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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.

Inactivation by paraoxon of free and immobilized acetylcholinesterase, in the presence of various amounts of different organic solvents, and with choline oxidase immobilized on an oxygen electrode was studied. With acetylcholinesterase in solution it was found that 5% cyclohexane enhances paraoxon detection when compared to the detection without solvent. In these conditions, as low as 10 −9 m paraoxon can be detected. Free acetylcholinesterase was more sensitive to paraoxon than immobilized acetylcholinesterase, depending on the nature and concentration of solvent (500-fold in the presence of 5% cyclohexane). The stability of the biosensor in continuous use and stored at room temperature was at least 3–4 weeks with triton X100 and bovine serum albumin addition. The sensitivity of paraoxon detection with free acetylcholinesterase in the presence of 5% cyclohexane corresponds to 0·2 ppb paraoxon, 10-fold below the legal limit admitted in the European Economic Community. The same approach could be applied for detection of low amounts of different organophosphorus or carbamate pesticides.

Covalent immobilization of L-lactate oxidase (LOD) with L-lactate dehydrogenase (LDH) on a film tightly bound to an oxygen electrode, for rapid and sensitive L-lactate measurements, is described. Regeneration of L-lactate by substrate recycling provided an amplification of the sensor response, making it possible to decrease the detection limit of L-lactate from 10 μM to 20 nM. The apparent K m for L-lactate with the LOD-LDH coupled reaction was 1 μM, compared with 3 mM when utilizing only LOD. Linearity was obtained from 20 to 300 nM with both enzymes, whereas with LOD alone it was from 10 μM to 1 mM. Optimization of the biosensor was obtained with an increase in LOD and LDH film loading and low L-lactate concentration. The enzymes covalently bound to the film stabilized the biosensor (half life 8 weeks) for over 400 measurements. Low L-lactate excreted by E. coli bacteria metabolism can be assayed in turbid culture medium without pretreatment by the amplified L-lactate detection.

Immobilized oxidases were bound on the surface of an affinity membrane and mounted on an oxygen electrode. These biosensors were used for heavy metal salt measurements. After immobilization on the enzymes, first order kinetics of inactivation were observed. Surface immobilization increases the sensitivity by a factor of 10, compared to reticulation of the enzyme in a gelatin matrix. After immobilization, 50% inactivation was observed with 20 μ m HgCl 2 for L-glycerophosphate oxidase and 50 n m for pyruvate oxidase. Restoration of activity after HgCl 2 treatment is feasible, but neither complete nor reproducible. To reuse the biosensor, L-lactate dehydrogenase (LDH) from rabbit muscle in solution was coupled to immobilized L-lactate oxidase (insensitive to heavy metal salts). LDH (particularly inexpensive) was replaced after each measurement. The I 50 in phosphate buffer was 1 μ m for HgCl 2 and 0·1 μ m for AgNO 3 ; with other heavy metal salts, no inhibition was observed below 500 μ m . In Tris buffer, the I 50 was 10 μ m for CdCl 2 and ZnCl 2 , 50 μ m for Pb-acetate and 250 μ m for CuSO 4 . The use of different enzymes and buffers may allow measurement of specific heavy metal salts.

Coimmobilization of l -lactate oxidase (LOD) and l -lactate dehydrogenase (LDH) on a film bound to an oxygen electrode makes it possible, by recycling l -lactate and pyruvate, to lower their detection limit to 10 nM. With LOD immobilized alone and LDH in solution, easy LDH measurements were performed. The detection limit was 1 IUl−1. Similarly, this mixed biosensor, using an LDH sensitive to mercurials, was used for p-chloromercuribenzoate (PCMB) detection. Coimmobilization of LOD + LDH with an NAD+-dependent dehydrogenase allows the measurements of all dehydrogenases. NAD+ is recycled. The detection limit was 50 nM. Applications can be found in medicine, the food industry, and the environment.

Contrary to the generl view point that respiratory control does not exist in Escherichia coli, we have been able to demonstrate this phenomenon in this micro-organism. E. coli cells growing aerobically on an oxidizable carbon source respire at a high rate, which cannot be increased by the addition of uncouplers of oxidative phosphorylation. Upon removal of the carbon source, the rate of respiration decreases progressively to reach, after 10 min, less that 1/20 of its previous value. Under these conditions the addition of an uncoupler increases the residual respiration by a factor of 3 to 9. This increase is due to the release of respiratory control. Release of respiratory control cannot be observed upon addition of ADP which does not penetrate across the membrane. Respiratory control can also be shown in the presence of non-limiting respiratory substrate provided that it is not a carbon source. This was shown by measuring the rate of oxidation of ascorbate and phenazine methosulfate. NADH oxidase activity of membrane vesicles obtained by high pressure disintegration was increased by a factor of about 1.5 upon the addition of uncouplers and also upon addition of ADP plus phosphate. In contrast, membrane vesicles depleted from BF1 ATPase (a soluble bacterial coupling factor presenting ATPase activity) did not exhibit respiratory control. It could be restored either by rebinding of BF1 ATPase or by addition of dicyclohexylcarbodiimide, an ATPase inhibitor.

1. Exposure of submitochondrial particles to phospholipases impaired the rate of oxidation as well as phosphorylation. Phospholipase A and phospholipase C were more inhibitory to phosphorylation and to NADH oxidation than to succinate oxidation. 2. At low concentrations of phospholipase A the inhibition of phosphorylation was prevented to a considerable extent by the presence of large amounts of bovine serum albumin preferably during both digestion and assay. At larger concentrations of phospholipase A only slight protection was observed by addition of serum albumin. 3. Exposure of submitochondrial particles to phospholipase C from Clostridium welchii in the presence of Mg++ or Mn++ inhibited the 32Pi-ATP exchange and the P:O ratio by about 30 to 60%. Addition of coupling factors restored phosphorylation associated with oxidation in these particles. Additional exposure of the digested particles to phospholipase C from Bacillus cereus in the presence of Zn++ further depressed the 32Pi-ATP exchange which was no longer restored by coupling factors. Addition of phospholipids partially restored the 32Pi-ATP exchange in these extensively digested particles. Similar effects were noted after prolonged exposure to phospholipase C from B. cereus alone, provided both Mn++ and Zn++ were present. 4. Oxidative phosphorylation was also severely impaired after exposure of submitochondrial particles to phospholipase C from B. cereus in the presence of Mn++ and Zn++. A partial restoration of the P:O ratio was achieved by reconstitution with phospholipids.

1. Exposure of bovine heart mitochondria to purified preparations of phospholipase A or phospholipase C resulted in losses of phosphorylation capacity, whereas respiration was not impaired. 2. The inhibitory effects of low concentrations of phospholipase A on phosphorylation were prevented or reversed by bovine serum albumin. At higher concentrations of phospholipase A neither bovine serum albumin nor lecithin were effective in restoring phosphorylation. 3. Exposure of mitochondria to phospholipase C resulted in a loss of Ca++ translocation, a partial depression of oxidative phosphorylation, and a partial loss of organically bound phosphorus. 4. Phospholipase C-treated mitochondria were resistant to the uncoupling action of Ca++ or K+ plus valinomycin. Addition of nigericin together with valinomycin and K+ was required to abolish phosphorylation associated with oxidation in phospholipase C-treated mitochondria. These responses resemble those of submitochondrial particles rather than those of mitochondria. In electron micrographs, phospholipase C-treated mitochondria looked like submitochondrial particles packaged in mitochondria. 5. A partial restoration of Ca++ translocation after phospholipase C digestion was achieved by addition of either egg lecithin or of large amounts of bovine serum albumin.