Amino Acid Adducts Research Articles

Formation of peroxide- and globin-derived radicals from the reaction of methaemoglobin and metmyoglobin with t-butyl hydroperoxide: an ESR spin-trapping investigation.

The reaction of human methaemoglobin and horse metmyoglobin with t-butyl hydroperoxide (t-BuOOH) was investigated with the ESR spin-trapping technique. With the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) the formation of peroxyl, alkoxyl and methyl radicals derived from t-BuOOH could be detected. The relative contributions of these radicals were determined at various DMPO concentrations by computer simulation. From these data it could be concluded that the alkoxyl radical was the initial radical produced, which indicates that the hydroperoxide is cleaved homolytically. Further investigations, with the nitroso spin trap 2-methyl-2-nitrosopropane (MNP), showed the formation of globin-centred radicals. Non-specific proteolysis of the MNP adducts revealed isotropic three-line spectra, which means that the radical adducts were centred on a tertiary carbon with no bonds to a hydrogen or nitrogen. Comparison with MNP adducts of several amino acids indicated that in methaemoglobin the radical adduct was most probably located on a valine residue. With metmyoglobin the same adduct was obtained, whereas an additional adduct could be assigned to a tyrosyl radical. These protein radicals most probably resulted from hydrogen abstraction by the metal-oxo species, formed by heterolytic cleavage of the hydroperoxide. These results therefore show that homolytic cleavage of the hydroperoxide leads to the formation of peroxide-derived radicals, whereas concurrent heterolytic cleavage results in protein-derived radicals.

Synthesis and mass spectrometric identification of the major amino acid adducts formed between sulphur mustard and haemoglobin in human blood.

As part of a program to develop methods for the verification of alleged exposure to sulphur mustard, we synthesized and characterized three amino acid adducts presumably formed by alkylation of haemoglobin: 4-(2-hydroxyethylthioethyl)-L-aspartate, 5-(2-hydroxyethylthioethyl)L-glutamate and N1- and N3-(2-hydroxyethylthioethyl)-L-histidine. Suitable derivatization methods for GC/MS analysis were developed for these adducts as well as for the cysteine and the N-terminal valine adduct. Incubation of human blood with [35S]sulfur mustard in vitro followed by acidic hydrolysis of isolated globin and derivatization with Fmoc-Cl afforded three radioactive peaks upon HPLC analysis, one of which coeluted with the synthetic Fmoc derivative of N1/N3-(2-hydroxyethylthioethyl)-L-histidine. After pronase digestion of globin the adducts of histidine, glutamic acid, aspartic acid, cysteine and N-terminal valine could be tentatively identified and quantitated. Final identification was obtained from GC/MS analysis. The most abundant adduct, N1/N3-(2-hydroxyethylthioethyl)-L-histidine, could not be sensitively analysed by GC/MS. A convenient LC-tandem MS procedure was developed for this compound, enabling the detection of exposure of human blood to 10 microM sulphur mustard in vitro.

