) 1998 Elsevier Science S.A. All rights reserved.