Reduced Benzyl Viologen Research Articles

Characterization of a corticosteroid 21-dehydroxylase from the intestinal anaerobic bacterium, Eubacterium lentum.

An oxygen-sensitive corticosteroid 21-dehydroxylase has been characterized in cell extracts of Eubacterium lentum. The enzyme was highly specific for corticosteroids containing and alpha-ketol structure and required FMNH2 or reduced benzyl viologen for activity. The enzyme used deoxycorticosterone, deoxycortisol, dehydrocorticosterone, and corticosterone as substrates. Substrate saturation kinetics using [3H]corticosterone yielded an apparent Km of 7.35 microM and a Vmax of 15.4 nmol (11 beta-[3H]hydroxyprogesterone) formed per hr x mg protein-1. 21-Dehydroxylase activity was inhibited by both water-soluble and lipophilic metal ion chelators. NADH: flavin oxidoreductase and 21-dehydroxylase activities were separated by anaerobic DEAE-cellulose and Sepharose 6B chromatography. 21-Dehydroxylase had a relative weight of 582,000 as determined by Sepharose 6B chromatography. There was a 7-fold increase in the rate of 21-dehydroxylation of [3H]deoxycorticosterone in whole cell suspensions of E. lentum sparged with H2 as compared to argon gas.

The role of the membrane-bound hydrogenase in the energy-conserving oxidation of molecular hydrogen by Escherichia coli.

H2-dependent reduction of fumarate and nitrate by spheroplasts from Escherichia coli is coupled to the translocation of protons across the cytoplasmic membrane. The leads to H+/2e- stoicheiometry (g-ions of H+ translocated divided by mol of H2 added) is approx. 2 with fumarate and approx. 4 with nitrate as electron acceptor. This proton translocation is dependent on H2 and a terminal electron acceptor and is not observed in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone and the respiratory inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide. H2-dependent reduction of menadione and ubiquinone-1 is coupled to a protonophore-sensitive, but 2-n-heptyl-4-hydroxy-quinoline N-oxide-insensitive, proton translocation with leads to H+/2e- stoicheiometry of approx. 2. H2-dependent reduction of Benzyl Viologen (BV++) to its radical (BV+) liberates protons at the periplasmic aspect of the cytoplasmic membrane according to the reaction: H2 + 2BV++ leads to 2H+ + 2BV+. It is concluded that the effective proton translocation observed in the H2-oxidizing segment of the anaerobic respiratory chain of Escherichia coli arises as a direct and inevitable consequence of transmembranous electron transfer between protolytic reactions that are spatially separated by a membrane of low proton-permeability.

Formate dehydrogenase mutants of Salmonella typhimurium: A new medium for their isolation and new mutant classes

We have designed a new medium for the differentiation of mutants of Salmonella typhimurium defective in the ability to reduce nitrate with formate, and have characterized 24 formate dehydrogenase (FDH) mutants isolated on this medium. The mutants were assayed for the ability to use formate to reduce benzyl viologen and phenazine methosulfate, and were mapped by means of conjugation and P22-mediated transduction. Mutants lacking the ability to reduce either dye were found to map at three distinct sites: at a site co-transducible with xyl (presumably fdhA), at a site or sites between 13U and 33U, but not co-transducible with aroA, bio, purB, pyrC, or pyrD (near, but not identical with fdhB), and at asite 10-20% co-transducible with pyrE, for which we suggest the designation fdhC. Six mutant isolates reduced benzyl viologen, but not phenazine methosulfate. They retained the ability to produce nitrite during growth with nitrate. They mapped between 83U and 89U, but no co-transduction was found with metE, glnA, metB, or argH. The combined biochemical and genetic data suggest the existence of a gene in this area which is essential for the reduction of nitrate with formate, but not for formate hydrogenlyase activity or for nitrate reductase activity.

Regulation of 2-oxoglutarate dehydrogenase synthesis in Citrobacter freundii by traces of oxygen in commercial nitrogen gas and by glutamate.

