Comparison of three types of reagents for detection of Bacteroides fragilis by direct immunofluorescence

Three different methods were used to prepare conjugates for the detection of rods of the Bacteroides fragilis group by direct immunofluorescence. Lyophilized conjugates were prepared. Three sets (five in each) of monovalent conjugates against serotype strains of B. fragilis (including conjugate E/E1 + E2) and polyvalent conjugate (A + B + C + D + E1 + E2) were obtained. Each conjugate was prepared in two variants: 1. unabsorbed, 2. absorbed with tissue powder prior to lyophilization. Conjugates obtained by precipitation of sera with 50% ethanol and direct coupling of gammaglobulins with stain were found to meet the requirement for good fluorescence reagents and are well suited for the detection of B. fragilis by direct immunofluorescence. Absorption of the conjugates with tissue powder before lyophilization did not affect their quality.     

Hydrophobized triphenyl phosphonium derivatives for the preparation of mitochondriotropic liposomes: choice of hydrophobic anchor influences cytotoxicity but not mitochondriotropic effect

Context: Nanocarrier-based strategies to achieve delivery of bioactives specifically to the mitochondria are being increasingly explored due to the importance of mitochondria in critical cellular processes.

Objective: To test the ability of liposomes modified with newly synthesized triphenylphosphonium (TPP)–phospholipid conjugates and to test their use in overcoming the cytotoxicity of stearyl triphenylphosphonium (STPP)-modified liposomes when used for delivery of therapeutic molecules to the mitochondria.

Methods: TPP–phospholipid conjugates with the dioleoyl, dimyristoyl or dipalmitoyl lipid moieties were synthesized and liposomes were prepared with these conjugates in a 1?mol% ratio. The subcellular distribution of the liposomes was tested by confocal microscopy. Furthermore, the liposomes were tested for their effect on cell viability using a MTS assay, on cell membrane integrity using a lactate dehydrogenase assay and on mitochondrial membrane integrity using a modified JC-1 assay.

Results: The liposomes modified with the new TPP–phospholipid conjugates exhibited similar mitochondriotropism as STPP-liposomes but they were more biocompatible as compared to the STPP liposomes. While the STPP-liposomes had a destabilizing effect on cell and mitochondrial membranes, the liposomes modified with the TPP–phospholipid conjugates did not demonstrate any such effect on biomembranes.

Conclusions: Using phospholipid anchors in the synthesis of TPP–lipid conjugates can provide liposomes that exhibit the same mitochondrial targeting ability as STPP but with much higher biocompatibility.     

Synthesis, characterization, and in vitro activity of dendrimer-streptokinase conjugates

Dendrimer conjugation with low molecular weight drugs has been of increasing interest recently for improving pharmacokinetics, targeting drugs to specific sites, and facilitating cellular uptake. Opportunities for increasing the performance of relatively large therapeutic proteins such as streptokinase (SK) using dendrimers are being explored in this study. Using the active ester method, a series of streptokinase-poly(amido amine) (PAMAM) G3.5 conjugates were synthesized with varying amounts of dendrimer-to-protein molar ratios. Characterization of these conjugates by GPC, IEC, and native-PAGE suggested that the conjugation reaction was successful, resulting in relatively pure SK-dendrimer conjugates. The conjugate made with an equimolar ratio of dendrimer to streptokinase (1:1) exhibited the highest enzymatic activity retention ( approximately 80% retained) that has been reported so far for conjugated streptokinase with macromolecules such as PEG or dextran. SK conjugates with higher streptokinase-to-dendrimer molar ratios (1:10 and 1:20) exhibited lower initial enzymatic activities. However, these conjugates showed sustained thrombolytic activity in plasma, perhaps due to the release of SK from the conjugate. All of the SK conjugates displayed significantly improved stability in phosphate buffer solution, compared to free SK. The high coupling reaction efficiencies and the resulting high enzymatic activity retention achieved in this study could enable a desirable way for modifying many bioactive macromolecules with dendrimers.     

