Determination of 1,3-dichloropropanol in soy and related sauces by using gas chromatography/mass spectrometry.

A gas chromatography/mass spectrometry method for 3-MCPD in foods and food ingredients was modified for the determination of 1,3-DCP in soy and related sauces. The method was validated by using a blank soy sauce. The detection limit, quantitation limit and recoveries were determined, and identities were confirmed by mass spectrometry on the basis of analyses of test portions spiked with 1,3-DCP at 10, 25, 50 and 100 ng g(-1). The spiked test portions were quantitated by using an internal standard calibration curve. For the spiked test portions, the mean internal standard-corrected recovery for 1,3-DCP was 100% with a relative standard deviation of 1.32%. The limits of detection and quantitation were determined as 0.055 and 0.185 ng g(-1), respectively. The method also was compared with a headspace GC/MS method recently developed by the UK's Central Science Laboratory. Results from the method comparison showed that the recoveries for the spiked test portions, as well as the amounts of 1,3-DCP found in the retail products, were comparable.

Survey of chloropropanols in soy sauces and related products.

A survey of soy sauces and related products available in the USA was conducted to determine the levels of 3-monochloropropane-1,2-diol (3-MCPD) and 1,3-dichloro-2-propanol (1,3-DCP) in these products. Fifty-five retail samples were purchased and analysed for 3-MCPD. 3-MCPD determinations were made according to a gas chromatography/mass spectrometry method validated by a collaborative trial. Eighty-five per cent of the samples analysed contained greater than the detection limit of 0.005 ppm (microg g(-1)) for 3-MCPD. Thirty-three per cent contained greater than 1 ppm; the highest level was 876 ppm 3-MCPD. Thirty-nine of the samples analysed for 3-MCPD also were analysed for 1,3-DCP by using a modified method developed and validated in-house. Fifty-six per cent of the samples analysed for 1,3-DCP contained greater than the detection limit of 0.055 ppb (ng g(-1)) for 1,3-DCP; the highest level was 9.8 ppm 1,3-DCP. Products manufactured in Asia contained the highest chloropropanol levels.

The C-terminus of dUTPase: observation on flexibility using NMR.

The dynamics of the C-terminus of the dUTPases from Escherichia coli and equine infectious anaemia virus (EIAV) were studied by 1H-(15)N nuclear magnetic resonance spectroscopy. The two enzymes differ with regard to flexibility in the backbone of the 15 most C-terminal amino acid residues, some of which are conserved and essential for enzymic activity. In the bacterial enzyme, the residues closest to the C-terminus are highly flexible and display a correlation time in the nanosecond time range. No similar high flexibility could be detected for the C-terminal part of EIAV dUTPase, indicating a different time range of flexibility.

Flavobacterium psychrophilum, invasion into and shedding by rainbow trout Oncorhynchus mykiss.

The infection route of Flavobacterium psychrophilum into rainbow trout Oncorhynchus mykiss was studied using bath and cohabitation challenges as well as oral challenge with live feed as a vector. Additionally, the number of bacterial cells shed by infected fish into the surrounding water was determined in the cohabitation experiment and in challenge experiments at 3 different water temperatures. The experiments showed that skin and skin mucus abrasion dramatically enhanced the invasion of F. psychrophilum into the affected fish in bath and cohabitation challenges. Disruption of the skin is discussed as an important invasion route for F. psychrophilum into the fish. The shedding rate of F. psychrophilum by infected fish was associated with water temperature and the mortality of the infected fish. High numbers of F. psychrophilum cells were released into the water by dead rainbow trout during a long time period compared to the numbers of cells shed by live fish. The results emphasise the importance of removing dead and moribund fish from rearing tanks in order to diminish the infection pressure against uninfected fish in commercial fish farms. In immunohistochemical examinations of organs and tissues of orally infected fish, F. psychrophilum cells were detected in only 1 fish out of 31 studied. Mortality of the orally challenged fish was not observed in the experiment.

Transient kinetics of ligand binding and role of the C-terminus in the dUTPase from equine infectious anemia virus.

Transient kinetics of the equine infectious anemia virus deoxyuridine 5'-triphosphate nucleotide hydrolase were characterized by monitoring the fluorescence of the protein. Rate constants for the association and dissociation of substrate and inhibitors were determined and found to be consistent with a one-step mechanism for substrate binding. A C-terminal part of the enzyme presumed to be flexible was removed by limited trypsinolysis. As a result, the activity of the dUTPase was completely quenched, but the rate constants and fluorescent signal of the truncated enzyme were affected only to a minor degree. We conclude that the flexible C-terminus is not a prerequisite for substrate binding, but indispensable for catalysis.

