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PubMed1998

Determination of a variety of chemical classes of pesticides in surface and ground waters by off-line solid-phase extraction, gas chromatography with electron-capture and nitrogen-phosphorus detection, and high-performance liquid chromatography with post-column derivatization and fluorescence detection.

Octadecyl (C18)-bonded porous silica was evaluated for the extraction of triazines, organochlorine, carbamates and acidic pesticides from surface and ground water. Gas chromatography with selected detection methods (electron-capture detection, nitrogen-phosphorus detection, mass spectrometry) and liquid chromatography-post-column derivatization fluorescence detection was employed for the determination of 32 pesticides. Recoveries varied from 52 to 102%. The recoveries of triazines obtained using C18 extraction cartridges and conventional liquid-liquid extraction (LLE) are compared. The limit of detection for seventeen organochlorine compounds was better than 0.003 microgram/l and the limit of detection for other 15 analytes was better than 0.06 microgram/l. The proposed analytical methodology was applied to analyze pesticides in surface and ground-water samples of the Lassithi Plateau, Crete, Greece.

Chromatography, Gas↗
PubMed1997

Assessment of affinity constants by rapid solid phase detection of equilibrium binding in a flow system.

We present a method for the determination of affinity constants based on equilibrium binding between an analyte and an antibody in liquid phase by a heterogeneous phase detection scheme. Equilibrium concentration of free antibody binding sites was probed kinetically by direct optical detection of specific binding to an immobilised analyte derivative. The additional binding signal due to dissociation of the analyte-antibody complex during detection was minimised by the use of fast flow-through conditions. The concentration of free antibody binding sites was titrated by adding increasing analyte concentrations. The affinity constant was derived from the titration curve by a non-linear least square fit of a model function. The affinity of monoclonal triazine antibodies to several s-triazine pesticides and a relevant metabolite was investigated. Kinetic determination of equilibrium concentration of free binding sites was carried out by reflectometric interference spectroscopy (RIfS) using flow injection analysis. The capabilities of the model were investigated using different analyte-antibody pairs and various antibody concentrations. Both bivalent IgG and monovalent Fab fragments were used to compare different binding models. The applied model corresponds well to the titration curves for affinity constants of 10(7) M(-1) and higher. For lower affinity constants significant deviations due to dissociation of the analyte-antibody complex during detection were observed.

Animals↗
PubMed2002

Inhibition of coral photosynthesis by the antifouling herbicide Irgarol 1051.

International regulation of organotin compounds for use in antifouling paints has led to the development and increased use of replacement compounds, notably the s-triazine herbicide Irgarol 1051. Little is known about the distribution of Irgarol 1051 in tropical waters. Nor has the potential impact of this triazine upon photosynthesis of endosymbiotic microalgae (zooxanthellae) in corals been assessed. In this study Irgarol 1051 was detected in marinas, harbours and coastal waters of the Florida Keys, Bermuda and St. Croix, with concentrations ranging between 3 and 294 ng 1(-1). 14C incubation experiments with isolated zooxanthellae from the common inshore coral Madracis mirabilis showed no incorporation of H14CO3- from the sea water medium after 4-8 h exposure to Irgarol 1051 concentrations as low as 63 ng 1(-1). Reduction in net photosynthesis of intact corals was found at concentrations of l00 ng 1(-1) with little or no photosynthesis at concentrations exceeding 1000 ng 1(-1) after 2-8 h exposure at all irradiances. The data suggest Irgarol 1051 to be both prevalent in tropical marine ecosystems and a potent inhibitor of coral photosynthesis at environmentally relevant concentrations.

Animals↗
PubMed2000

Herbicides and herbicide degradates in shallow groundwater and the Cedar River near a municipal well field, Cedar Rapids, Iowa.