Synthesis, solution conformation, and antibody recognition of oligotuftsin-based conjugates containing a beta-amyloid(4-10) plaque-specific epitope

One possible therapeutic approach to treat or prevent Alzheimer's disease (AD) is immunotherapy. On the basis of the identification of Abeta(4-10) (FRHDSGY) as the predominant B-cell epitope recognized by therapeutically active antisera from transgenic AD mice, conjugates with defined structures containing the epitope peptide attached to a tetratuftsin derivative as an oligopeptide carrier were synthesized and their structure characterized. To produce immunogenic constructs, the Abeta(4-10) epitope alone or flanked by alpha- or beta-alanine residues was attached through an amide bond to the tetratuftsin derivative (Ac-[TKPKG]4-NH2) or to a carrier peptide elongated by a promiscuous T-helper cell epitope (Ac-FFLLTRILTIPQSLD-[TKPKG]4-NH2). The conformational preferences of the carrier and conjugates were examined by CD spectroscopy in water and in 1:1 and 9:1 TFE:water mixtures (v/v). We found that the presence of flanking dimers in the conjugates had no effects on the generally unordered solution conformation of the conjugates. However, conjugates with an elongated peptide backbone exhibited CD spectra indicative for a partially ordered secondary structure in the presence of TFE. Comparative ELISA binding studies, using monoclonal antibody raised against the beta-amyloid (1-17) peptide, showed that conjugates with T-helper cell epitope in the carrier backbone exhibited decreased monoclonal antibody recognition. However, we found that this effect was compensated in conjugates comprising the Abeta(4-10) B-cell epitope with the beta-alanine dimer flanking regions at both N- and C-termini. Results suggest that modification of the B-cell epitope peptide from Abeta with rational combination of structural elements (e.g. conjugation to carrier, introduction of flanking dimers) can result in synthetic antigen with preserved antibody recognition.     

Reduced immunogenicity of beta-lactoglobulin by conjugating with chitosan

Bovine beta-lactoglobulin (beta-LG) was conjugated with chitosan (CHS) by means of a water-soluble carbodiimide to reduce the immunogenicity of beta-LG. Each beta-LG-CHS conjugate was purified by ion-exchange chromatography and hydrophobic chromatography. The conjugation between beta-LG and CHS was confirmed by SDS-PAGE, the isoelectric point of the conjugate being higher than that of beta-LG. Two types of the beta-LG-CHS conjugate were obtained with molar ratios of beta-LG to CHS of 1:1 (F1) and 1:2 (F2). Structural analyses by fluorescence measurement, ELISA with monoclonal antibodies and retinol-binding activity indicated that the conjugates had almost maintained the native structure of beta-LG. The antigenicity of the beta-LG-CHS conjugates was similar to that of beta-LG in C3H/He mice. Reduction of the immunogenicity of beta-LG was achieved by conjugation with CHS. In particular, F2 showed very low immunogenicity. B cell epitopes of beta-LG and the conjugates recognized in C3H/He mice were determined with 15-mer multi-pin peptide; the linear epitope profiles of the conjugates were found to be similar to those of beta-LG, while the antibody response to each epitope was dramatically reduced. Conjugation of beta-LG with chitosan was effective for reducing the immunogenicity of beta-LG.     

