The mechanism of the peroxynitrite-mediated oxidation of myoglobin in the absence and presence of carbon dioxide

Among the cellular components that can react directly with peroxynitrite in the presence of physiological CO(2) concentrations are sulfur-, selenium-, and metal-containing proteins, in particular hemoproteins. We have previously shown that the reactions of peroxynitrite with oxymyoglobin (MbFeO(2)) and oxyhemoglobin proceed via the corresponding ferryl species, which, in a second step, are reduced to the iron(III) forms of the proteins (metMb and metHb). In this study, we have investigated the influence of the concentration of added CO(2) on the kinetics and the mechanism of the peroxynitrite-mediated oxidation of MbFeO(2). We found that this reaction proceeds in two steps via the formation of MbFe(IV)=O also in the presence of millimolar amounts of CO(2). As compared to the values measured in the absence of added CO(2), the second-order rate constant for the first reaction step in the presence of 1.2 mM CO(2) is significantly larger [(4.1 +/- 0.7) x 10(5) M(-1) s(-1), at pH 7.5 and 20 degrees C], whereas that for the peroxynitrite-mediated reduction of MbFe(IV)=O to metMb is almost unchanged [(3.2 +/- 0.2) x 10(4) M(-1) s(-1), at pH 7.5 and 20 degrees C]. Finally, we show that because of the parallel decay of peroxynitrite, 8-25 equiv are needed to completely oxidize MbFeO(2) to metMb, with larger amounts required to oxidize diluted MbFeO(2) solutions in the presence of CO(2). Simulation of the reaction in the absence and presence of CO(2) was carried out to get a better understanding of the mechanism. The results suggest that CO(3)*(-) and NO(2)* may be involved in the reaction and interact with MbFeO(2) and MbFe(IV)=O, respectively.     

Kinetic constraints for the thiolysis of 4-methyl-5-(pyrazin-2-yl)-1,2-dithiole-3-thione (oltipraz) and related dithiole-3-thiones in aqueous solution

This report summarizes an investigation of the reactions of biological and other thiols with the cancer chemopreventive oltipraz and other dithiolethiones. Analysis of the kinetics of reaction of 4-methyl-5-(pyrazin-2-yl)-1,2-dithiole-3-thione (oltipraz) 1 with monothiols and dithiols in the range of 0.75-20 mM in aqueous 15% ethanol, at pH 7.5 (0.1 M Tris buffer) and at 37 degrees C has been undertaken. A plot of k(obsd) against [thiol] shows that reactions of mono- and dithiols are first order in thiol concentration. The dependence on pH of these reactions shows that the active species is the thiolate anion. Specific second-order rate constants, k(2) (M(-1) s(-1)) for reaction of the thiolate anions with oltipraz have been determined to be cysteine, 0.040 +/- 0.001; 2-mercaptoethanol, 2.0 +/- 0.02; glutathione, 0.099 +/- 0.001; mercaptoacetic acid anion, 4.0 +/- 0.01; dithiothreitol, 1.33 +/- 0.02; 1,3-propanedithiol, 10 +/- 0.5; 1-mercaptopropane-3-ol, 6.5 +/- 0.1; 1-mercaptopropane-2,3-diol, 1.26 +/- 0.05. A plot of pK(a) against log k(2) for monothiols shows a linear dependence of k(2) on pK(a), beta(nuc) 1.1 +/- 0.07, which accounts for most of the reportedly enhanced reactivity of dithiols over monothiols. The pseudo-first-order rate constant for the solvolysis of oltipraz has been measured as 2.2 (+/-0.2) x 10(-8) s(-1). The kinetics of reaction of three other dithiole-3-thiones with glutathione has also been studied for comparison with oltipraz. The specific second-order rate constants, k(2) (M(-1) s(-1)) are 5-phenyl-1,2-dithiole-3-thione, 4.7 x 10(-)(4); 5-(4-methoxyphenyl)-1,2-dithiole-3-thione, 4.1 x 10(-4); and 1,2-dithiole-3-thione 0.08. Important implications for the mode of biological action of these compounds and the nature of the putative biological targets of the compounds are discussed.     