Reactions of 4 methylphenyl isocyanate with amino acids

Arylisocyanates are important intermediates in the chemical industry. Amongst the main damage after low levels of isocyanate exposure are lung sensitization and asthma. Protein adducts of isocyanates might be involved in the aetiology of sensitization reactions. Blood protein adducts are used as dosimeters for modifications of macromolecules in the target organs where the disease develops. To develop methods for the quantitation of protein adducts we reacted 4 methylphenyl isocyanate 4MPI with the tripeptide valyl glycyl glycine and with single amino acids yielding N 4 methylphenyl carbamoyl L valyl glycyl glycine 4MPI Val Gly Gly, N 4 methylphenyl carbamoyl L valine 4MPI Val, N 4 methylphenyl carbamoyl L aspartic acid 4MPI Asp, N acetyl S 4 methylphenyl carbamoyl L cysteine 4MPI AcCys, N acetyl N 4 methylphenyl carbamoyl lysine 4MPI AcLys, N acetyl O 4 methylphenyl carbamoyl tyrosine 4MPI AcTyr and N acetyl O 4 methylphenyl carbamoyl D,L serine 4MPI AcSer. The hydrolysis of the adducts was tested under acidic and basic conditions, to obtain the maximum yield of 4 methylaniline 4MA. The isocyanates were hydrolysed for 1 h, 3h and 24h at 100 C with 6 M HCl in and or 0.1 M NaOH at room temperature, following methods applied for the analyses of biological samples of arylisocyanate exposed workers. In addition, we applied a new protocol: the adducts were hydrolyzed for 1-24 h in 0.3 M NaOH at 100 C. The hydrolysates were analysed using HPLC with UV detection and quantified against the internal standard, 4 fluoroaniline or 4 chloroaniline. 4MA was obtained with the best yields using 0.3M NaOH; after 24 h all amino acid adducts were cleaved under these conditions. Acid hydrolysis of 4MPI Val and 4MPI Asp yielded the respective hydantoins 3 4 methylphenyl 5 isopropyl 1,3 imidazoline 2,4 dione and 2 1 4 methylphenyl 2,5 dioxoperhydro 4 imidazolyl acetic acid. For future studies, we propose to hydrolyse biological samples with 0.3 M NaOH at 100 C to release the maximum amount of 4MA from the adducts. However, in biological samples from workers, hydrolysable adducts can also result from arylamine exposure. Therefore, we propose to analyse the N terminal adducts of isocyanates with blood protein to distinguish between arylamine and arylisocyanate exposure.

4-Hydroxy-2-nonenal–Protein Adducts and Apoptosis in Murine Lung Cells after Acute Ozone Exposure

Ozone is a photochemically generated pollutant that can cause acute pulmonary inflammation and induce cellular injury and may contribute to the development or exacerbation of chronic lung diseases. Despite extensive investigation, the mechanisms of ozone and oxidant-induced cellular injury are still uncertain. Ozone has been reported to cause the formation of aldehydes in biological fluids that could explain many of the cellular effects caused by ozone. One of the most toxic aldehydes formed during oxidant-induced lipid peroxidation is 4-hydroxy-2-nonenal (HNE). HNE reacts primarily with Cys and secondarily with Lys and His amino acids, altering protein function and forming protein adducts that can be detected using specific adducts. In this study, we investigated whether ozone could cause the formation of HNE by assaying for HNE–protein adducts in cells isolated by lung lavage from C3H/HeJ mice exposed to 2.0 and 0.25 ppm ozone for 3 hr. Since oxidative stress and HNE have been shown to cause apoptosis we also examined the lung lavage cells for evidence of apoptosis following ozone exposure. Using a specific polyclonal antibody against HNE–amino acid adducts, two principle HNE–protein adducts were detected by Western analysis in cells obtained after ozone exposure at approximately 86–90 and 32 kDa. In addition to cell necrosis, apoptosis of lung cells was significant 3 hr after ozone exposure as detected using a Cell Death ELISA procedure and confirmed with DNA ladder and morphological analysis. The apoptotic cell injury peaked at 6 hr postexposure and decreased by 24 hr. Taken together, these results demonstrate that HNE is formedin vivofollowing ozone exposure and HNE appears to form specific protein adducts in lung cells. Furthermore, ozone can cause lung cell injury by an apoptotic mechanism in addition to a necrotic mechanism. Since HNE is toxic to cells and is formed as a result of ozone exposure, it may contribute to the lung cell injury following ozone exposure.

Characterization of neutral fragments in tandem mass spectrometry: a unique route to mechanistic and structural information.

The neutral species eliminated upon fragmentation of fast-moving mass-selected ions can be directly identified by collisional ionization and detection in neutral fragment reionization (Nf R) mass spectra. Establishment of the identity of neutral fragments yields valuable insight into the decomposition mechanism of a precursor ion, as demonstrated for fullerene and alkali metal iodide cluster ions as well as metal ion adducts of amino acids. In addition, neutral fragment reionization also provides structural information that may not be available from the complementary ionic fragments alone; this is illustrated in the differentiation of isomeric mononucleotides. The parameters influencing the appearance of Nf R spectra are discussed and the scope and general applicability of the method are briefly evaluated.

