Kinetic study of heavy metal salt effects on the activity of L-lactate dehydrogenase in solution or immobilized on an oxygen electrode.
A sensitive and convenient biosensor for detection of heavy metal salts has been developed. The method is based on the effects of heavy metal salts on the catalytic activity of L-lactate dehydrogenase (LDH) in solution or coimmobilized with L-lactate oxidase (LOD) on an oxygen electrode. At metal concentrations below 100 microM, the kinetic behavior, with the LDH substrate NADH, showed a competitive inhibition with high affinity during the first 10 s. With increased incubation time, irreversible first order inactivation with respect to enzyme concentration was observed. This irreversible inactivation of LDH in solution was dose dependent. The efficiencies obtained for the different heavy metal salts were: HgGl2 > AgNO3 > Pb(COOCH3)2 > CuSO4 > ZnCl2. HgCl2 and AgNO3 were effective in the nanomolar range while the other metal salts acted at the micromolar level. LDH is protected by saturating amounts of substrate NADH against the effects of the heavy metal salts studied. The pKs for LDH catalytic activity and inactivation by heavy metal salts were similar. The results suggest binding of the heavy metal salts to the enzyme active site. Except for lead acetate, all heavy metal detection was in the range of European norms. For AgNO3, CuSO4 and HgCl2, the sensor limit of detection reached the European norm values whereas with ZnCl2 it was well below. The immobilization of LDH considerably decreased the amount of enzyme consumed by permitting repetitive assays. The efficiency of inactivation by the heavy metal salts was reduced in comparison with LDH in solution. Restoration of activity of the inactivated immobilized enzyme was obtained with DTT, EDTA, KCN and NADH treatment. This opens up possibilities for detection of toxic compounds using simple procedures suitable for assays in a variety of monitoring conditions in environmental and food pollution control.
Fennouh
S
S
Laboratoire de Technologies des Enzymes et des Biomembranes-Case 7124, Université Paris 7-2, France.
Casimiri
V
V
Geloso-Meyer
A
A
Burstein
C
C
D015374:Biosensing Techniques; D007770:L-Lactate Dehydrogenase; D019216:Metals, Heavy; D010100:Oxygen / Q000032:analysis*
D019216:Metals, Heavy; D007770:L-Lactate Dehydrogenase; D010100:Oxygen