Synthesis of bisphosphonate derivatives of ATP by T4 DNA ligase, ubiquitin activating enzyme (E1) and other ligases

T4 DNA ligase and the ubiquitin activating enzyme (E1), catalyze the synthesis of ATP beta,gamma-bisphosphonate derivatives. Concerning T4 DNA ligase: (i) etidronate (pC(OH)(CH(3))p) displaced the AMP moiety of the complex E-AMP in a concentration dependent manner; (ii) the K(m) values and the rate of synthesis k(cat) (s(-1)), determined for the following compounds were, respectively: etidronate, 0.73+/-0.09 mM and (70+/-10)x10(-3) s(-1); clodronate (pCCl(2)p), 0.08+/-0.01 mM and (4.1+/-0.3)x10(-3) s(-1); methylenebisphosphonate (pCH(2)p), 0.024+/-0.001 mM and (0.6+/-0.1)x10(-3) s(-1); tripolyphosphate (P(3)) (in the synthesis of adenosine 5'-tetraphosphate, p(4)A), 1.30+/-0.30 mM and (6.2+/-1.1)x10(-3) s(-1); (iii) in the presence of GTP and ATP, inhibition of the synthesis of Ap(4)G was observed with clodronate but not with pamidronate (pC(OH)(CH(2)-CH(2)-NH(3))p). Concerning the ubiquitin activating enzyme (E1): methylenebisphosphonate was the only bisphosphonate, out of the ones tested, that served as substrate for the synthesis of an ATP derivative (K(m)=0.36+/-0.09 mM and k(cat)=0.15+/-0.02 s(-1)). None of the above bisphosphonates were substrates of the reaction catalyzed by luciferase or by acyl-CoA synthetase. The ability of acetyl-CoA synthetase to use methylenebisphosphonate as substrate depended on the commercial source of the enzyme. In our view this report widens our knowledge of the enzymes able to metabolize bisphosphonates, a therapeutic tool widely used in the treatment of osteoporosis.     

Gibbs energy of formation of peroxynitrite

A standard Gibbs energy of formation of 16.6 kcal mol(-)(1) has been reported for peroxynitrite [Merényi, G., and Lind, J. (1998) Chem. Res. Toxicol. 11, 243-246]. This value is based on the rate constants for the forward and backward rate constants of the equilibrium O2*- + NO* if ONOO(-). A rate constant of 0.017 s(-)(1) for the backward rate constant was determined by observing the formation of C(NO(2))(3)(-) when peroxynitrite was mixed with C(NO(2))(4). However, a similar rate constant is also observed in the presence of NO(*), which indicates that formation of C(NO(2))(3)(-) is due to a process other than the reduction of C(NO(2))(4) by O2*-. Additionally, copper(II) nitrilotriacetate enhances the decay of ONOO(-) at pH 9.3, without reduction of copper(II). The preferred thermodynamic values are therefore as follows: Delta(f)H degrees (ONOO(-)) = -10 +/- 2 kcal mol(-)(1), Delta(f)G degrees (ONOO(-)) = 14 +/- 3 kcal mol(-)(1), S degrees (ONOO(-)) = 31 eu, and E degrees '(ONOOH/NO(2)(*), H(2)O) = 1.6 V at pH 7 [Koppenol, W. H., and Kissner, R. (1998) Chem. Res. Toxicol. 11, 87-90].     

Alkaline hydrolysis of oxaliplatin--isolation and identification of the oxalato monodentate intermediate

The alkaline degradation of the chemotherapeutic agent oxaliplatin has been studied using liquid chromatography. The oxalato ligand is lost in two consecutive steps. First, the oxalato ring is opened, forming an oxalato monodentate intermediate, as identified by electrospray ionization mass spectrometry. Subsequently, the oxalato ligand is lost and the dihydrated oxaliplatin complex is formed. The observed rate constants for the first step (k(1)) and the second step (k(2)) follow the equation k(1) or k(2) = k(0) + k(OH(-) )[OH(-)], where k(0) is the rate constant for the degradation catalyzed by water and k(OH(-) ) represents the second-order rate constant for the degradation catalyzed by the hydroxide ion. At 37 degrees C the rate constants for the first step are k(OH(-) ) = 5.5 x 10(-2) min(-1) M(-1) [95% confidence interval (CI), 2.7 x 10(-2) to 8.4 x 10(-2) min(-1) M(-1)] and k(0) = 4.3 x 10(-2) min(-1) (95% CI, 4.0 x 10(-2) to 4.7 x 10(-2) min(-1)). For the second step the rate constants are k(OH(-) ) = 1.1 x 10(-3) min(-1) M(-1) (95% CI, -1.1 x 10(-3) to 3.3 x 10(-3)) min(-1) M(-1) and k(0) = 7.5 x 10(-3) min(-1) (95% CI, 7.2 x 10(-3) to 7.8 x 10(-3) min(-1)). Thus, the ring-opening step is nearly six times faster than the step involving the loss of the oxalato ligand.     