Characterization of Epitopes Recognized by 4-Hydroxy-2-nonenal Specific Antibodies

In the present study, we have raised anti-peptide antibodies directed to the major membrane lipid peroxidation product 4-hydroxy-2-nonenal (HNE) attached covalently to histidine, and their specificities were compared with those of the polyclonal antibodies (anti-HNE–protein antibodies) raised against HNE-treated keyhole limpet hemocyanin (K. Uchidaet al.(1993)Proc. Natl. Acad. Sci. USA90, 8742–8746). The anti-HNE-histidyl peptide antibodies (anti-HNE–histidine antibodies) were prepared by immunizing rabbits with a HNE-conjugated heptapeptide (Gly3-His-Gly3amide) coupled to the carrier protein. The antisera were purified on an affinity gel prepared by covalent attachment of a HNE-conjugated heptapeptide (Ala3-His-Ala3amide). Among the structurally defined 4-hydroxy-2-alkenal–amino acid adducts tested, binding of anti-HNE–histidine antibodies to the HNE-treated protein was not only inhibited by HNE–histidine, HNE–cysteine, and HNE–lysine, but also by 4-hydroxy-2-octenal–histidine and 4-hydroxy-2-decenal–histidine adducts. Cross-reactivity studies revealed that both anti-HNE–protein antibodies had the highest affinity for the HNE-treated protein and that neither of the antibodies cross-reacted with the protein treated with aldehydes including malondialdehyde, 1-hexanal, 2-hexenal, or 2-nonenal. These results suggest that the dominant epitope recognized by antibodies is the 2-CH3(CH2)n-5-hydroxytetrahydrofuran (n≥ 3) moiety of the Michael adducts. The immunohistochemical analysis of atherosclerotic lesions of human aorta demonstrated that these antibodies reacted strongly with granular cytoplasmic elements of foam cells and weakly with elements in the surrounding sclerotic stroma.

Covalent binding of ethylene dibromide and its metabolites to albumin

The present study was undertaken to determine covalent binding of [1,2- 14C]ethylene dibromide (EDB) to albumin under in vivo and in vitro conditions. For the in vivo covalent binding, 25 mg kg body weight of [1,2- 14C]EDB was given daily to male rats for 12 consecutive days and the animals were sacrificed at 24 h following the last dose. Blood was withdrawn from inferior vena cava in heparinized tubes and plasma was separated, dialyzed against ice-cold 10 mM phosphate buffer (pH 7.4) and then subjected to size-exclusion high-performance liquid chromatography (SE-HPLC). A major radioactive peak eluted at an elution volume corresponding to 65 000 dalton molecular mass was found to be associated to albumin at a level of 0.14 nmol equivalent EDB mg protein. For the in vitro covalent binding, human plasma or purified albumin was incubated with [1,2- 14C]EDB in the presence of phenobarbital-treated rat liver microsomes and NADPH-generating system for 2 h at 37°C. The 100 000 × g supernatant of the incubation mixture was dialyzed extensively and analyzed as described for the in vivo studies. Approximately 0.28 nmol equivalent EDB mg protein was found to be associated to albumin (about 2-fold higher than the in vivo binding). Binding of 14C-label to albumin under in vivo and in vitro conditions was further supported by the affinity chromatography of albumin fraction isolated by SE-HPLC. Reversed-phase HPLC analysis of pronase digest of the albumin obtained from in vitro studies indicated formation of several amino acid adducts of EDB and/or its metabolites. Structure elucidation of such amino acid adducts will be helpful in developing a relatively non-invasive method of measuring the EDB exposure.