Glutamate induced the synthesis of 2-oxoglutarate dehydrogenase 50-fold during anaerobic growth of Citrobacter freundii and, in the absence of glutamate, this enzyme was even more active in cultures sparged with N2/CO2(95:5, v/v). Enzyme synthesis was partially repressed when the inlet gas was passed through heated copper but totally repressed when the inlet gas was passed through alkaline pyrogallol and reduced benzyl viologen (a treatment which would remove CO2 as well as O2). Fumarate hydratase activity also decreased but alcohol dehydrogenase and the sum of the succinate dehydrogenase and fumarate reductase activities increased when residual O2 was removed from the sparging gas. Soluble cytochromes a1 and c552.5 were detected in rigorously anaerobic cultures. Thus traces of O2 which contaminate commercial compressed N2 are sufficient to induce 2-oxoglutarate dehydrogenase synthesis and to affect significantly the synthesis and incorporation of respiratory chain components into the cytoplasmic membrane.

Comparison of the membrane-bound and detergent-solubilised hydrogenase from Paracoccus denitrificans. Isolation of the hydrogenase

The hydrogenase from Paracoccus denitrificans is an integral membrane protein and has been solubilised by Triton X-100. The membrane-bound and detergent-solubilised forms of the enzyme have been compared. Both forms of the enzyme show a pH optimum for reduction of benzyl viologen at pH 8.5–9.0 and are both inhibited by concentrations of NaCl greater than 30 mM. An Arrhenius plot of the activity of hydrogenase in the membrane shows no ‘break’. The form of the Arrhenius plot and the activation energy are not significantly changed on solubilisation of the enzyme. The K m and V values for benzyl viologen, methyl viologen and H 2 are unaltered when the enzyme is extracted from the membrane. Therefore, solubilisation of hydrogenase from the membrane by Triton X-100 is unlikely to disrupt the native conformation of the enzyme. The detergent-solubilised hydrogenase has subsequently been purified using ammonium sulphate precipitation, sucrose density gradient centrifugation and chromatography on hydroxyapatite. The overall yield of activity is 23%, with a final purification of over 100-fold.

Nitrate reductase and glutamate dehydrogenase of the red alga Porphyridium aerugineum

Assimilatory nitrate reductase and glutamate dehydrogenase of the red alga Porphyridium aerugineum have been studied. Nitrate reductase exhibits a pH optimum at approx. 7.75; it is functional with reduced benzyl viologen, FMN; FAD, NADH and is not functional with NADPH. In crude extracts of cells grown on nitrate as the sole nitrogen source, a partially inactive form of nitrate reductase occurs. This inactive enzyme can be activated by heating at 50°C. Glutamate dehydrogenase of P. aerugineum is strictly dependent on NADPH.

Solubilization and properties of a particulate hydrogenase from Methanobacterium strain G2R.

Mechanical disruption of cells of Methanobacterium strain G2R resulted in a 78-fold increase in the specific activity of the hydrogenase as measured by the benzyl viologen reduction assay. Approximately 50% of the activity in disrupted cells was associated with the particulate fraction. Between 69 and 85% of the particulate hydrogenase was released by treatment with the detergents Triton X-100, deoxycholate, and octyl-beta-d-glucopyranoside. The relative electrophoretic mobilities of the soluble hydrogenases were identical, indicating that G2R possessed a single electrophoretically distinct hydrogenase. The particulate enzyme was inactivated by oxygen and could be reactivated with dithionite or glucose plus glucose oxidase. The enzyme had a pH optimum of 8.5 and resisted heating at 52 but not 77 degrees C. A number of nonspecific dyes, flavin adenine dinucleotide, and riboflavin 5'-phosphate were effective electron acceptors; oxidized nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, and factor 420 were apparently not reduced. Hydrogenase activity was inhibited by p-hydroxymercuribenzoate, cyanide, chloroform, and chloramphenicol. The molecular weight of the solubilized enzyme was 900,000, with subunits of molecular weights 38,500, 50,700, and approximately 80,000. It is suggested that, in intact cells of G2R, the large hydrogenase complex is loosely bound to the cell wall or membrane.

