Formate dehydrogenase (EC 1.2.1.2) from Pseudornonas oxalaticus has been isolated and characterized. The enzyme (molecular weight 31 5 000) is a complex flavoprotein containing 2 FMN, 18-25 non-heme iron atoms and 15-20 acid-labile sulphides. In the last step of the purification, a sucrose gradient centrifugation, a second catalytically active species has been found apparently originating from a dissociation of the enzyme into two equal subunits. The enzyme is specific toward its natural substrate formate. It transfers electrons to NAD', oxygen, ferricyanide, and a lot of nonphysiological acceptors (dyes). In addition electrons are transferred from NADH to these acceptors.The (reversible) removal of FMN requires a reduction step. Reincorporation has been followed by the reappearance of the reactivity against formate and by fluorescence titration. The deflavo enzyme also binds FAD and riboflavin. The resulting enzyme species show characteristic catalytic abilities. Activity against formate is peculiar to the FMN species.When the aerobic bacterium Pseudornonas osalati-L'US is grown on formate as the main carbon source, the oxidation of formate to COZ is the key process for obtaining energy and reducing equivalents. The key enzyme for the latter is formate dehydrogenase, a complex flavoprotein containing FMN, iron and labile sulphur. It catalyzes the reaction HCOF + NAD' COz + NADH. (1) The C02 formed is incorporated predominantly into the ribulose 1,5-bisphosphate cycle as in autotrophic organisms [I, 21. The observation that a cosubstrate (pyruvate or acetate) is needed for growth on formate [3] has not been related to the carbon metabolism of the organism so far.In an earlier paper [4] we reported that the attainment of the thermodynamically expected equilibrium (1) is catalyzed by the enzyme in both directions and that COZ is reduced to formate by NADH under catalysis of the enzyme. COZ (not bicarbonate) has been identified as the active species in this equilibrium.As observed for all other complex flavoproteins, formate dehydrogenase catalyzes several other nonphysiological oxidation/reduction reactions because its chromophoric groups interact with various electron donors and acceptors : dyes, oxygen, ferricyanide. The resulting reactions can be separated into two groups,

The direct reduction of C02 to formate is catalysed by formate : NAD oxidoreductase in the presence of substrate amounts of NADH. Proof for this reaction is supplied by the detection of a CO2-dependent NADH oxidation, and by the identification of ['4C]formate as the product of a NADH-dependent reduction of ['4C]carbonate.The enzyme-catalysed COZ reduction by NADH attains the equilibrium predicted by thermodynamic considerations, a state which is also reached from the formate side. The Michaelis constant for COZ is about 40 mM indicating the low affinity of the enzyme for this substrate. The corresponding value for formate is 0.1 mM. Under the special conditions employed the enzyme catalyses the formate oxidation about 30 times faster than the CO2 reduction. That C02 and not HCOT is the active species in the reduction was shown by comparing the pH dependency of the velocities of the forward and back reactions and by observing the kinetics of COz reduction during the simultaneous attainment of the COz-HCOT equilibrium.The physiological pathway of reductive CO2 assimilation was long thought to be indirect, using an acceptor molecule : carboxylation and afterwards reduction of the carboxyl group as in the reductive pentose phosphate pathway [1,2] or in the reductive carboxylic acid cycle [3,4]. The first C02-reduction leading to formate as an intermediate was found by Thauer et al. in Clostridium kluyveri [5,6]. This socalled reductive monocarboxylic acid cycle includes a ferredoxin-dependent reductive carboxylation of acetyl-CoA to pyruvate and the CoA-dependent cleavage of pyruvate to acetyl-CoA and formate. All these COZ reductions are indirect.Reduced ferredoxin (G = -0.40 V, see [7]), the most likely reductant for C02 (C02/formate: Ed = -0.42 V, see [S]), was found to act in this way in Clostridium pusteuriunum [9, lo]. The impossibility of using it in substrate amounts was the main reason for coupling it with a reducing regenerating system which contained hydrogen (PI,, = 1 atm) and ferredoxin hydrogenase. Its conditions approximate those of the standard hydrogen electrode, so that the real reduction potential was about -0.43 V. Under these conditions the equilibrium of the reduction of CO;? to formate by hydrogen had been shifted towards the formate side compared to a reduction by ferredoxin itself under standard conditions. Enw-ymr. Formate dchydrogenase or formate: NAD oxidoreductase (EC 1.2.1.2).Until 1970 many authors believed NAD(P)H to be out of question as a reductant for C02 because of its standard reduction potential (G = -0.32 V) (for instance Ljungdahl and Wood [ll]). Later Thauer proved that NADPH acts physiologically as a reductant for C02 in Clostridium therrnouceticum [I 2,131. In examining this reaction a NADPH regenerating system similar to that mentioned above was used, so that in this case COZ was also reduced by hydrogen. This means that C02 reduction did not work under the standard conditions ( Thermodynamically, the COZ reduction by substrate amounts of NAD(P)H is to be expected. The ...

The present paper describes a multicentre evaluation of a one step enzyme-immunoassay for the determination of cortisol in serum or plasma. Data from the investigation were analysed in terms of imprecision, detection limit, and correlation with other test methods.Within-run and between-run imprecisions (coefficient of variation) of Enzymun-Test® Cortisol were less than 8% and 12%, respectively. The detection limit was 30 nmol/1 (11 g/l). With the exception of prednisolone, only low interference was found with other endogenous steroids. A good correlation between Enzymun-Test® Cortisol and HPLC, LIA, FPIA and RIA was registered, although the latter two methods showed a scattering of regression lines from the different evaluators.The results show that Enzymun-Test® Cortisol can be recommended as an alternative for the measurement of cortisol. As the method is calibrated against isotope dilution-mass spectrometry, results obtained with Enzymun-Test® Cortisol are in agreement with the reference method.

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This enzyme immunoassay (EIA) of human prolactin (hPRL) involves incubation of sample and anti-hPRL antibodies conjugated to horseradish peroxidase (EC 1.11.1.7) in tubes coated with a second antibody to hPRL. The test can be performed within 60 min. No reaction of the antibodies with human placental lactogen and human somatotropin is detectable. The presence of detergent allows assay of both serum and plasma. Precision was improved by including polyethylene glycol in the reaction mixture. To optimize analytical recovery, we added protease inhibitor. Assay of the EIA standards shows good correlation with results for World Health Organization reference preparations. The measurable range is 1 to 400 micrograms/L. Intra- and interassay CVs are about 5%. Comparisons with two RIAs and two other EIAs show reasonably good correlations. The components of our EIA are stable for 18 months.