Water samples were collected near a Cedar Rapids, Iowa municipal well field from June 1998 to August 1998 and analyzed for selected triazine and acetanilide herbicides and degradates. The purpose of the study was to evaluate the occurrence of herbicides and herbicide degradates in the well field during a period following springtime application of herbicides to upstream cropland. The well field is in an alluvial aquifer adjacent to the Cedar River. Parent herbicide concentrations generally were greatest in June, and decreased in July and August. Atrazine was most frequently detected and occurred at the greatest concentrations; acetochlor, cyanazine and metolachlor also were detected, but at lesser concentrations than atrazine. Triazine degradate concentrations were relatively small (< 0.50 microg/l) and generally decreased from June to August. Although the rate of groundwater movement is relatively fast (approx. 1 m per day) in the alluvial aquifer near the Cedar River, deethylatrazine (DEA) to atrazine ratios in groundwater samples collected near the Cedar River indicate that atrazine and DEA probably are gradually transported into the alluvial aquifer from the Cedar River. Deisopropylatrazine (DIA) to DEA ratios in water samples indicate most DIA in the Cedar River and alluvial aquifer is produced by atrazine degradation, although some could be from cyanazine degradation. Acetanilide degradates were detected more frequently and at greater concentrations than their corresponding parent herbicides. Ethanesulfonic-acid (ESA) degradates comprised at least 80% of the total acetanilide-degradate concentrations in samples collected from the Cedar River and alluvial aquifer in June, July and August; oxanilic acid degradates comprised less than 20% of the total concentrations. ESA-degradate concentrations generally were smallest in June and greater in July and August. Acetanilide degradate concentrations in groundwater adjacent to the Cedar River indicate acetanilide degradates are transported into the alluvial aquifer in a manner similar to that indicated for atrazine and DEA.

Acetamides↗
PubMed1999

Effects of the chlorotriazine herbicide, cyanazine, on GABA(A) receptors in cortical tissue from rat brain.

Chlorotriazine herbicides disrupt luteinizing hormone (LH) release in female rats following in vivo exposure. Although the mechanism of action is unknown, significant evidence suggests that inhibition of LH release by chlorotriazines may be mediated by effects in the central nervous system. GABA(A) receptors are important for neuronal regulation of gonadotropin releasing hormone and LH release. The ability of chlorotriazine herbicides to interact with GABA(A) receptors was examined by measuring their effects on [3H]muscimol, [3H]Ro15-4513 and [35S]tert-butylbicyclophosphorothionate (TBPS) binding to rat cortical membranes. Cyanazine (1-400 microM) inhibited [3H]Ro15-4513 binding with an IC50 of approximately 105 microM (n=4). Atrazine (1-400 microM) also inhibited [3H]Ro15-4513 binding, but was less potent than cyanazine (IC50 = 305 microM). However, the chlorotriazine metabolites diaminochlorotriazine, 2-amino-4-chloro-6-ethylamino-s-triazine and 2-amino-4-chloro-6-isopropylamino-s-triazine were without significant effect on [3H]Ro15-4513 binding. Cyanazine and the other chlorotriazines were without effect on [3H]muscimol or [35S]TBPS binding. To examine whether cyanazine altered GABA(A) receptor function, GABA-stimulated 36Cl- flux into synaptoneurosomes was examined. Cyanazine (50-100 microM) alone did not significantly decrease GABA-stimulated 36Cl- flux. Diazepam (10 microM) and pentobarbital (100 microM) potentiated GABA-stimulated 36Cl- flux to 126 and 166% of control, respectively. At concentrations of 50 and 100 microM, cyanazine decreased potentiation by diazepam to 112 and 97% of control, respectively, and decreased potentiation by pentobarbital to 158 and 137% of control (n = 6). Interestingly, at lower concentrations (5 microM), cyanazine shifted the EC50 for GABA-stimulated 36Cl- flux into synaptoneurosomes from 28.9 to 19.4 microM, respectively (n = 5). These results suggest that cyanazine modulates benzodiazepine, but not the muscimol (GABA receptor site) or TBPS (Cl- channel), binding sites on GABA(A) receptors. Furthermore, at low concentrations, cyanazine may slightly enhance function of GABA(A) receptors, but at higher concentrations, cyanazine antagonizes GABA(A) receptor function and in particular antagonizes the positive modulatory effects of diazepam and pentobarbital.

Animals↗
PubMed2001

Use of a Salmonella microsuspension bioassay to detect the mutagenicity of munitions compounds at low concentrations.