Identification of a cysteinyl adduct of oxidized 3-methylindole from goat lung and human liver microsomal proteins.

3-Methylindole is a selective pneumotoxin that is oxidized by cytochrome P-450 enzymes to a reactive intermediate. 3-Methyleneidolenine, a methylene imine electrophile, is the postulated reactive intermediate, and it binds to proteins, a reaction that probably initiates the pneumotoxicity of 3-methylindole. Thioether adducts of this electrophile are formed with glutathione in vitro, but the identity of the adducted electrophile with amino acid residues of microsomal proteins had not previously been determined. 3-Methylindole was incubated with NADPH and goat lung or human liver microsomal proteins, and the proteins were hydrolyzed. 3-(Cystein-S-ylmethyl)indole was isolated and identified as the major amino acid adduct of 3-methyleneindolenine, demonstrating that cysteine thiols preferentially attack the exocyclic methylene position and result in a covalently (thioether) attached 3-methylindole residue to these pulmonary and hepatic proteins. These results demonstrate that the putative methylene imine intermediate is indeed the active electrophile that binds to proteins and presumably initiates the toxic events.

Tissue distribution of monochloroacetic acid and its binding to albumin in rats.

Distribution of monochloroacetic acid (CA) was studied in rats given a single oral dose of 0.1 mmole/kg body weight [1-14C]CA, by gavage. The animals were sacrificed at 4, 8, 12, 24 and 48 hr following the treatment. The distribution of 14C-label, determined in different tissues, suggests that CA is rapidly absorbed and eliminated from the body. The elimination phase appears to be fast for intestine and kidney as compared to other tissues. Maximum radioactivity was detected in intestine and kidney at 4 and 8 hr following the treatment which was followed by liver, spleen, testes, lung, brain and heart in a decreasing order. A group of rats treated with a single oral dose of 1 mmole/kg [1-14C]CA, by gavage, was also sacrificed at 24 hr following the exposure to study the effect of a higher dose on distribution of [1-14C]CA. The distribution of 14C-label at both dose levels indicates that toxicokinetic properties of CA are dose-dependent. Another group of rats administered 1 mmole/kg [1-14C] CA daily for three days was also sacrificed at 24 hr following the last dose to evaluate the bioaccumulating properties of CA and/or its metabolites in the tissues. As compared to the number of doses given, the accumulation of 14C-label was not as large as expected. 14C-Label determined in the dialyzed plasma, suggests an in vivo binding of 14C-label to plasma proteins where albumin accounted for about 65% as determined by affinity chromatography. Isolation and identification of the adducts of amino acids will help in understanding the mechanism of CA toxicity and development of a suitable biomarker of CA exposure.

In vivo detection and characterization of protein adducts resulting from bioactivation of haloethene cysteine S-conjugates by 19F NMR: chlorotrifluoroethene and tetrafluoroethene.

Several haloalkenes are selective nephrotoxins. The bioactivation of nephrotoxic haloalkenes involves hepatic glutathione S-conjugate formation, peptidase-catalyzed metabolism of the glutathione S-conjugates to the corresponding cysteine S-conjugates, uptake of cysteine S-conjugates by the kidneys, and renal cysteine conjugate beta-lyase-catalyzed beta-elimination of a thiol. The haloalkyl and haloalkenyl thiols thus released are unstable and yield reactive intermediates whose interactions with cellular constituents are though to contribute to the observed toxicity of S-conjugates. Tetrafluoroethene and chlorotrifluoroethene are metabolized to the cysteine S-conjugates S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC) and S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine (CTFC), respectively. Administration of TFEC (1.0 mmol/kg) or CTFC (1.0 mmol/kg) to rats resulted in acylation of renal proteins, as demonstrated with 19F nuclear magnetic resonance spectroscopy. Single, broad resonances near 41 or 56 ppm were found in spectra of renal proteins from TFEC- or CTFC-treated rats, respectively, and these resonances were not lost on dialysis. Renal protein incubated with 2-chloro-1,1,2-trifluoroethyl-2-nitrophenyl disulfide, a proreactive intermediate that yields 2-chloro-1,1,2-trifluoroethanethiol, showed the same 19F NMR spectrum as was found with CTFC-treated rats. In vitro incubation of various N alpha-blocked amino acids with this proreactive intermediate indicated that only lysine is stably adducted, whereas histidine is transiently acylated. In each case, proteolysis of modified protein converted a single broad NMR resonance to a doublet with little change in chemical shift and with clearly resolved, characteristic H-F couplings. The single, stable amino acid adduct formed with renal proteins of rats given CTFC or TFEC was N epsilon-(chlorofluorothioacetyl)lysine and N epsilon-(difluorothioacetyl)lysine, respectively.