A robust method for the preparation and purification of antibody/streptavidin conjugates

The many uses of antibody-protein conjugates, especially antibody-streptavidin conjugates, give rise to the need for a reliable conjugation method offering reasonable yields and reproducible quality. We describe a method for preparing antibody-streptavidin conjugates that has consistently produced conjugates of quality and in sufficient quantity to be used in the clinical development and evaluation of the Pretarget delivery system. In this method antibody disulfides are reduced to generate reactive thiols, and maleimides are linked to streptavidin with the heterobifunctional cross-linking agent, SMCC. The two activated proteins are then mixed briefly before the conjugation is terminated with an oxidizing agent that reforms disulfides from unreacted thiols. The preponderance of the conjugate produced is 1:1 and 1:2 Ab:SA conjugate. This fraction is isolated from unconjugated proteins and high molecular weight byproduct by iminobiotin affinity and ion-exchange chromatography. The resulting conjugate is at least 90% 1:1 + 1:2 Ab:SA conjugate, contains no SA or Ab, and is produced reproducibly in 37% yield.     

The effect of condition of oxidation of the carbohydrate component of peroxidase on the composition and properties of insulin-peroxidase conjugate

The influence of sodium metaperiodate concentration on kinetics and conversion degree of peroxidase carbohydrate moiety as well as the effect of the oxidation degree of the carbohydrate moiety on the composition, structure and properties of insulin-peroxidase conjugates were studied. The initial rate of peroxidase's oxidation is directly proportional to the periodate concentration; the oxidation rate constant of peroxidase carbohydrate moiety is 1.23 x 10(-3) M-1 min-1. At the molar ratio of metaperiodate to peroxidase 150:1 or higher, the maximal quantity of aldehyde groups (62 +/- 2) in the peroxidase molecule is formed and the oxidation of each carbohydrate chain leads to the formation of eight aldehyde groups. The molecular mass composition of the insulin-peroxidase conjugates was studied by HPLC. The conjugates proved to be multicomponent mixtures of oligomers (53, 83, 128, 174, 268, 440 kD and higher). The insulin-peroxidase molar ratio in the fractions of the conjugates with molecular masses higher than 83 kD is 8:1. It was shown that the affinity of insulin-peroxidase conjugates to antibodies depends on the oxidation degree of peroxidase used for production of conjugates.     

氨甲喋呤-人血清白蛋白-单抗79结合物的制备及其体外细胞毒作用

 本研究采用间接连接法,应用异型双功能连接剂SPDP,形成以酰胺键和二硫键连接的氮甲喋吟-人血清白蛋白-单抗79(MTX-HSA-McAb79)结合物。其中McAb79与HSA克分子比有效地控制在1:1～2,且结合物的纯度及产率均较高,有一定的制备价值。 对靶细胞Nalm-6的体外细胞毒试验结果表明:MTX-HSA-McAb 79的杀伤活性显著强于MTX-HSA-nIgG,但较游离MTX的杀伤活性为弱。且MTX-HSA-McAb 79的杀伤作用依赖于McAb 79与靶抗原CALLA的抗原抗体反应。     

Direct determination of anabolic steroid conjugates in human urine by combined high-performance liquid chromatography and tandem mass spectrometry

A novel screening procedure for the sulfate and glucuronide conjugates of testosterone (T) and epitestosterone (E) in human urine was developed based on liquid-solid extraction and microbore high-performance liquid chromatography combined on-line with ion-spray tandem mass spectrometry. Confirmation of the sulfate and glucuronide conjugates of testosterone and epitestosterone isolated frrm normal human urine was acheived by selected reaction monitoring of characteristic product ions of the parent compounds. Endogenous levels of the steroid conjugates are detected in normal male urine and an increase is observed when the sample is fortified with authentic analytical standards of the conjugates. Calibration curves of all steroid conjugates in urine are linear over a range of twenty. Deuterated internal standards of testosterone glucuronide and epitestosterone sulfate were used for quantitation of the endogenous conjugates. T/E ratios were determined based on the glucuronide fractions of six replicates from a normal male and were shown to be statistically reproducible and below the accepted T/E threshold of 6:1. Sulfate conjugates were shown to be present at significantly lower levels in the urine. The method has potential as an alternative for monitoring anabolic steroid conjugates in human urin.     