Past production and handling of munitions has resulted in soil contamination at various military facilities. Depending on the concentrations present, these soils pose both a reactivity and toxicity hazard and the potential for groundwater contamination. Many munitions-related chemicals have been examined for mutagenicity in the Ames test, but because the metabolites may be present in low environmental concentrations, a more sensitive method is needed to elucidate the associated mutagenicity. RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine), TNT (2,4,6-trinitrotoluene), tetryl (N-methyl-N-2,4,6-tetranitroaniline), TNB (1,3,5-trinitrobenzene) and metabolites were examined for mutagenicity in a microsuspension modification of the Salmonella histidine reversion assay with and without metabolic activation. TNB and tetryl were positive in TA98 (32.5, 5.2revertants/nmole) and TA100 (7.4, 9.5revertants/nmole) without metabolic activation and were more potent than TNT (TA98, 0.3revertants/nmole; TA100, 2.4revertants/nmole). With the exception of the tetranitroazoxytoluene derivatives, TNT metabolites were less mutagenic than TNT. RDX and two metabolites were negative in both strains, however, hexahydro-1,3,5-trinitroso-1,3,5-triazine was positive in TA100 with and without S9. Microsuspension bioassay results tend to correlate well with published Ames test data, however, there are discrepancies among the published data sets and the microsuspension assay results.

Aniline Compounds↗
PubMed2000

Application of the thermally tuned tandem column concept to the separation of several families of environmental toxicants.

Separations of several families of environmental toxicants were optimized by means of the thermally tuned tandem column (T3C) concept. We use a tandem combination of an octadecylsilane (ODS) and a carbon-coated zirconia (C-ZrO2) column; and tune the selectivity by independently adjusting the isothermal temperatures of the two columns. This results in the change in the contribution that each column makes to the overall retention and selectivity. The separation was optimized by locating the optimum pair of column temperatures which give the best separation of the critical solute pair. For both triazine herbicides and carbamate pesticides samples, dramatically different selectivities and different critical pairs were observed for the two types of phases. Although neither individual phase gave adequate separation, the T3C approach provided baseline separations using only four preliminary trial separations. We also showed that, for the triazine samples, the T3C approach gave a better separation than did conventional mobile phase optimization with an ODS column. The combination of superior selectivity of T3C and high flow rate allows the baseline separation of complex mixtures in just a few minutes.

Carbamates↗
PubMedDate not supplied

Influence of coupling method on the luminescence properties, coupling efficiency, and binding affinity of antibodies labeled with europium (III) chelates.

A series of chelating 4-(phenylethynyl)pyridines having various 1,3,5-triazin-2-ylamino groups at the para position of the phenyl ring was synthesized. Their europium chelates were coupled to antibodies and the properties of antibody conjugates analyzed by fluorometry and in time-resolved fluorometric immunoassay. The substituents in the triazine ring were observed to have various effects on the chelate luminescence, the labeling properties of the chelates, and the immunoreactivity of labeled antibodies. The series of substituted triazinyl derivatives serves as a model of bioreactive groups that can be applied when certain properties are searched for, such as improved chelate solubilities, minimized internal quenching, different effects on the ligand triplet state, and stipulated coupling reactivities.

Antibodies↗
PubMed1989

Characterization of four herbicide-resistant mutants of Rhodopseudomonas viridis by genetic analysis, electron paramagnetic resonance, and optical spectroscopy.

Herbicides of the triazine class block electron transfer in the photosynthetic reaction centers of purple bacteria and PSII of higher plants. They are thought to act by competing with one of the electron acceptors, the secondary quinone, QB, for its binding site. Several mutants of the purple bacterium Rhodopseudomonas viridis resistant to terbutryn [2-(methylthio)-4-(ethylamino)-6-(tert-butylamino)-s-triazine] have been isolated by their ability to grow photosynthetically in the presence of the herbicide. Sequence analysis of the genes coding for the L and M subunits of the reaction center showed that four different mutants were obtained, two of them being double mutated: T1 (SerL223----Ala and ArgL217----His), T3 (PheL216----Ser and ValM263----Phe), T4 (TyrL222----Phe), and T6 (PheL216----Ser). The residues L223 and L216 are involved in binding of QB, whereas L217 and L222 are not. M263 is part of the binding pocket of the primary quinone, QA. The affinity of the reaction centers for terbutryn and the electron transfer inhibitor o-phenanthroline, determined via the biphasic charge recombination after one flash, is decreased for all mutants. The affinity for ubiquinone 9 is also decreased, except in T1. Characterization by EPR spectroscopy showed that the QB.-Fe2+ signal of T4, having a g = 1.93 peak, is different from the signals obtained with the wild type and the other mutants but very similar to those of Rhodospirillum rubrum and PSII. The results obtained by the combination of these different techniques are discussed with respect to the three-dimensional structure of the wild type and the mode of binding of ubiquinone, terbutryn, and o-phenanthroline as determined by X-ray structure analysis.