Thermodynamics of solubility, sublimation and solvation processes of parabens.

Saturated vapor pressures for a number of parabens (methyl- (MePB); ethyl- (EtPB), n-propyl- (PrPB) and n-butyl- (BuPB)) were obtained and from their respective temperature dependences the sublimation enthalpy, DeltaH(sub)( degrees ), and sublimation entropy, DeltaS(sub)(degrees), as well as their respective relative fractions in the process calculated. The sublimation enthalpies are: DeltaH(sub)(degrees)(MePB)=98.8+/-0.8; DeltaH(sub)(degrees)(EtPB)=100.9+/-0.7; DeltaH(sub)(degrees)(PrPB)=123.7+/-0.6; DeltaH(sub)(degrees)(BuPB)=108.4+/-0.8 kJmol(-1). The obtained values are discussed with regard to X-ray data from the literature. Theoretical calculations of the respective crystal lattice energies were carried out and compared to the experimental data. The following parameters were analyzed: (a) energetic contribution of van der Waals forces and hydrogen bonding to the total packing energy of the crystals; (b) contributions of the different fragments of the paraben molecules to the packing energy; (c) influence of bias of the supposed C-H distances on the result of the calculation procedure. Enthalpies of evaporation were estimated from the measured enthalpies of sublimation and enthalpies of fusion, and compared with literature data. Moreover, the thermodynamic functions of solvation of the molecules in water and in a number of n-alcohols were evaluated. The thermodynamic terms (Gibbs energy, enthalpy and entropy) of the solvation process were split up in their respective specific and nonspecific fraction, and these values compared for all combinations of parabens and solvents. The influence of mutual saturation of the phases in the water-octanol system on the partitioning process of the molecules is also discussed.     

Kinetics and mechanism of the hydrolytic degradation of indinavir: intramolecular catalysis

The pH-rate profile of first-order rate constants for the lactonization of Indinavir in aqueous solutions with ionic strength I = 1 (KCl) at 40 degrees C is reported. The lactonization reaction is a subject of strong buffer catalysis with a nonlinear dependence of the first-order rate constants on the concentration of the buffer. The pH-rate profile is more complex than the pH-rate profiles for the hydrolysis of simple peptides and for the intramolecular OH-catalyzed hydrolysis of gamma-hydroxyamides. This complexity appears unique to Indinavir and is a result of the cis-aminoindanol leaving group. The mechanistic pathways for the lactonization are discussed. The buffer catalysis data are consistent with kinetic general acid catalysis. The second-order rate constant for the specific-acid catalyzed hydrolysis of Indinavir at 40 degrees C (k(H) = 2.2 x 10(-4) M(-1) min(-1)) is similar to that for a simple peptide indicating similar interactions in the rate limiting transition state for both reactions.     

Stability of andrographolide in powdered Andrographis herb under accelerated conditions

The stability of andrographolide in powdered Andrographis Herb--the aerial part of Andrographis paniculata (Burm. f.) Nees (Acanthaceae)--was determined using a heat-accelerated experiment to reveal a second-order kinetics of degradation. The fast decomposition was observed regardless of the method of analysis. The rate constant of the decomposition of andrographolide at 25 degrees C ( K(25)( degrees C)), predicted from the Arrhenius plot, was 6.58 x 10 (-6) d (-1).     

Crotoxin acceptor protein isolated from Torpedo electric organ: binding properties to crotoxin by surface plasmon resonance.