Cytotoxic action of conjugates of alpha-fetoprotein and epidermal growth factor with photoheme, chlorines, and phthalocyanines

Covalently bound conjugates of alpha-fetoprotein (AFP) and epidermal growth factor (EGF) with photoheme (PH), 3-desvinyl-3-formylchlorine p6 (Chl p6), chlorine e6 (Chl e6), aluminum disulfochloride phthalocyanine (PC(Al)), and cobalt octa-4,5-carboxyphthalocyanine (teraphthal, TP(Co)) were synthesized. Their molar ratios were 1:4 for AFP-cytotoxin conjugates (cf. 1:10 for AFP-TP(Co)) and 1:2 for EGF conjugates (cf. 1:1 for EGF-PC(Al)). Dark toxicity of both protein conjugates with PH, chlorines, and PC(Al) was much lower than their phototoxicity. Studies on phototoxicity demonstrated that PC(Al) conjugates with AFP and EGF and also EGF-Chl p6 were the most effective. The cytotoxic activity (CTA) of AFP-PC(Al) and EGF-Chl p6 was 80% and of EGF-PC(Al) 64% higher than the CTA of the free drugs. Conjugates with TP(Co) were much more toxic on their activation with ascorbic acid (AA): in the presence of AA the CTA of AFP-TP(Co) and of EGF-TP(Co) was 19 and 61.1% higher, respectively, than the CTA of the free TP(Co).     

Biodistribution of the chimeric monoclonal antibody U36 radioiodinated with a closo-dodecaborate-containing linker. Comparison with other radioiodination methods

We have evaluated the applicability of the [(4-isothiocyanatobenzylammonio)undecahydro-closo-dodecaborate (1-)] (DABI) linker molecule for antibody radiohalogenation and compared it to radiohalogenation using the linker N-succinimidyl 4-iodobenzoate (PIB) and to direct radiohalogenation using Chloramine T. These studies were performed to assess the potential of DABI conjugates and to optimize the biological properties of halogen-labeled cMAb U36. The three conjugates were evaluated in vitro for their specificity and affinity and in vivo for their biodistribution patterns in normal mice at 1.5, 6, 24, and 96 h pi. Labeling efficiencies of direct CAT labeling, indirect PIB labeling, and indirect DABI labeling were 90-95%, 60%, and 68%, respectively. This resulted in a PIB:cMAb U36 molar ratio of 1.8-2.5 and a DABI:cMAb U36 molar ratio of 4.1. The in vitro data demonstrated specific binding for all conjugates and similar affinities with values around 1 x 10(8) M(-)(1). However, the in vivo data revealed accumulation of the radioiodine uptake in thyroid for the directly labeled conjugate, with a value 10 times higher than the indirectly labeled conjugates 96 h pi. Both the (125)I-PIB-cMAb U36 and (125)I-DABI-cMAb U36 conjugates yielded a low thyroid uptake with no accumulation, indicating different catabolites for these conjugates. This may favor the use of the indirectly labeled conjugates for future studies. Apart from the specific results obtained, these findings also demonstrate how the right linker molecule will provide additional opportunities to further improve the properties of an antibody-radionuclide conjugate.     

5'-bis-pyrenylated oligonucleotides displaying excimer fluorescence provide sensitive probes of RNA sequence and structure

Oligonucleotide conjugates bearing two pyrene residues attached to 5′-phosphate through a phosphoramide bond were synthesised. Fluorescence spectra of the conjugates show a peak typical of monomer emission (λ_max 382 nm) and a broad emission peak with λ_max 476 nm, which indicates the excimer formation between the two pyrene residues. Conjugation of these two pyrene residues to the 5′-phosphate of oligonucleotides does not affect the stabilities of heteroduplexes formed by conjugates with the corresponding linear strands. A monomer fluorescence of the conjugates is considerably affected by the heteroduplex formation allowing the conjugates to be used as fluorescent hybridisation probes. The 5′-bis-pyrenylated oligonucleotides have been successfully used for investigation of affinity and kinetics of antisense oligonucleotides binding to the multidrug resistance gene 1 (PGY1/MDR1) mRNA. The changes of excimer fluorescence of the conjugates occurring during hybridisation depended on the structure of the binding sites: hybridisation to heavily structured parts of RNA resulted in quenching of the excimer fluorescence, while binding to RNA regions with a loose secondary structure was accompanied by an enhancement of the excimer fluorescence. Potentially, these conjugates may be considered as fluorescent probes for RNA structure investigation.     