Application of newly synthesized detergents in the side chain degradation of plant sterols by Mycobacterium fortuitum.

Newly synthesized detergents of polyoxyethylene polyoxypropylene co-polymers (Co-EOPO), polyoxyethylene polyoxypropylene adducts of the diethylene triamine (DETA-EOPO), and steroidal detergents (SDD) stimulate significantly side chain degradation of plant sterols referring to both the sterol degradation and the formation of the product, 9 alpha-hydroxy-androsta-4-ene-3,17-dione (9 OH-AD), by Mycobacterium fortuitum NRRL-B-8119. Highest stimulative effects were observed with derivatives of the DETA-EOPO group. Using compounds of the Co-EOPO group optimal sterol transformation rates were found for polymers with a polyoxypropylene domain of 1400-2000 in molecular weight and with a polyoxyethylene content of 20-50%. Mostly efficient steroidal detergents tested were the 4-cholesten-3-(O-carboxymethyl)-oxime and the amino acid adducts of sterol-3 beta-chlorocarbonates. Detergents were favourably applied as pre-formed sterol-detergent complexes (9:1/w:w) in submers fermentation procedures. By treating harvested resting cells of M. fortuitum with derivatives of Co-EOPO or DETA-EOPO a considerable activation of the cells takes place concerning the sterol degradation and the formation of 9 OH-AD at a high yield in subsequent transformation process on buffer. In solubilization experiments we revealed that there is no correlation between the detergent mediated solubilization of the hydrophobic sterol substrate in the aqueous medium and the transformation activity of the bacterial cells. The detergents are assumed to interact with a multicomponent mesophase (FMCM) which is placed between cells and sterol surface and mediates the transport.

Reactivity and adduct formation of a polyaromatic hydrocarbon, 7-bromomethylbenz[ a]anthracene, with chromatin histone proteins

The alkylation of histones by the direct-acting carcinogen 7-bromomethylbenz[ a]anthracene was demonstrated both in vivo and in vitro. The relative molar reactivity for mouse liver histones in vivo was H3>H1>H2b>H4>H2a. The in vitro modification of histone H3 was examined in detail. Amino acid adducts stable to acid hydrolysis were separated after acetylation by reversed-phase high-performance liquid chromatography and characterized using ultraviolet absorbance spectra and synthetic amino acid adduct standards. Three major adducts were observed and tentatively identified as cysteinyl, lysyl and histidinyl adducts of histone H3.

Binding of Amino Acids with a Bifunctional Metalloporphyrin via Concurrent Metal-Coordination and Electrostatic Interactions

Abstract The Cl–Rh(III) complex of 5,15-bis [2-(N,N-dimethylaminomethyl-1-phenyl]octaethylporphyrin undergoes intermolecular coordination to give the dimer in CDCl3, which, upon treatment with an aqueous amino acid solution, readily forms a monomeric amino acid adduct via a two-point interaction involving the metal coordination and electrostatic interaction or salt formation.