Crotoxin, a potent neurotoxin from the South American rattlesnake Crotalus durissus terrificus, is a heterodimeric phospholipase A(2) (EC 3.1.1.4), which blocks the release of acetylcholine from peripheral neurons. We previously have suggested the existence of a 48 kDa crotoxin-binding protein in the presynaptic membranes of the electric organ of Torpedo marmorata. Here, we report the purification and characterization of this protein that we called the crotoxin acceptor protein from Torpedo (CAPT). The membranes of electric organs from Torpedo were solubilized with a detergent (4% (w/v) Triton X-100) and CAPT was isolated by affinity chromatography on a crotoxin column. SDS-PAGE showed that the purified protein was homogeneous and cross-linking studies with radioiodinated crotoxin confirmed that it had retained its toxin-binding properties. The purified CAPT has similar molecular mass as crocalbin, a crotoxin-binding protein isolated from porcine brains, yet anti-crocalbin antiserum failed to recognize CAPT. Surface plasmon resonance biosensor technology was used to measure the specific interaction between crotoxin and solubilized CAPT. Using this method, it was possible to follow CAPT throughout the purification procedure. As well, an apparent dissociation constant (K(d)(app)) of 3.4 nM was calculated for the interaction of pure CAPT and crotoxin from the dissociation rate constant (k(off)=1.2 x 10(-2)s(-1)) and the association rate constant (k(on)=3.5 x 10(6)M(-1)s(-1)).     

Reactivity of perhydroxyl (HOO.) with 1,4-cyclohexadiene (model for allylic groups in biomembranes)

The electron-transfer reduction of molecular oxygen yields superoxide ion (O2.-), which reacts with proton sources to form HO2.. In water the latter species disproportionates via reaction with O2.- (kbi, 10(8) M-1 s-1) and itself (kd, 10(6) M-1 s-1). The rate constants (kd) for the homolytic disproportionation process (HO2. + HO2.----H2O2 + O2), which have been determined from stopped-flow spectrophotometric decay data for HO2. at 25 degrees C, are (1.7 +/- 0.5) x 10(4) M-1 s-1 in dimethyl sulfoxide (Me2SO), (5.3 +/- 0.5) x 10(4) M-1 s-1 in dimethylformamide (DMF), and approximately 10(7) M-1 s-1 in acetonitrile. With limiting fluxes of protons to control the rate of formation of HO2. from O2.-, the rate of decay of HO2. is enhanced by reaction with the allylic hydrogens of excess 1,4-cyclohexadiene (RH). On the basis of such data the apparent second-order process (HO2. + RH----R. + H2O2) has a rate constant (kox) of (1.6 +/- 0.6) x 10(2) M-1 s-1. The reactivity of HO2. decreases as its solvation energy increases.     

Kinetics of hypobromous acid-mediated oxidation of lipid components and antioxidants

Hypohalous acids are generated from the oxidation of halide ions by myeloperoxidase and eosinophil peroxidase in the presence of H2O2. These oxidants are potent antibacterial agents, but excessive production can result in host tissue damage, with this implicated in a number of human pathologies. Rate constants for HOCl with lipid components and antioxidants have been established. Here, the corresponding reactions of HOBr have been examined to determine whether this species shows similar reactivity. The second-order rate constants for the reaction of HOBr with 3-pentenoic acid and sorbate, models of unsaturated lipids, are 1.1x10(4) and 1.3x10(3) M(-1) s(-1), respectively, while those for reaction of HOBr with phosphoryl-serine and phosphoryl-ethanolamine are ca. 10(6) M(-1) s(-1). The second-order rate constants (M(-1) s(-1)) for reactions of HOBr with Trolox (6.4x10(4)), hydroquinone (2.4x10(5)), and ubiquinol-0 (2.5x10(6)) were determined, as models of the lipid-soluble antioxidants, alpha-tocopherol, and ubiquinol-10; all of these rate constants are ca. 50-2000-fold greater than for HOCl. In contrast, the second-order rate constants for the reaction of HOBr with the water-soluble antioxidants, ascorbate and urate, are ca. 10(6) M(-1) s(-1) and closer in magnitude to those for HOCl. Kinetic models have been developed to predict the sites of HOBr attack on low-density lipoproteins. The data obtained indicate that HOBr reacts to a much greater extent with fatty acid side chains and lipid-soluble antioxidants than HOCl; this has important implications for HOBr-mediated damage to cells and lipoproteins.     