Amino Acid Sequence↗
PubMed2002

Biotransformation of hexahydro-1,3,5-trinitro-1,3,5-tiazine catalyzed by a NAD(P)H: nitrate oxidoreductase from Aspergillus niger.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) can be efficiently mineralized with anaerobic domestic sludge, but the initial enzymatic processes involved in its transformation are unknown. To test the hypothesis that the initial reaction involves reduction of nitro group(s), we designed experiments to test the ability of a nitrate reductase (EC 1.6.6.2) to catalyze the initial reaction leading to ring cleavage and subsequent decomposition. A nitrate reductase from Aspergillus niger catalyzed the biotransformation of RDX most effectively at pH 7.0 and 30 degrees C under anaerobic conditions using NADPH as electron donor. LC/MS (ES-) chromatograms showed the formation of hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and methylenedinitramine as key initial products of RDX, but neither the dinitroso neither (DNX) nor trinitroso (TNX) derivatives were observed. None of the above detected products persisted, and their disappearance was accompanied by the accumulation of nitrous oxide (N20), formaldehyde (HCHO), and ammonium ion (NH4+). Stoichiometric studies showed that three NADPH molecules were consumed, and one molecule of methylenedinitramine was produced per RDX molecule. The carbon and nitrogen mass balances were 96.14% and 82.10%, respectively. The stoichiometries and mass balance measurements supported a mechanism involving initial transformation of RDX to MNX via a two-electron reduction mechanism. Subsequent reduction of MNX followed by rapid ring cleavage gave methylenedinitramine which in turn decomposed in water to produce quantitatively N2O and HCHO. The results clearly indicate that an initial reduction of a nitro group by nitrate reductase is sufficient for the decomposition of RDX.

Aspergillus niger↗
PubMed2003

Alkaline hydrolysis of the cyclic nitramine explosives RDX, HMX, and CL-20: new insights into degradation pathways obtained by the observation of novel intermediates.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX, I) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) hydrolyze at pH > 10 to form end products including NO2-, HCHO, HCOOH, NH3, and N2O, but little information is available on intermediates, apart from the tentatively identified pentahydro-3,5-dinitro-1,3,5-triazacyclohex-1-ene (II). Despite suggestions that RDX and HMX contaminated groundwater could be economically treated via alkaline hydrolysis, the optimization of such a process requires more detailed knowledge of intermediates and degradation pathways. In this study, we hydrolyzed the monocyclic nitramines RDX, MNX (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine), and HMX in aqueous solution (pH 10-12.3) and found that nitramine removal was accompanied by formation of 1 molar equiv of nitrite and the accumulation of the key ring cleavage product 4-nitro-2,4-diazabutanal (4-NDAB, O2NNHCH2NHCHO). Most of the remaining C and N content of RDX, MNX, and HMX was found in HCHO, N2O, HCOOH, and NH3. Consequently, we selected RDX as a model compound and hydrolyzed it in aqueous acetonitrile solutions (pH 12.3) in the presence and absence of hydroxypropyl-beta-cyclodextrin (HP-beta-CD) to explore other early intermediates in more detail. We observed a transient LC-MS peak with a [M-H] at 192 Da that was tentatively identified as 4,6-dinitro-2,4,6-triaza-hexanal (O2NNHCH2NNO2CH2NHCHO, III) considered as the hydrolyzed product of II. In addition, we detected another novel intermediate with a [M-H] at 148 Da that was tentatively identified as a hydrolyzed product of III, namely, 5-hydroxy-4-nitro-2,4-diaza-pentanal (HOCH2NNO2CH2NHCHO, IV). Both III and IV can act as precursors to 4-NDAB. In the case of the polycyclic nitramine 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20), denitration (two NO2-) also led to the formation of HCOOH, NH3, and N2O, but neither HCHO nor 4-NDAB were detected. The results provide strong evidence that initial denitration of cyclic nitramines in water is sufficient to cause ring cleavage followed by spontaneous decomposition to form the final products.