Amino acid profiling using HRGC-FTIR oftert.-Butyldimethylsilyl derivatives

The use of N, O (S)-tert.-Butyldimethylsilyl derivatives of amino acids for capillary gas chromatography and FTIR identification of amino acids is described. Rapid identification is achieved by computerised comparison of FTIR spectra. Derivatives that are characterised by identical sets of ions in gas chromatography-mass spectrometry experiments are clearly distinguished and identified by means of their infrared spectra. This, for example, is the case with alanine, β-alanine and sarcosine, which norvaline and valine, and with norleucine, leucine and isoleucine. Unknows are also promptly detected. This is especially useful in screening biological samples for less common amino acids. Screening of hemoglogin hydrolysates for amino acid adducts is exemplified.

High-performance liquid chromatography with photochemical fluorimetric detection of tryptophan based on 4-fluoro-7-nitrobenzo-2-oxa- 1,3-diazole : Total protein amino acid analysis

The simultaneous determination of amino acids including trytophan is described. The NBD- F forms a single adduct with tryptophan as with other amino acids, but the adduct lacks intrinsic fluorescence. After ultraviolet irradiation, the adduct fluoresces (pale-green); the fluorescence intensity increases with increasing irradiation time at pH 2-10, Under the same conditions, the other amino acid adducts are slowly decomposed. When the tryptophan adduct, separated on a Nucleosil ODS column (150×4.6 mm, 6 μm), is irradiated in an on-line photochemical reactor (310 nm), its fluorescence peak appears between those of the phenylalanine and lysine adducts. The detection limit for tryptophan by the proposed method is 3 pmol; the limits for other amino acids are 10–100 fmol.

Covalent binding of 4-nitrobenzyl mercaptan S-sulfate to the sulfhydryl groups of hepatic cytosolic proteins and bovine serum albumin with mixed disulfide bond formation

4-Nitrobenzyl [ 35S]mercaptan S-sulfonic acid ([ 35S]NBM S-sulfate), a new type of reactive metabolite of the thiol [ 35S]NBM in rat liver cytosol fortified with 3′-phosphoadenosine 5′-phosphosulfate, bound rapidly and covalently at pH 7.4 and 37°C to the sulfhydryl groups of rat liver cytosolic proteins with formation of disulfide bonds. From the radioactive proteins was isolated and identified the sole amino acid adduct, S-([ 35S]NBM)cysteine, after their acid hydrolysis under the anaerobic conditions. Bovine serum albumin (BSA), a model protein with a single SH group, also reacted readily with radioactive NBM S-sulfate to form a disulfide bond in stoichiometric manner. S-([ 35S]NBM)cysteine was also isolated and identified as the sole amino acid adduct from the well-washed, radioactive BSA after the same anaerobic acid hydrolysis. A normal hepatic level of GSH not only retarded the BSA-NBM adduct formation completely, but also detached the radioactivity from BSA by the reduction of the disulfide bond with formations of [ 35S]NBM and its disulfide. Of twenty-one amino acids examined at pH 7.4 and 37°C, only cysteine reacted with NBM S-sulfate and afforded S-(NBM)cysteine with concomitant formations of S-sulfocysteine, cystine, NBM, and its disulfide.

Photochemical coupling of 5-bromouracil to tryptophan, tyrosine and histidine, peptide-like derivatives in aqueous fluid solution.

AbstractDirect irradiation of 5‐bromouracil (BU) in aqueous fluid solution in the presence of tryptophan (trp), tyrosine (tyr) or histidine (his) derivatives using a XeCl excimer laser at 308 nm yielded photocoupling of BU to the aromatic ring of each amino acid. Irradiation of BU at 308 nm most likely results in excitation of the n‐φ* transition, intersystem crossing to the triplet manifold, and coupling via electron transfer from the aromatic amino acid. The coupling observed was regiospecific between the 5‐position of uracil (U) and the 2‐position of the indole and phenol rings and the 5‐position of the imidazole ring of the respective amino acids. Quantum yields of photocoupling to BU ranged from 1 × 10‐3 to 7 × 10‐3 and paralleled known rates of electron transfer and ionization potentials of the aromatic rings. The photocoupling between BU and some of the aromatic amino acid peptide‐like derivatives possibly mimics photocrosslinking of BU‐DNA to associated proteins, a potentially useful photoreaction for studying nucleic acid‐protein interactions. Formation of crosslinks of the type proposed here might be detected by the characteristic fluorescence emission of the uracil amino acid adducts.