Mechanism of nickel assault on the zinc finger of DNA repair protein XPA

Xeroderma pigmentosum group A complementing protein (XPA) is a member of the protein complex of the nucleotide excision repair (NER) pathway of DNA repair, participating in the assembly of the incision complex. The 4S zinc finger domain of XPA is involved the interactions with other NER proteins. As demonstrated previously, the activity of XPA is compromised by several metal ions implicated in DNA repair inhibition, including Ni(II), Cd(II), and Co(II) (Asmuss, M., Mullenders, L. H. F., Elker, A., and Hartwig, A. (2000) Carcinogenesis 21, 2097-2104). To study the possible molecular mechanisms of XPA inhibition, we investigated Zn(II) and Ni(II) interactions with the synthetic 37 peptide (XPAzf), representing the XPA zinc finger sequence AcDYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDKHKam. The binding constants were determined using fluorescence and UV-vis spectroscopies, structural insights were provided by CD, and oxidative damage to XPAzf was studied with HPLC. The binding constants for Zn(II) and Ni(II) are (8.5 +/- 1.5) x 10(8) (log value 8.93(7)) and (1.05 +/- 0.07) x 10(6) M(-)(1) (6.02(3)), respectively, in 10 mM phosphate buffer, pH 7.4, and (6 +/- 4) x 10(9) (9.8(2)) and (2.9 +/- 0.5) x 10(6) M(-)(1) (6.46(8)) in 50 mM phosphate buffer, pH 7.4, yielding binding constant ratios Zn(II)/Ni(II) of 800 +/- 100 and 2300 +/- 500, respectively. The Ni(II) ion forms a square planar complex with the sulfurs of XPAzf, opposed to the tetrahedral structure of the native Zn(II) complex. Consequently, the overall zinc finger structure is lost in the Ni(II)-substituted peptide. Zn(II)-saturated XPAzf is remarkably resistant to air oxidation and is only slowly oxidized by 0.01 mM, 0.1 mM, and 1 mM H(2)O(2) in a concentration-dependent fashion. However, the presence of just 10-fold molar excess of Ni(II) is sufficient to accelerate this process for all three H(2)O(2) concentrations tested. Overall, our results indicate that XPAzf can undergo Ni(II) assault in specific conditions.     

Absolute rate constants for the reaction of hypochlorous acid with protein side chains and peptide bonds

Hypochlorous acid (HOCl) is a potent oxidant, which is produced in vivo by activated phagocytes. This compound is an important antibacterial agent, but excessive or misplaced production has been implicated in a number of human diseases, including atherosclerosis, arthritis, and some cancers. Proteins are major targets for this oxidant, and such reaction results in side-chain modification, backbone fragmentation, and cross-linking. Despite a wealth of qualitative data for such reactions, little absolute kinetic data is available to rationalize the in vitro and in vivo data. In this study, absolute second-order rate constants for the reactions of HOCl with protein side chains, model compounds, and backbone amide (peptide) bonds have been determined at physiological pH values. The reactivity of HOCl with potential reactive sites in proteins is summarized by the series: Met (3.8 x 10(7) M(-1) x s(-1)) > Cys (3.0 x 10(7) M(-1) x s(-1)) > cystine (1.6 x 10(5) M(-1) x s(-1)) approximately His (1.0 x 10(5) M(-1) x s(-1)) approximately alpha-amino (1.0 x 10(5) M(-1) x s(-1)) > Trp (1.1 x 10(4) M(-1) x s(-1)) > Lys (5.0 x 10(3) M(-1) x s(-1)) > Tyr (44 M(-1) x s(-1)) approximately Arg (26 M(-1) x s(-1)) > backbone amides (10-10(-3) M(-1) x s(-1)) > Gln(0.03 M(-1) x s(-1)) approximately Asn (0.03 M(-1) x s(-1)). The rate constants for reaction of HOCl with backbone amides (peptide bonds) vary by 4 orders of magnitude with uncharged peptide bonds reacting more readily with HOCl than those in a charged environment. These kinetic parameters have been used in computer modeling of the reactions of HOCl with human serum albumin, apolipoprotein-A1 and free amino acids in plasma at different molar excesses. These models are useful tools for predicting, and reconciling, experimental data obtained in HOCl-induced oxidations and allow estimations to be made as to the flux of HOCl to which proteins are exposed in vivo.     