Crystal structure of dUTPase from equine infectious anaemia virus; active site metal binding in a substrate analogue complex.

The X-ray structures of dUTPase from equine infectious anaemia virus (EIAV) in unliganded and complexed forms have been determined to 1.9 and 2.0 A resolution, respectively. The structures were solved by molecular replacement using Escherichia coli dUTPase as search model. The exploitation of a relatively novel refinement approach for the initial model, combining maximum likelihood refinement with stereochemically unrestrained updating of the model, proved to be of crucial importance and should be of general relevance.EIAV dUTPase is a homotrimer where each subunit folds into a twisted antiparallel beta-barrel with the N and C-terminal portions interacting with adjacent subunits. The C-terminal 14 and 17 amino acid residues are disordered in the crystal structure of the unliganded and complexed enzyme, respectively. Interactions along the 3-fold axis include a water-containing volume (size 207 A3) which has no contact with bulk solvent. It has earlier been shown that a divalent metal ion is essential for catalysis. For the first time, a putative binding site for such a metal ion, in this case Sr2+, is established. The positions of the inhibitor (the non-hydrolysable substrate analogue dUDP) and the metal ion in the complex are consistent with the location of the active centre established for trimeric dUTPase structures, in which subunit interfaces form three surface clefts lined with evolutionary conserved residues. However, a detailed comparison of the active sites of the EIAV and E. coli enzymes reveals some structural differences. The viral enzyme undergoes a small conformational change in the uracil-binding beta-hairpin structure upon dUDP binding not observed in the other known dUTPase structures.

The refined structure of dUTPase from Escherichia coli.

Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase, E.C. 3.6. 1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate and is involved in nucleotide metabolism and DNA synthesis. A crystal of the recombinant E. coli enzyme, precipitated from polyethylene glycol mixtures in the presence of succinate at pH 4.2, was used to collect synchrotron diffraction data to 1.9 A resolution, in space group R3, a = b = 86.62, c = 62.23 A. Mercury and platinum derivative data were collected at wavelengths to optimize the anomalous contribution. The resulting 2.2 A MIRAS phases differed from the final set by 40 degrees on average and produced an excellent map which was easy to interpret. The model contains 132 water molecules and refined to an R value of 13.7%. 136 residues have clear electron density out of 152 expected from the gene sequence. The 16 C-terminal residues are presumably disordered in the crystal lattice. The monomer is a 'jelly-roll' type, containing mostly beta-sheet and only one short helix. The molecule is a tight trimer. A long C-terminal arm extends from one subunit and encompasses the next one within the trimer contributing to its beta-sheet. Conserved sequence motifs common among dUTPases, previously suggested to compose the active site and confirmed in a recent study of the dUDP complex, are located at subunit-subunit interfaces along the threefold axis, in parts of the beta-sheet and in loop regions. A similar molecular architecture has recently been found in two other trimeric dUTPases.

The complete triphosphate moiety of non-hydrolyzable substrate analogues is required for a conformational shift of the flexible C-terminus in E. coli dUTP pyrophosphatase.

The molecular mechanism of substrate analogue interaction with Escherichia coli dUTPase was investigated, using the non-hydrolyzable 2'-deoxyuridine 5'-(alpha,beta-imido)triphosphate (alpha,beta-imido-dUTP). Binding of this analogue induces a difference in the far UV circular dichroism (CD) spectrum arguing for a significant change in protein conformation. The spectral shift is strictly Mg2+-dependent, does not appear with dUDP instead of alpha,beta-imido-dUTP and is not elicited if the flexible C-terminal arm is deleted from the protein by limited tryptic digestion. Involvement of the C-terminal arm in alpha,beta-imido-dUTP binding is consistent with the finding that this analogue protects against tryptic hydrolysis at Arg-141. Near UV CD of ligand-enzyme complexes reveals a characteristic difference in the microenvironments of enzyme-bound dUDP and alpha,beta-imido-dUTP, a difference not observable in C-terminally truncated dUTPase. The results suggest that (i) closing of the active site during the catalytic cycle, through the movement of the C-terminal arm, requires the presence of the complete triphosphate moiety of the substrate in complex with Mg2+, and (ii) after catalytic cleavage the active site pops open to facilitate product release.