Resonance Raman spectroscopy of pyridoxal Schiff bases.

Resonance Raman (RR) spectra are reported for amino acid and amine adducts of pyridoxal 5'-phosphate (PLP) and 5'-deoxypyridoxal (5'-dPL) in aqueous solution. For the valine adducts, a detailed study has been carried out on solutions at pH and pD 5, 9, and 13, values at which the pyridine and imine protons are successively ionized, and on the adducts formed from 15N-valine, alpha-deuterovaline, and N-methyl-PLP. Good quality spectra were obtained, despite the strong fluorescence of pyridoxal Schiff bases, by adding KI as a quencher, and by exciting the molecules on the blue side of their absorption bands: 406.7 nm (cw Kr+ laser) for the pH 5 and 9 species (lambda max = 409 and 414 nm), and 354.7 nm (pulsed YAG laser, third harmonic) for the pH 13 species (lambda max = 360 nm). A prominent band at 1646 cm-1 is assigned to the imine C=N stretch via its 13 cm-1 15N shift. A 12 cm-1 down-shift of the band in D2O confirms that the Schiff base linkage is protonated at pH 9. Deprotonation at pH 13 shifts VC = N from 1646 to 1629 cm-1, values typical of conjugated Schiff bases. The strongest band in the spectrum, at 1338 cm-1, shifts to 1347 cm-1 upon pyridine protonation at pH 5, and is assigned to a ring mode with a large component of phenolate C-O stretch. A shoulder on its low-frequency side is assigned to the C4-C4' stretch. Large enhancements of these modes can be understood qualitatively in terms of the dominant resonance structures contributing to the ground and resonant excited states. A number of weaker bands are observed, and assigned to pyridine ring modes. These modes gain significantly in intensity, while the exocyclic modes diminish, when the spectra are excited at 266 nm (YAG laser, fourth harmonic) in resonance with ring-localized electronic transitions.

A revised structure of the antitumor drug elliptinium — Amino(acid) adducts

Using simple organic reactions and spectrometric techniques (nuclear magnetic resonance and mass spectrometry), we propose a revised structure of the adducts obtained through the H2O2-peroxidase oxidation of the antitumor drug ellipticinium and aliphatic amino(acid) compounds. These derivatives display an oxazole ring structure between the O(9)- and N(10)-atoms.

Structural characterization of the major covalent adduct formed in vitro between acetaminophen and bovine serum albumin

The structure of the covalent adduct formed in vitro between [14C]-acetaminophen ([14C]APAP) and bovine serum albumin (BSA) has been investigated with the aid of new analytical methodology. The APAP-BSA adduct, isolated from mouse liver microsomal incubations to which the radiolabeled drug and BSA had been added, was cleaved using a combination of specific (cyanogen bromide) and non-specific (acid hydrolysis) procedures, following which the mixture of amino acids obtained was derivatized, in aqueous solution, with ethyl chloroformate. The resulting ethoxycarbonyl derivatives were recovered by extraction into ethylacetate, methylated and subjected to profile analysis using both reverse-phase and normal-phase HPLC techniques. In each HPLC step, one major radioactive amino acid adduct was detected and was identified by mass spectrometry as the derivative of 3-cystein-S-yl-4-hydroxyaniline. Based on this finding, and with a knowledge of the behavior under acidic hydrolysis conditions of the 3-cysteinyl conjugate of APAP, it could be concluded that the major APAP-BSA adduct is one in which the drug is bound, via a thioether linkage at the C-3 position, to a sulfhydryl group on the protein. Furthermore, it could be established that this -SH function almost certainly is that associated with the cys-34 residue of BSA.