Nitrogen dioxide as an oxidizing agent of 8-oxo-7,8-dihydro-2'-deoxyguanosine but not of 2'-deoxyguanosine

The redox reactions of guanine and its widely studied oxidation product, the 8-oxo-7,8-dihydro derivative, are of significant importance for understanding the mechanisms of oxidative damage in DNA. Employing 2'-deoxyguanosine 5'-monophosphate (dGMP) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in neutral aqueous solutions as model systems, we have used nanosecond laser flash photolysis to demonstrate that neutral radicals, dGMP(-H)(*), derived by the one-electron oxidation and deprotonation of dGMP, can oxidize nitrite anions (NO2(-)) to the nitrogen dioxide radical (*)NO2. In turn, we show that (*)NO2 can give rise to a one-electron oxidation of 8-oxo-G, but not of dGMP. The one-electron oxidation of dGMP was initiated by a radical cation generated by the laser pulse-induced photoionization of a pyrene derivative with enhanced water solubility, 7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene (BPT). The dGMP(-H)(*) neutral radicals formed via deprotonation of the dGMP(*)(+) radical cations and identified by their characteristic transient absorption spectrum (lambda(max) approximately 310 nm) oxidize nitrite anions with a rate constant of (2.6 +/- 0.3) x 10(6) M(-1) s(-1). The 8-oxo-dG is oxidized by (*)NO2 with a rate constant of (5.3 +/- 0.5) x 10(6) M(-1) s(-1). The 8-oxo-dG(-H)(*) neutral radicals thus generated are clearly identified by their characteristic transient absorption spectra (lambda(max) approximately 320 nm). The rate constant of 8-oxo-dG oxidation (k(12)) by the (*)NO2 one-electron oxidant (the (*)NO2/NO2(-) redox potential, E degrees approximately 1.04 V vs NHE) is lower than k(12) for a series of oxidizing aromatic radical cations with known redox potentials. The k(12) values for 8-oxo-dG oxidation by different aromatic radical cations derived from the photoionization of their parent compounds depend on the redox potentials of the latter, which were in the range of 0.8-1.6 V versus NHE. The magnitude of k(12) gradually decreases from a value of 2.2 x 10(9) M(-1) s(-1) (E degrees = 1.62 V) to 5.8 x 10(8) M(-1) s(-1) (E degrees = 1.13 V) and eventually to 5 x 10(7) M(-1) s(-1) (E degrees = 0.91 V). The implications of these results, including the possibility that the redox cycling of the (*)NO2/NO2(-) species can be involved in the further oxidative damage of 8-oxo-dG in DNA in cellular environments, are discussed.     

Kinetic analysis of the reactions of 4-hydroperoxycyclophosphamide and acrolein with glutathione, mesna, and WR-1065.

The kinetics of the reactions of glutathione (GSH) with 4-hydroperoxycyclophosphamide (4OOH-CP) and acrolein, a metabolite of 4OOH-CP, were investigated in a cell-free medium (pH approximately 7.5) and peripheral blood mononuclear cells. The ability of the thiol drugs, sodium 2-mercaptoethane sulfonate (mesna) and S-2-(3-aminopropylamino)ethanethiol (WR-1065), to affect the reactions of cellular GSH with the alkyalting agents was also studied. The amount of unreacted thiols in the various reactions was determined by derivatization with monobromobimane, followed by separation of fluorescent-labeled thioether adducts using high-pressure liquid chromatography. The second-order rate constants (k(2)) for reactions of GSH, mesna, and WR-1065 with 4OOH-CP in solution were 38 +/- 5, 25 +/- 5, and 880 +/- 50 M(-1) s(-1), respectively. The corresponding k(2) for reactions of GSH, mesna, and WR-1065 with acrolein were 490 +/- 100, 700 +/- 150, and >2000 M(-1) s(-1), respectively. The apparent rate constants for reactions of cellular GSH with acrolein and 4OOH-CP were smaller than those obtained in solution. Assuming that the k(2) is the same inside and outside cells, we estimate the first-order rate constant (k(1)) for transfer of 4OOH-CP and acrolein across the cell membrane as approximately 0.01 and approximately 0.04 s(-1), respectively. WR-1065 was more effective than mesna in blocking depletion of cellular GSH (because it passes into the cell more quickly and has higher reaction rates with the alkylators than the latter compound). When WR-1065 and mesna were used together, the protection against cellular depletion of GSH was additive. Our results are relevant to the administration of thiol drugs with high-dose alkylating agents.     