Reduced immunogenicity of beta-lactoglobulin by conjugation with carboxymethyl dextran

We prepared two beta-lactoglobulin (beta-LG)-carboxymethyl dextran (CMD) conjugates (Conj. 10A and Conj. 10B) by using a water-soluble carbodiimide to decrease the immunogenicity of beta-LG. The molar ratios of beta-LG to CMD in the conjugates were 5:1 (Conj. 10A) and 2:1 (Conj. 10B). The beta-LG-CMD conjugates maintained the retinol-binding activity of native beta-LG. Intrinsic fluorescence study indicated that shielding of the surface of beta-LG by CMD occurred in each conjugate, which was eminent in Conj. 10B. A local conformational change around (125)Thr-(135)Lys (alpha-helix) in each conjugate was detected by ELISA with monoclonal antibodies. The denaturation temperature of beta-LG evaluated by differential scanning calorimetry was greatly enhanced in each conjugate. The anti-beta-LG antibody response was markedly reduced after immunization with the beta-LG-CMD conjugates in BALB/c, C57BL/6, and C3H/He mice. We determined the B cell epitopes of beta-LG and each conjugate recognized in these mice and found that the linear epitope profiles of the beta-LG-CMD conjugates were similar to those of beta-LG, while the antibody response for each epitope was dramatically reduced. The reduced immunogenicity of beta-LG was most marked in the case of Conj. 10B, which contained more CMD than Conj. 10A, and was effectively shielded by CMD. We concluded that masking of epitopes by CMD is responsible for the decreased immunogenicity of the beta-LG in these conjugates.     

Properties of Bioconjugates of Streptokinase with Anionic Polyamidoamine Dendrimers of Various Generations

Covalent conjugates of streptokinase (SK) with polyamidoamine (PAMAM) dendrimers G1.5, G2.5, and G3.5 (SK–G1.5, SK–G2.5, and SK–G3.5) with the protein–polymer molar ratios of (1: 1), (1: 5), and (1: 10) were obtained and their properties were studied as compared to the properties of free SK. It was shown that the initial rates of formation of the modified Pm. SK complex, activation of plasminogen, and lysis of the plasma clot under the action of SK–dendrimer conjugates decreased with increasing number of bound dendrimers (from 1 to 10) and increased with increasing dendrimer generation (from G1.5 up to G3.5). Conjugates SK–G3.5 (1: 1) and (1: 5) were the most active compared to other conjugates. It was found that the catalytic efficiency of plasminogen activation (k_Pg/K_Pg) by conjugates SK–G3.5 (1: 1) (0.15 μM^–1 min^–1) and SK–G3.5 (1: 5) (0.12 μM^–1 min^–1) was comparable to the efficiency of free SK (0.18 μM^–1 min^–1). Probably, small in size, soft, and easily deformable dendrimers G1.5 and G2.5 are able to penetrate into the internal shielded cavities of the native SK molecule and there modify amino groups that are important for the effective formation of the Pm · SK complex. By contrast, the larger and more rigid molecule of dendrimer G3.5 modifies, mainly, exposed lysine residues in the SK molecule, without affecting the latent internal lysines. Conjugates SK–G3.5 (1: 1) and (1: 5), which had the maximum activator activity, retained up to 85% of thrombolytic activity compared to the activity of free SK. In addition, due to modification of the exposed lysines—most sensitive to proteolysis in the SK molecule—with dendrimer G3.5, which has the highest density of negative charge on its surface, SK–G3.5 (1: 1) and (1: 5) conjugates were more stable in plasma and caused less exhaustion of plasma levels of plasminogen, α2-antiplasmin, and fibrinogen than free SK in vitro. Thus, thrombolytic activity of the SK–dendrimer conjugates depends on the degree of modification of the amino groups of SK, size, stiffness, and density of the negative charge on the surface of the PAMAM dendrimer. Conjugates SK–G3.5 (1: 1) and (1: 5) are potential candidates for the development of a new thrombolytic agent.     