Azocines↗
PubMed2005

Supramolecular helical mesomorphic polymers. Chiral induction through H-bonding.

The work described here concerns a challenge of general interest in supramolecular chemistry: the achievement of chiral helical organizations with controlled structures. This work provides a strategy to obtain supramolecular polymers in which a chiral helical conformation has been induced by a noncovalent association, that is, through hydrogen bonding. Polycatenar 2,4,6-triarylamino-1,3,5-triazines, which organize into columnar mesophases and are susceptible to H-bonding interactions, were chosen as a starting point to build up the chiral supramolecular structure. The stacking of these mesogens has been forced to wind in a helical way by means of H-bond association with (R)-3-methyladipic acid, within the mesophase. The optically active columnar organization has been studied in depth by optical microscopy, differential scanning calorimetry (DSC), X-ray diffraction, and circular dichroism. Formation of stable complexes between the triazine units and (R)-3-methyladipic acid has also been investigated by means of NMR diffusion-ordered spectroscopy (DOSY) experiments in chloroform.

Adipates↗
PubMed2002

Studies on bound (14)C-chlorsulfuron residues in soil.

The cause for phytotoxicity of bound residues of chlorsulfuron (2-chloro-N-[[4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino] carbonyl]benzenesulfonamide) to rotational crops is unknown. This study was conducted to determine the formation of nonextractable (bound) residues of chlorsulfuron in soil, and the distribution of bound residues in different organic matter fractions. The results showed that over 150 days, the extractable fraction of (14)C-residues decreased to 25.1% of applied chlorsulfuron, while bound residues concurrently increased to 47.1%. The distribution of (14)C-bound residues in soil organic matter fractions followed an order of humic acid (HA) < humin < fulvic acid (FA). Although the most bound residues were detected in the FA fraction, the amount associated with the humin fraction increased with time. After soil treatment by autoclaving, it was found that bound (14)C-chlorsulfuron residues became available again in the soil. One of the released products was 2-amino-4-hydroxyl-6-methyl-1,3,5-triazine (identified by GC-MS), which is a degradation product of chlorsulfuron.

Benzopyrans↗
PubMed2003

Preparation of antibodies and development of an enzyme-linked immunosorbent assay for determination of dealkylated hydroxytriazines.

The development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for dealkylated hydroxytriazines is reported here for the first time. The assay uses polyclonal antibodies raised against N-(4-amine-6-hydroxy-[1,3,5]triazin-2-yl)-4-aminobutanoic acid (hapten 2g) conjugated to keyhole limpet hemocyanin by the active ester method. The immunizing hapten was synthesized by first introducing the amino group to the triazine ring in a protected form in order to increase its solubility in organic media. Subsequent steps consisted of reacting this compound with an appropriate spacer arm, followed by removal of the protecting group in acidic media. The resulting assay uses a homologous competitor hapten coupled to conalbumin by the mixed anhydride method. Coating antigens prepared using a homologous covalent coupling procedure failed to produce competitive immunoassays. The assay tolerates media with high ionic strength (up to 70 mS cm(-)(1)) and basic pH values (7.5-9.5 units). Under the optimized conditions, this ELISA is specific for dealkylated hydroxytriazines, reaching suitable limits of detection.

Animals↗
PubMed2000

Triflusulfuron-methyl dissipation in water and soil.

This paper reports laboratory studies of the behavior and fate of triflusulfuron-methyl in aqueous buffer and soils. Aqueous hydrolysis was pH-dependent and fast in acidic buffer solutions. In basic buffers, the hydrolysis rate variation was low between pH 7 and pH 10. The degradation pathway in the range of pH 4-10 was via cleavage of the sulfonylurea bridge to form two transformation products: 2-amino-4-(dimethylamino)-6-(2,2,2-trifluoroethoxy)-1,3, 5-triazine (2) and 6-methyl-2-methylcarboxylate benzene sulfonamide (3). Comparison of transformation rates in sterile and nonsterile soils indicates that chemical and microbial processes are important in soil degradation. The former is more important in acidic soils, and the latter is more important in basic soils. A biphasic model fits well with dissipation of triflusulfuron-methyl in soil. The triazine formed during the first step of transformation was degraded more rapidly in basic soils than in acidic soils.

Benzoates↗
PubMed2000

Analytical method for the determination of cyromazine and melamine residues in soil using LC-UV and GC-MSD.