Kinetics and mechanistic studies of the hydrolysis of diisocyanate-derived bis-thiocarbamates of cysteine methyl ester

Diisocyanates (dNCOs) are the most commonly reported cause of chemically induced occupational asthma, but the ultimate antigenic form is unknown. Reactions of the three most common monomeric dNCOs, hexamethylene dNCO (HDI), methylene diphenylisocyanate (MDI), and toluene dNCO (TDI), with cysteine methyl ester (CME) gave the corresponding bis-dithiocarbamates (HDI-CME, TDI-CME, and MDI-CME). The dissociation kinetics of these bis-thiocarbamates, in aqueous conditions, was followed spectrophotometrically under varying pH and temperature conditions. Reaction of the adducts with methylamine or human serum albumin (HSA) produced diurea, monourea, and diamine products, and this was consistent with the base-catalyzed elimination reaction (E1cB) pathway being the dominant, but not exclusive, dissociation mechanism. The hydrolysis of the adducts was first-order with respect to OH(-) concentration and overall second-order (HDI-CME, k = 3.36 x 10(2) M(-)(1) min(-)(1); TDI-CME, k = 2.49 x 10(4) M(-)(1) min(-)(1); and MDI-CME, k = 5.78 x 10(4) M(-)(1) min(-)(1) at pH 7.4) with deviation from second-order when the dNCO had an aromatic functional group. Arrhenius plots gave activation energies (HDI-CME, E(a) = 70.6 kJ/mol; TDI-CME, E(a) = 46.1 kJ/mol; and MDI-CME, E(a) = 44.5 kJ/mol) that were consistent with the following order of stability: HDI-CME > TDI-CME > MDI-CME. Therefore, the stability of different dNCO-derived thiocarbamates in aqueous environments can vary greatly. Thiocarbamate dissociation rates and type of products formed may potentially influence antigenicity and subsequent hypersensitivity/toxic reactions following dNCO exposures.     

Hypochlorous acid-mediated oxidation of lipid components and antioxidants present in low-density lipoproteins: absolute rate constants,product analysis,and computational modeling

Oxidation of low-density lipoproteins (LDL) is believed to contribute to the increased uptake of LDL by macrophages, which is an early event in atherosclerosis. Hypochlorous acid (HOCl) has been implicated as one of the major oxidants involved in these processes. In a previous study, the rates of reaction of HOCl with the reactive sites in proteins were investigated (Pattison, D. I., and Davies, M. J. (2001) Chem. Res. Toxicol. 14, 1453-1464). The work presented here expands on those studies to determine absolute second-order rate constants for the reactions of HOCl with various lipid components and antioxidants in aqueous solution (pH 7.4). The reactions of HOCl with phosphoryl-serine and phosphoryl-ethanolamine are rapid (k approximately 10(5) M(-)(1) s(-)(1)) and of comparable reactivity to many of the protein sites. The major products formed in these reactions are chloramines, which decay to give both nitrogen- and carbon-centered radicals. Subsequent reactions of these species may induce oxidation of the LDL lipid component. In contrast, phosphoryl-choline reacted much more slowly (k < 10(-)(2) M(-)(1) s(-)(1)). Reaction of HOCl with 3-pentenoic acid was used as a model of lipid double bonds and yielded k = 9 M(-)(1) s(-)(1). The reactions of the lipid-soluble antioxidants, alpha-tocopherol and ubiquinol-10, with HOCl were investigated with model compounds. For the reactions of HOCl with both Trolox and ubiquinol-0, k = 1.3 x 10(3) M(-)(1) s(-)(1); thus, these lipid soluble antioxidants are relatively ineffective as direct scavengers for HOCl as compared to water soluble antioxidants (e.g., ascorbate, k ca. 10(6) M(-)(1) s(-)(1)). The reaction of HOCl with hydroquinone (a simple model for ubiquinol-10) was also investigated both in aqueous solution (k = 45 M(-)(1) s(-)(1)) and in a less polar environment (k approximately 0.5 M(-)(1) s(-)(1) in THF). A computational model was developed using these kinetic parameters to predict which LDL targets are oxidized with varying molar excesses of HOCl, in both the absence and the presence of added ascorbate. The results from these models compare well with experimental data and can be used to predict the effects of HOCl-mediated oxidation on LDL composition.     