Anaphylactogenicity of dextran-conjugated serum globulins

Active anaphylaxis in 238 guinea-pigs has revealed a decrease in the anaphylactogenic activity of horse blood serum IgG conjugates with dextran and of serum treated with dextran according to Diaferm method. The conjugates were used for a challenge injection. The sensitizing activity of dextran-conjugated proteins was higher than that of native proteins. The effect was most pronounced with 150,000 D dextran used as a matrix. A lower increase in sensitizing protein activity and a decrease in anaphylactogenic activity were achieved with dextran matrix of the molecular weight of 35-50 D and protein/dextran ratio from 1:6 to 1:9.     

Preparation of immunoenzyme conjugates of beta-lactamase from Bacillus licheniformis 749/c and horseradish peroxidase with human antibodies to HIV-1]

By using three different linkage methods with carbodiimide, glutaraldehyde and periodite, immunoenzyme conjugates of beta-lactamase from Bacillus licheniformis 749/c and horse radish peroxidase with human antibodies to HIV-1 were prepared. The human antibodies were purified by the affinity procedure on Protein-A-Sepharose 6B. The conjugates were tested in a solid phase immunoenzymatic system for the HIV-1 antigen. It was shown that the conjugates prepared by the carbodiimide linkage method had the highest titer, the beta-lactamase conjugate being superior by its titer to the respective peroxidase conjugate. In the lyophilized state the conjugates prepared with the carbodiimide linkage method were stable.     

LPS-based conjugate vaccines composed of O-polysaccharide from Pseudomonas aeruginosa IATS 6 and 11 bound to a carrier protein

Lipopolysaccharide (LPS) is the major surface antigen of Pseudomonas aeruginosa and elicits protective antibodies in animals. No cross reaction was observed between LPSs of P. aeruginosa International Antigenic Typing Scheme (IATS) 6 and 11 strains using rabbit polyclonal antibodies raised against the whole cells. The O-polysaccharides (O-PSs) from IATS 6 and 11, the antigenic determinant of LPS, were directly coupled to bovine serum albumin (BSA) by carbodiimide mediated condensation reaction. The molar ratios of saccharide repeating units to BSA in the prepared conjugates were 15:1 and 26:1 for IATS 6 and 11 conjugates, respectively. Mice were immunized with the conjugates emulsified with monophosphoryl lipid A (MPL), Freund, and Alum adjuvants. The conjugates emulsified with MPL adjuvant elicited the highest IgM antibody, followed by Freund. While both MPL and Freund adjuvants elicited high IgG antibody. Good correlation was observed between the IgG and IgM levels with the bactericidal activities of the sera against homologous strains. In addition, immunization of mice with the prepared conjugates emulsified with MPL and Freund adjuvants provided high protection against ten times the LD₅₀ of P. aeruginsoa IATS 6 and 11, which showed a good correlations with the IgG titer.     

The structures of ubiquitin conjugates of yeast Iso-2-cytochrome c.