Kinetic properties and stereospecificity of the monomeric dUTPase from herpes simplex virus type 1.

Kinetic properties of the monomeric enzyme dUTPase from herpes simplex virus type 1 (HSV) were investigated and compared to those previously determined for homotrimeric dUTPases of bacterial and retroviral origins. The HSV and Escherichia coli dUTPases are equally potent as catalysts towards the native substrate dUTP with a kcat/K(M) of about 10(7) M(-1) s(-1) and a K(M) of 0.3 microM. However, the viral enzymes are less specific than the bacterial enzyme. The HSV and E. coli dUTPases show the same stereospecificity towards the racemic substrate analogue dUTPalphaS (2'-deoxyuridine 5'-(alpha-thio)triphosphate), suggesting that they have identical reaction mechanisms.

dUTPase from the retrovirus equine infectious anemia virus: specificity, turnover and inhibition.

The kinetic properties of dUTPase from equine infectious anemia virus (EIAV) were investigated. K(M) (1.1 +/- 0.1 microM) and k(cat) (25 s(-1)) were found to be independent of pH in the neutral pH range. Above pH 8.0, K(M) increases slightly. Below pH 6.0, the enzyme is rapidly deactivated. Detergent was found to enhance activity, leaving K(M) and k(cat) unaffected. Compared to the Escherichia coli dUTPase, the EIAV enzyme is equally potent in hydrolyzing dUTP, but less specific. Inhibition of the viral enzyme by the nucleotides dTTP, dUMP and a synthetic analogue, 2'-deoxyuridine 5'-(alpha,beta-imido)triphosphate, is stronger by one order of magnitude.

Ethyl carbamate levels resulting from azodicarbonamide use in bread.

Azodicarbonamide (ADA), a dough conditioner, is an additive approved in the US up to a maximum of 45 mg/kg in flour. The addition of 45 mg/kg of ADA was investigated and found to increase the ethyl carbamate (EC) content of commercially prepared breads by 1-3 micrograms/kg. A similar increase in EC was observed in breads baked in the laboratory with a bread machine. The increase in EC levels appears to depend on a variety of factors, most notably the concentration of ADA added and the time of fermentation. The addition of 20 mg/kg ADA caused only a slight increase, if any, in commercial products but a 2.3 micrograms/kg increase of EC in breads baked with a bread machine. When 100 mg/kg of ascorbic acid was added along with ADA, smaller EC increases were observed. Addition of urea was also found to enhance the EC content of the bread. Toasting, which was previously shown to increase EC levels, caused even larger increases when ADA or urea had been added.

The dUTPases from herpes simplex virus type 1 and mouse mammary tumour virus are less specific than the Escherichia coli enzyme.

The enzyme dUTPase catalyses the hydrolysis of dUTP to dUMP and pyrophosphate, thereby suppressing incorporation of uracil into DNA and providing a pool of dUMP, the precursor of dTTP. Hydrolysis of other nucleotides similar in structure to dUTP would conceivably be physiologically detrimental and high specificity of the reaction seems to be necessary. In this work, we characterize the substrate specificity of the dUTPases from herpes simplex virus type 1 (HSV-1) and mouse mammary tumour virus (MMTV) in comparison to the Escherichia coli enzyme. We tested dCTP, dTTP, UTP and dUDP as substrates. Significantly higher reactivity was observed for the HSV-1 enzyme with dCTP and dTTP and for the MMTV enzyme with dTTP and UTP. The lower substrate specificity of the two virus enzymes compared with the bacterial enzyme is discussed in relation to the DNA precursor metabolism during virus replication.

Crystallization and preliminary X-ray analysis of human dUTPase.

Human dUTPase, expressed in Escherichia coli, has been crystallized. Single crystals were obtained by the vapour-diffusion technique using 2-propanol and PEG 4000 as precipitants. The enzyme was co-crystallized with the substrate dUTP and a metal chelator EDTA to prevent hydrolysis of the substrate. The crystals belong to the orthorombic space group P2(1)2(1)2(1) with cell dimensions a = 67.51, b = 68.26 and c = 91.00 A. The crystallographic asymmetric unit contains one trimer of identical subunits.