Nitric oxide- and nitric oxide donors-induced relaxation and its modulation by oxidative stress in piglet pulmonary arteries.

Inhaled nitric oxide (iNO) is widely used in the treatment of pulmonary hypertension while inhaled NO donors have been suggested as an alternative therapy. The differential susceptibility to inactivation by oxidative stress and oxyhaemoglobin of NO and two NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetyl-penicillamine (SNAP) were analysed in isolated endothelium-denuded pulmonary arteries from 2-week-old piglets stimulated with U46619. NO, SNAP and SNP relaxed the arteries (pIC(30)=7.73+/-0.12, 7.26+/-0.17 and 6.43+/-0.13, respectively) but NO was not detected electrochemically in the bath after the addition of SNP and only at concentrations at which SNAP produced more than 50% relaxation. The sGC inhibitor ODQ (10(-6) M) or the sarcoplasmic Ca(2+)-ATPase thapsigargin (2x10(-6) M) markedly inhibited the relaxation induced by NO, SNAP and SNP. Addition of oxyhaemoglobin (3x10(-7) M) or diethyldithiocarbamate (1 mM) markedly inhibited NO- (pIC(30)=6.88+/-0.07 and 6.92+/-0.18, respectively), weakly inhibited SNAP- and had no effect on SNP-induced relaxation. Xanthine oxidase (5 mu ml(-1)) plus hypoxanthine (10(-4) M) markedly inhibited NO- (pIC(30)=6.96+/-0.12) but not SNAP- or SNP-induced relaxation. Superoxide dismutase (SOD), MnCl(2), diphenileneiodonium and exposing the luminal surface of the rings outwards (inversion) potentiated the relaxant responses of NO (pIC(30)=8.52+/-0.16, 8.23+/-0.11, 8.01+/-0.11 and 8.20+/-0.10, respectively). However, SOD did not modify the NO detected by the electrode and had no effect on SNAP- or SNP-induced relaxation. Therefore, the kinetics and local distribution of NO release of NO donors influence the susceptibility to the scavenging effects of oxyhaemoglobin and superoxide.     

Binding of cationic porphyrin to human serum albumin studied using comprehensive spectroscopic methods

The interaction between cationic porphyrin, a potential valuable anti-tumor and antibiotic drug, and human serum albumin (HSA) was investigated using spectroscopy methods. The binding constants were obtained using fluorescence quenching method (K(SV) = (3.24 +/- 0.29) x 10(4) M(-1)) and surface plasmon resonance (SPR) spectroscopy (K(A) = (6.287 +/- 0.407) x 10(4) M(-1)). The association rate constant (k(a) = 1622 +/- 72.9 M(-1) s(-1)) and dissociation rate constant (K(d) = 0.02589 +/- 0.0024 s(-1)) of the binding process were also calculated. Compared with the two results, it was known that one of the binding sites was near the tryptophan residue and also there existed other binding sites. The Fourier transform infrared (FT-IR) spectroscopy and circular dichroism (CD) spectroscopy indicated that the confirmation of HSA was nearly not affected with the addition of porphyrin.     

Transport properties and association behaviour of the zwitterionic drug 5-aminolevulinic acid in water. A precision conductometric study.

The behavior of the hydrochloride salt of 5-aminolevulinic acid (ALA-HCl) with respect to transport properties and dissociation in aqueous solution at 25 degrees C has been studied using precision conductometry within the concentration range 0.24-5.17mM. The conductivity data are interpreted according to elaborated conductance theory. The carboxyl group appears to be, in practice, undissociated. The dissociation constant, K(a), of the NH(3)(+) form of the amino acid molecules is determined to 6.78x10(-5) (molarity scale); pK(a)=4.17. The limiting molar conductivity of the ALA-H(+) ion, lambda(0)=33.5cm(2)Omega(-1)mol(-1); electric mobility u=3.47x10(-4)cm(2)V(-1)s(-1), is close to the electric mobilites of the acetate and benzoic ions.