Hydrogen-Dependent Growth of Escherichia coli in Anaerobic Respiration and the Presence of Hydrogenases with Different Functions

E. coli K10 was found to grow anaerobically on molecular hydrogen by reducing nitrate, fumarate, and trimethylamine N-oxide when peptone was added to the culture medium. Molar growth yields based on consumed hydrogen estimated from the amounts of reduction products were all 7.8 g cells/mol, suggesting that 1 mol of ATP was produced in the oxidation of 1 mol of hydrogen. Hydrogenase activity measured in terms of hydrogen evolution was several times higher in cells grown on glucose than in cells grown on hydrogen in the presence of fumarate and trimethylamine N-oxide, while hydrogenase activity measured in terms of hydrogen uptake was unchanged in both cases. The ratio of hydrogenase activities measured in terms of hydrogen uptake and evolution was also high in the extract and centrifugal fractions from cells grown in hydrogen. The soluble fraction and trypsin digest of the precipitate at 100,000 X g were subjected to polyacrylamide disc gel electrophoresis and hydrogenase bands were stained by reduction of benzyl viologen with hydrogen and by oxidation of reduced methyl viologen. The resulting patterns suggest that multiple forms of hydrogenase are present and that the amounts of forms functioning in hydrogen evolution were greatly decresed in cells grown on hydrogen in the presence of acceptors.

Nitrate Reductase Deficient Mutants of Chlamydomonas reinhardii. Biochemical Characteristics

Two mutants of Chlamydomonas reinhardii that cannot grow with nitrate as nitrogen source have been examined biochemically. Each mutant differs from the wild-type strain by a single mutation in a Mendelian gene. One mutant (14/15) carries a mutation in a gene designated nitB. This organism took up nitrate and converted nitrate-nitrogen to insoluble-nitrogen; nevertheless no enzymic activities associated with nitrate reductase (NADH-nitrate reductase, reduced benzyl viologen nitrate reductase or inducible NADH-cytochrome c reductase) could be demonstrated in frozen/thawed cells or cell-free extracts. At high nitrate concentrations (5 mM) the rate of nitrate uptake by this mutant [V max 59 nmol (mg dry wt)–1 h–1] was considerably lower than that of wild-type [V max 920 nmol (mg dry wt)–1 h–1]. It is concluded that nitB is probably a regulatory gene for nitrate reductase. The other mutant (17/4) has a mutation in a gene designated nitA. This organism did not take up nitrate and had no NADH-nitrate reductase or inducible NADH-cytochrome c reductase activities. Frozen/thawed cells and cell-free extracts had reduced benzyl viologen nitrate reductase activity. The enzyme from the nitA mutant eluted from a Sepharose 4B column later than wild-type enzyme; it was therefore probably of lower molecular weight. The nitA mutation of C. reinhardii closely resembles the nit-3 mutation of Neurospora crassa and, like this mutation, it probably results in loss of the NADH combining sub-unit of nitrate reductase. Strain 137c of C. reinhardii had no enzymic activities associated with nitrate reductase and did not take up nitrate. This organism may have mutations in both the nitA and nitB loci.

Reactivity of Desulfovibrio gigas hydrogenase toward artificial and natural electron donors or acceptors

A purified preparation of hydrogenase from D. gigas was inactive toward ferredoxin, flavodoxin or rubredoxin in the absence of cytochrome c3 (M.W. 13,000), in an atmosphere of hydrogen, although direct reduction of benzyl viologen or FMN was possible. The hydrogen evolution reaction from dithionite was possible with methyl viologen. The same reaction also occured with cytochrome c3 (M.W. 13,000) or cytochrome c3 (M.W. 26,000). Addition of either ferredoxin or flavodoxin did not accelerate the reaction.

Fumarate reductase of Clostridium formicoaceticum

When Clostridium formicoaceticum was grown on fumarate or L-malate crude cell extracts contained a high fumarate reductase activity. Using reduced methyl viologen as electron donor the specific activity amounted to 2-3.5 U per mg of protein. Reduced benzyl viologen, FMNH2 and NADH could also serve as electron donors but the specific activities were much lower. The NADH-dependent activity was strictly membrane-bound and rather labile. Its specific activity did not exceed 0.08 U per mg of particle protein. Fumarate reductase activity was also found in cells of C. formicoaceticum grown on fructose, gluconate, glutamate and some other substrates. The methyl viologen-dependent fumarate reductase activity could almost completely be measured with intact cells whereas only about 25% of the cytoplasmic acetate kinase activity was detected with cell suspensions. The preparation of spheroplasts from cells of C. formicoaceticum in 20 mM HEPES-KOH buffer containing 0.6 M sucrose and 1 mM dithioerythritol resulted in the specific release of 88% of the fumarate reductase activity into the spheroplast medium. Only small amounts of the cytoplasmic proteins malic enzyme and acetate kinase were released during this procedure. There results indicate a peripheral location of the fumarate reductase of C. formicoaceticum on the membrane.