Kinetic characterization of dUTPase from Escherichia coli.

The enzyme dUTPase catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate, thereby preventing a deleterious incorporation of uracil into DNA. The best known dUTPase is that from Escherichia coli, which, like the human enzyme, consists of three identical subunits. In the present work, the catalytic properties of the E. coli dUTPase were investigated in the pH range 5-11. The enzyme was found to be highly specific for dUTP and discriminated both base and sugar as well as the phosphate moiety (bound dUDP was not hydrolyzed). The second best substrate among the nucleotides serving as building blocks for DNA was dCTP, which was hydrolyzed an astonishing 10(5) times less efficiently than dUTP, a decline largely accounted for by a higher Km for dCTP. With dUTP.Mg as substrate, kcat was found to vary little with pH and to range from 6 to 9 s-1. Km passed through a broad minimum in the neutral pH range with values approaching 10(-7) M. It increased with deprotonation of the uracil moiety of dUTP and showed dependence on two ionizations in the enzyme, exhibiting pKa values of 5.8 and 10.3. When excess dUTPase was reacted with dUTP middle dotMg at pH 8, the two protons transferred to the reaction medium were released in a concerted mode after the rate-limiting step. The Mg2+ ion enhances binding to dUTPase of dUTP by a factor of 100 and dUDP by a factor of 10. Only one enantiomer of the substrate analog 2'-deoxyuridine-5'-(alpha-thio)-triphosphate was hydrolyzed by the enzyme. These results are interpreted to favor a catalytic mechanism involving magnesium binding to the alpha-phosphate, rate-limiting hydrolysis by a shielded and activated water molecule and a fast ordered desorption of the products. The results are discussed with reference to recent data on the structure of the E. coli dUTPase.UDP complex.

Screening method for the gas chromatographic/mass spectrometric determination of microgram/litre levels of bromate in bottled water.

Bromate can be formed as a by-product of ozone treatment that is sometimes used for the disinfection of municipal water supplies and bottled waters. The US Environmental Protection Agency has proposed a maximum contaminant level (MCL) of 10 micrograms/l for bromate in public drinking water. Should the proposed MCL for bromate become final, it may then be considered for adoption as a bottled water quality standard by the US Food and Drug Administration. This paper reports the development of a gas chromatographic/ mass spectrometric (GC/MS) method for the determination of parts-per-billion (microgram/l) levels of bromate (BrO3-) in bottled water. The GC/MS method was validated by using distilled and deionized Milli-Q water; detection limits, quantitation limits, and recoveries were determined and identities were confirmed by MS on the basis of analyses of test portions fortified with BrO3- at 0.8, 3.8, 7.7, 15, and 46 micrograms/l. The method also was evaluated on the basis of recoveries determined for two commercial brands of bottled water fortified with BrO3- at 3.8 and 7.7 micrograms/l and two commercial brands fortified at 0.8, 3.8, and 7.7 micrograms/l. For the Milli-Q water, recoveries ranged from 100 to 121%; for the fortified commercial products, recoveries ranged from 87 to 115%. The limits of detection and quantitation were determined to be 0.4 and 0.7 microgram/l, respectively. Several commercial brands of bottled water were analysed, and BrO3- was found in these products at levels ranging from none to 38 micrograms/l.

Determination of low nanogram/millilitre levels of volatile organic compounds in on-purpose and by-product hydrochloric acids.

Hydrochloric acid (HCl) is used in the food industry as a processing aid in the manufacture of certain foods. HCl is manufactured, either directly from inorganic sources, i.e. "on-purpose' or indirectly as a by-product from the production of organic compounds generally intended for non-food applications. By-product HCl potentially may contain traces of the manufactured product, its precursors, and other reaction by-products. Many of the potential contaminants are volatile organic compounds (VOCs) such as vinyl chloride and benzene. A study was conducted to determine whether there were differences between the levels of VOCs found in HCl samples collected from two on-purpose HCl manufacturers and two by-product HCl manufacturers. Process water used to manufacture the HCl also was analysed. Twenty-nine VOCs were determined by purge- and-trap concentration and capillary gas chromatography/mass spectrometry. Of these, nine were found in the process water and in both the by-product and on-purpose HCl samples at total VOC levels of less than 116 ng/ml. The levels of total VOCs identified in the two on-purpose HCl samples were 58 and 80 ng/ml; the levels of total VOCs identified in the two by-product samples were 41 and 115 ng/ml. The process water appeared to be the source of most of the contaminants. These results show that the levels of VOCs found in the two types of acid are comparable.