A method is reported for the determination of cyromazine and melamine residues in soil. Soil samples are extracted twice via mechanical shaking, each time with 70% acetonitrile/30% 0.050 M ammomium carbonate for 30 min. An aliquot portion of the pooled extracts is subjected to strong cation exchange (SCX) purification on AG 50W-X4 resin. Final analysis is accomplished using liquid chromatography-ultraviolet (LC-UV) detection at a wavelength of 214 nm. Confirmatory analyses can be performed using gas chromatography-mass selective detection (GC-MSD) in the selected ion monitoring (SIM) mode. The limit of detection (LOD) is 2.5 ng injected and the limit of quantification (LOQ) is 10 ppb when using LC-UV for the analysis of N-cyclopropyl-1,3,5-triazine-2,4, 6-triamine (cyromazine) and 1,3,5-triazine-2,4,6-triamine (melamine). The LOD is 0.050 ng injected and the LOQ is 10 ppb when using GC-MSD for confirmatory analyses. The mean procedural recoveries were 97 and 95% and the standard deviations were 16 and 11% for cyromazine and melamine, respectively (n = 24), when using LC-UV. The mean procedural recoveries were 107 and 92% and the standard deviations were 9.9 and 16% for cyromazine and melamine, respectively (n = 29), when using GC-MSD. The method validation study was conducted under U.S. EPA FIFRA Good Laboratory Practice Guidelines 40 CFR 160. The method also passed an Independent Laboratory Validation (ILV) as per U.S. EPA FIFRA Subdivision N.

Chromatography, Liquid↗
PubMed2000

Isolation of a novel protein tyrosine phosphatase inhibitor, 2-methyl-fervenulone, and its precursors from Streptomyces.

High-throughput screening identified an extract from Streptomyces sp. IM 2096 with inhibitory activity toward several protein tyrosine phosphatases (PTPs). Four 1,2,4-triazine compounds 2096A-D (1-4) were isolated from this extract and their structures elucidated by interpretation of spectroscopic data and confirmed by degradation and synthesis. The novel glycocyamidine derivatives 1 and 2 are diastereomers and may interconvert. Both are inactive in the PTP inhibition assay. Compounds 1 and 2 are unstable and partially decompose to 3 and glycocyamidine (5) at room temperature. Compound 3, known as MSD-92 or 2-methyl-fervenulone, is a broad-specificity PTP inhibitor with comparable potency to vanadate. The imidazo[4, 5-e]-1,2,4-triazine (4), inactive in the PTP-inhibition assay, may be a degradation product of 3.

Enzyme Inhibitors↗
PubMed2006

Identification of a novel hemoglobin adduct in Sprague Dawley rats exposed to atrazine.

Atrazine (2-chloro-4-[ethylamino]-6-[isopropylamino]-1,3,5-triazine) is one of the most commonly used herbicides in North America and is frequently detected in ground and surface waters. This research investigated possible covalent modifications of hemoglobin following in vivo exposures to atrazine in Sprague Dawley (SD) rats and in vitro incubations with diaminochlorotriazine. SD rats were exposed to 0, 10, 30, 100, and 300 (mg atrazine/kg)/day for 3 days via oral gavages, and blood was drawn at 0 h, 24 h, 72 h, 20 days, 1 month, and 2 months for globin analysis. Globin was purified from red blood cells, separated with high-performance liquid chromatography, and analyzed with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). An additional beta globin peak was seen in exposed animals during the HPLC and MALDI-TOF MS analysis with a mass 110 Da greater than the normal beta subunits. Tryptic digests of this beta peak contained a peptide of 1449.9 m/z that corresponded to a modified peptide of amino acids 121-132. Mass spectrometry sequencing of this peptide indicated a 110 Da addition to Cys-125 of the major beta globin chain, which corresponds to a nucleophilic substitution reaction with a diaminochlorotriazine. In vitro incubations of SD globin and diaminochlorotriazine also resulted in a peptide of 1449.6 m/z that was identical in sequence to the modified peptide seen in the in vivo digest, confirming the nucleophilic substitution mechanism of adduct formation. Exposures of SD rats to atrazine results in formation of an adduct that is easily detected and provides an analytical model for detection of triazine adducts in other macromolecules with sulfhydryl functional groups.

Amino Acid Sequence↗