Oxidoreductases involved in cell carbon synthesis of Methanobacterium thermoautotrophicum.

Cell-free extracts of Methanobacterium thermoautotrophicum were found to contain high activities of the following oxidoreductases (at 60 degrees C): pyruvate dehydrogenase (coenzyme A acetylating), 275 nmol/min per mg of protein; alpha-ketoglutarate dehydrogenase (coenzyme A acylating), 100 nmol/min per mg; fumarate reductase, 360 nmol/min per mg; malate dehydrogenase, 240 nmol/min per mg; and glyceraldehyde-3-phosphate dehydrogenase, 100 nmol/min per mg. The kinetic properties (apparent V(max) and K(M) values), pH optimum, temperature dependence of the rate, and specificity for electron acceptors/donors of the different oxidoreductases were examined. Pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase were shown to be two separate enzymes specific for factor 420 rather than for nicotinamide adenine dinucleotide (NAD), NADP, or ferredoxin as the electron acceptor. Both activities catalyzed the reduction of methyl viologen with the respective alpha-ketoacid and a coenzyme A-dependent exchange between the carboxyl group of the alpha-ketoacid and CO(2). The data indicate that the two enzymes are similar to pyruvate synthase and alpha-ketoglutarate synthase, respectively. Fumarate reductase was found in the soluble cell fraction. This enzyme activity coupled with reduced benzyl viologen as the electron donor, but reduced factor 420, NADH, or NADPH was not effective. The cells did not contain menaquinone, thus excluding this compound as the physiological electron donor for fumarate reduction. NAD was the preferred coenzyme for malate dehydrogenase, whereas NADP was preferred for glyceraldehyde-3-phosphate dehydrogenase. The organism also possessed a factor 420-dependent hydrogenase and a factor 420-linked NADP reductase. The involvement of the described oxidoreductases in cell carbon synthesis is discussed.

Attempts to increase viable count recovery of human supragingival dental plaque.

Studies of the microbiota of dental plaque have been hampered by an inability to cultivate all or even a majority of microorganisms present in this site. Failure to recover the microorganisms could be attributed to at least 3 types of losses; inadequate dispersion, adhesion to glassware used in dilution and spreading, and an inability of any single cultural environment to recover all of the resident microorganisms. Clumps of microorganisms could be more effectively dispersed by sonic oscillation than tissue grinders. Anaerobic sonication proved to be significantly more effective in maintaining viability of plaque isolates than aerobic sonication while dispersion in dilute salt solutions permitted recovery of greater numbers of organisms than dispersion in broth.About 5% of the organisms were lost due to adsorption to glassware as determined by pouring molten agar media over the used apparatus and counting the resultant colonies.Optimum recovery of plaque organisms occurred in this study when samples were dispersed by anaerobic sonic oscillation in pre‐reduced anaerobically sterilized 1/4 strength Ringer's solution supplemented with 1% sodium metaphosphate, 0.05% L cysteine, and 0.0001% resazurin. When the resulting suspension was anaerobically serially diluted, and plated on trypticase soy 5% sheep blood agar plates which were incubated in Brewer jars containing 80% N2, 10% H2. and 10% CO2, 60% of the total microscopic cell count could be recovered.The use of additional primary isolation environments including blood agar plates incubated aerobically, as well as trypticase soy and reduced benzyl viologen roll tubes increased the recovery an additional 15%. Thus an average of 75% of the microscopic count could be cultivated. An additional 5% was lost due to adsorption to glassware and an average of 10% of the microbiota remained undispersed in clumps.