Crystal structure of the Escherichia coli dUTPase in complex with a substrate analogue (dUDP).

We have determined the structure of the homotrimeric dUTPase from Escherichia coli, completed with an inhibitor and substrate analogue, dUDP. Three molecules of dUDP are found symmetrically bound per trimer, each in a shallow cleft between adjacent subunits, interacting with evolutionary conserved residues. The interactions of the uracil ring and the deoxypentose with the protein are consistent with the high specificity of the enzyme with respect to these groups. The positions of the two phosphate groups and adjacent water molecules are discussed in relation to the mechanism and kinetics of catalysis. The role that dUTPase plays in DNA metabolism makes the enzyme a potential target for chemotherapeutic drugs: the results presented here will aid in the design and development of inhibitory compounds.

A cluster of genes encoding major isozymes of lignin peroxidase and manganese peroxidase from the white-rot fungus Trametes versicolor.

A gene cluster from the white-rot basidiomycete Trametes (Coriolus) versicolor (Tv) PRL 572 containing three structural genes, LPGIII, LPGIV and MPGI, was characterized. The genes are arranged in the same transcriptional direction, within a 10-kb region, and found to encode quantitatively dominant isozymes of lignin peroxidase (LP) and manganese peroxidase (MP). The second gene in sequence, LPGIV, predicts a 346-amino-acid (aa) mature polypeptide (36.9 kDa, pI 4.31) which is identical with the partial aa sequence information available on the LP12 isozyme (43.1 kDa, pI 3.27). The first gene, LPGIII, encodes a 341-aa polypeptide (36.1 kDa, pI 3.93) which has not been identified at the protein level. However, the similarity of LPGIV would suggest that the predicted product is an LP-type enzyme. LPGIII and LPGIV are homologous to the tandemly arranged genes LPGII and LPGI, respectively, recently described by Jönsson and Nyman [Biochim. Biophys. Acta 1218 (1994) 408-412]. The homologous genes, LPGIII/LPGII and LPGIV/LPGI, are 99% and 96% identical in sequence, respectively, and are predicted to encode identical polypeptides, since base substitutions in the predicted exons are all synonymous. The third gene, MPGI, is different in intron-exon organization and predicted to be disrupted by five rather than six introns, as are the LP genes. The deduced polypeptide, 339 aa in size (35.9 kDa, pI 4.07), is identical with the partial aa sequence information available for isozyme MP2 (44.5 kDa, pI 3.09). The MPGI- and LPGIV-encoded polypeptides are 70% identical in sequence which suggests that MP and LP from Tv may be regarded as members of the same family within the plant peroxidase superfamily. Most importantly, this study identifies a gene encoding the MP2 isozyme, and further shows that genes encoding MP and LP can be closely linked on the chromosome and may be coordinately transcribed.

Synthesis of 2'-deoxyuridine 5'-(alpha,beta-imido) triphosphate: a substrate analogue and potent inhibitor of dUTPase.

The dUDP analogue, 2'-deoxyuridine 5'-(alpha,beta-imido)diphosphate (dUPNP) was synthesized. The corresponding triphosphate analogue (dUPNPP) was prepared by enzymic phosphorylation of dUPNP using the enzyme pyruvate kinase and phosphoenolpyruvate as the phosphate donor. This method was successful in phosphorylating the imidodiphosphate analogue of 2'-deoxythymidine (dTPNP) to 2'-deoxythymidine 5'-(alpha, beta-imido)triphosphate (dTPNPP), in contradiction to a previous report. The properties of dUPNPP have been tested using the enzyme dUTPase from Escherichia coli. This enzyme, having a crucial role in nucleotide metabolism, is strictly specific for its substrate (dUTP) and catalyzes the hydrolysis of the alpha, beta-bridge, resulting in dUMP and pyrophosphate. Replacement of the alpha, beta-bridging oxygen in dUTP with an imido group resulted in a nonhydrolyzable substrate analogue and a potent competitive inhibitor of dUTPase (Ki = 5 microM). The analogue prepared (dUPNPP) may be utilized in crystallographic studies of the active site of dUTPase to provide knowledge about specific interactions involved in substrate binding and as a parental compound in design of dUTPase inhibition for medical purposes.