Ubiquitin (Ub) conjugates to Saccharomyces cerevisiae iso-2-cytochrome c were formed in vitro in a rabbit reticulocyte extract (Fraction II). In the presence of ubiquitin-aldehyde, used to inhibit ubiquitin-protein isopeptidases in Fraction II, mono-, di-, and triubiquitinated cytochrome c conjugates accumulated in a 1.2:1.0:0.2 molar ratio. CNBr digestions showed that, in all three conjugates, Ub attachment was within the first 73 amino acids of the cytochrome c. For the two most abundant conjugates, this region was further narrowed to the first 30 residues by peptide mapping with Staphylococcus aureus V8 protease. N-terminal protein sequencing identified Lys-13 as the major ubiquitination site in each conjugate. For di- and triubiquitinated iso-2-cytochrome c, this suggested that Ub2 and Ub3 multiubiquitin chains extend from Lys-13. This conclusion was supported by a variation of protein sequencing in which polypeptides recovered after Edman degradation were analyzed to determine at which cycle(s) radiolabeled Ub or Ubn was cleaved from the conjugate. Because of the sensitivity afforded by the use of 125I-Ub in this "stutter-step" sequencing method, minor ubiquitination at Lys-8 also was detected. Thus, Ub2-iso-2-cytochrome c conjugates contain mostly Ub2 at Lys-13 with a small fraction of conjugates having single Ubs on 2 residues, Lys-8 and Lys-13. Similarly, Ub3-iso-2-cytochrome c predominantly has a Ub3 chain on Lys-13, although minor species with combinations of Ub1 and Ub2 distributed on Lys-8 and Lys-13 also may be present. This specificity is discussed in the context of iso-2-cytochrome c structure.     

Fixation of various aldehydic dextrans onto human hemoglobin: Study of conjugate stability

Formation and stability of different aldehydic dextran-hemoglobin conjugates were studied. Two types of polymers were used: sulfated or unsulfated oxidized dextrans and 4-carboxamidobenzaldehyde dextran. Periodate-oxidized dextran forms imine and ketoamine linkages by reaction with hemoglobin and the obtained conjugates are not completely stable, as their molecular size increases with time or decreases after incubation with lysine. The sulfated conjugates are more sensitive to lysine action than the unsulfated ones, which is consistent with the decreased possibilities of Amadori rearrangement. Therefore, this proves the importance of ketoamines for ensuring the cohesion of oxidized dextran-based conjugates. Carboxamidobenzaldehyde dextran forms only imine linkages with hemoglobin and the corresponding conjugates possess a marked instability in the absence of reductive treatment. The different types of conjugates could be stabilized by a sodium borohydride treatment in a satisfying manner.     

A Transient Study of Formation of Conjugates during TNT Metabolism by Plant Tissues

The formation of TNT-derived conjugates was investigated in hairy root tissue cultures of Catharanthus roseus and in aquatic plant systems of Myriophyllum aquaticum. The temporal profiles of four TNT-derived conjugates, TNT-1, 2A-1, TNT-2 and 4A-1, were determined over 3 to 16-day exposure durations. When axenic C. roseus roots were exposed separately to 2,4,6 trinitrotoluene, 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene, the array and levels of conjugates varied. Exposure of axenic roots to either 4-amino-2,6-dinitrotoluene or 2-amino-4,6-dinitrotoluene resulted in the formation of only 4A-1 and 2A-1, respectively, and not TNT-1 and TNT-2. However, amendment of previously unexposed roots with TNT produced all four conjugates. The conjugates were preferentially accumulated within the biomass phase of root cultures. Significantly, conjugates TNT-1 and TNT-2 were observed in the biomass phase of intact M. aquaticum plants exposed to TNT. The results clearly indicate the presence of common TNT transformation products in two diverse plants species and tissue type. The distribution of conjugates formed via monoamine derivatives of TNT, however, may be a function of several factors, including the starting xenobiotic type and/or level. Initial bulk rate constants for disappearance of 2,4,6 trinitrotoluene, 2-amino-4,6-dinitrotoluene, and 4-amino-2,6-dinitrotoluene were also determined. Their magnitude followed the order: TNT >> 4-A-2,6-DNT > 2-A-4,6-DNT.