NADPH- and NADH-nitrate reductases from soybean leaves

Two nitrate reductase activities from young first leaves of soybeans have been isolated and separated by DEAE-cellulose column chromatography. They have different mobilities when electrophoresed on polyacrylamide gels. These two enzymes have been designated NADH-nitrate reductase and NADPH-nitrate reductase because of their differing affinities for reduced pyridine nucleotides. Five to ten millimolar cysteine was required in the extraction medium to achieve maximum activity of the two nitrate reductases. NADPH-nitrate reductase was about 50% as active as NADH-nitrate reductase. NADPH-nitrate reductase was detected in extracts prepared without cysteine, whereas the NADH enzyme was not found in the absence of cysteine. Both nitrate reductases utilized NADH, NADPH, FADH 2 (reduced flavin-adenine dinucleotide), reduced methyl viologen, and reduced benzyl viologen as electron donors. When NADPH was used as the electron donor for NADPH-nitrate reductase, FAD (flavin-adenine dinucleotide) was required for maximum activity, but FAD did not enhance the activity of NADH-nitrate reductase. NADH-nitrate reductase had a molecular weight of 330,000 by gel filtration, a pH optimum of 6.5 with NADH, K m (KNO 3) of 0.11 m m, and K m (NADH) of 8.1 μ m. NADPH-nitrate reductase had a 220,000 molecular weight as determined by gel filtration, a pH optimum of 6.5 in phosphate and of 6.2 in morpholinoethane sulfonic acid buffer, a K m (KNO 3) of 4.5 m m, and a K m (NADPH) of 1.5 μ m. Potassium cyanide and sodium azide inhibited both nitrate reductases, while potassium fluoride did not inhibit either.

Isolation and some properties of NAD+ reductase of the green photosynthetic bacterium Prosthecochloris aestuarii.

NAD+ reductase of the green photosynthetic bacterium Prosthecochloris aestuarii was isolated and purified by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration. This enzyme is an FAD-containing flavoprotein and has absorption maxima at 485 (shoulder0 452, 411, and 385 nm (the 411 nm band is due to cytochrome). The molecular weight of the enzyme as determined by gel filtration using Sephadex G-200 is 119,000. The enzyme catalyzes the reduction of NAD+ and NADP+ by photoreduced spinach ferredoxin or reduced benzyl viologen...

Biochemical studies on the Nit mutants of Neurospora crassa

One allele at each of the five nit loci in Neurospora crassa together with the wild type strain have been compared on various nitrogen sources with regard to (i) their growth characteristics (ii) the level of nitrate reductase and its associated activities (reduced benzyl viologen nitrate reductase and cytochrome c reductase) (iii) the level of nitrate reductase and (iv) their ability to take up nitrite from the surrounding medium. Results are consistent with the hypothesis that nit-3 is the structural gene for nitrate reductase, nit-1 specifies in part of molybdenum containing moiety which is responsible for the nit-3 gene product dimerising to form nitrate reductase, nit-4 and nit-5 are regulator genes whose products are involved in the induction of both nitrate reductase and nitrite reductase and nit-2 codes for a generalised ammonium activated repressor protein. Studies on the induction of nitrate reductase (and its associated activities) and nitrite reductase in wild type, nit-1 and nit-3 in the presence of either nitrate or nitrite suggest that each enzyme may be regulated independently of the other and that nitrite could be true co-inducer of the assimilatory pathway. Nitrite uptake experiments with nit-2, nit-4 and nit-5 strains show that whereas nit-4 and nit-5 are freely permeable to this molecule, it is unable to enter the nit-2 mycelium.

Proton translocation and the respiratory nitrate reductase of Escherichia coli.

Stoicheometries and rates of proton translocation associated with respiratory reduction of NO3- have been measured for spheroplasts of Escherichia coli grown anaerobically in the presence of NO3-. Observed stoicheiometries [leads to H+/NO3- ratio; P. Mitchell (1966) Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation, Glynn Research, Bodmin] were approx. 4 for L-malate oxidation and approx. 2 for succinate, D-lactate and glycerol oxidation. Measurements of the leads to H+/2e- ratio with formate as the reductant and oxygen or NO3- as the oxidant were complicated by pH changes associated with formate uptake and CO2 formation. Nevertheless, it was possible to conclude that the site of formate oxidation is on the inner aspect of the cytoplasmic membrane, that the leads to H+/O ratio for formate oxidation is approx. 4, and that the leads to H+/NO3- ratio is greater than 2. Measurements of the rate of NO3- penetration into osmotically sensitive spheroplasts demonstrated an electrogenic entry of NO3- anion. The permeability coefficient for nitrate entry at 30 degrees C was between 10(-9) and 10(-10) cm- s(-1). The calculated rate of nitrate entry at the concentration typically used for the assay of nitrate reductase (EC 1.7.99.4) activity was about 0.1% of that required to support the observed rate of nitrate reduction by reduced Benzyl Viologen. Measurements of the distribution of nitrate between the intracellular and extracellular spaces of a haem-less mutant, de-repressed for nitrate reductase but unable to reduce nitrate by the respiratory chain, showed that, irrespective of the presence or the absence of added glucose, nitrate was not concentrated intracellularly. Osmotic-swelling experiments showed that the rate of diffusion of azid anion across the cytoplasmic membrane is relatively low in comparison with the fast diffusion of hydrazoic acid. The inhibitory effect of azide on nitrate reductase was not altered by treatments that modify pH gradients across the cytoplasmic membrane. It is concluded that the nitrate-reducing azide-sensitive site of nitrate reductase is located on the outer aspect of the cytoplasmic membrane. The consequences of this location for mechanisms of proton translocation driven by nitrate reduction are discussed, and lead to the proposal that the nitrate reductase of the cytoplasmic membrane is vectorial, reducing nitrate on the outer aspect of the membrane with 2H+ and 2e- that have crossed from the inner aspect of the membrane.

Nitrate reductase system in Staphylococcus aureus wild type and mutants.

Respiratory nitrate reductase with lactate as a hydrogen donor has been studied in cells and spheroplast preparations of wild type and heme-deficienct mutants of Staphylococcus aureus. The activity is rapidly induced when suspensions of aerobically grown cells are incubated without aeration in a complete medium with nitrate. In ruptured spheroplast preparations, the activity with lactate as the donor is located in the membrane fraction, whereas at least 50% of the activity assayed with reduced benzyl viologen is in the cytoplasm. The reductase is inhibited by azide and cyanide, and the lactate-linked system is also sensitive to oxamate, 2-heptyl-4-hydroxyquinoline-N-oxide, dicoumarol, and p-chloromercuribenzoate. An inactive form of the reductase is apparently made during induction with tungstate; this can be activated by subsequent incubation with molybdate in the presence of chloramphenicol. Nitrate reductase activity with reduced benzyl viologen as the donor is induced in suspensions of heme-deficient mutants in the presence or absence of heme. The proportion of cytoplasmic activity is increased in the absence of heme. The staphylococcal nitrate reductase has many of the characteristics commonly associated with the respiratory enzyme in other organisms, but the apparent predominance of cytoplasmic activity is unusual.

The effects of immune and non-immune rabbit sera on the various activities of spinach ( Spinacea oleracea L.) nitrate reductase

Antibodies have been raised in rabbit against nitrate reductase purified from spinach. The three activities of the enzyme, NADH-nitrate reductase, reduced benzyl viologen (BV°)-nitrate reductase and NADH-dehydrogenase were differentially inhibited by immune serum and γ-globulin at high concentrations but stimulated at low concentrations suggesting the presence of stimulating antibodies. Non-immune whole serum but not its γ-globulin stimulated all activities of a freshly prepared enzyme, particularly BV°-nitrate reductase. A standard enzyme was used to quantify the relative anti-nitrate reductase content of the whole serum and γ-globulin at 15.8 and 70.9 respectively.

The non-enzymic reduction of nitrite by benzyl viologen (free-radical) in the presence and absence of ammonium sulphate

Some similarity is inferred between the reaction or reduced benzyl viologen with undissociated nitrous acid, which is significant at pH values below 7 and that with the undissociated product of nitrite ion and ammonium sulphate; presumably ammonium nitrite. This would explain why the presence of ammonium sulphate appreciably offsets the effects of decreasing pH and also the exponential relationship between rate of nitrite loss and ammonium sulphate concentration. There are other features of the reaction which cannot be explained at present, especially with regard to the degree of reduction of benzyl viologen. It is nevertheless apparent that a complex non-enzymic reaction yielding several products occurs when ammonium sulphate is present and that the presence of likely residual quantities after its use in enzyme purification may cause serious errors in enzyme assay.