Intermediates in the reaction of substrate-free cytochrome P450cam with peroxy acetic acid. (1/77)

Freeze-quenched intermediates of substrate-free cytochrome 57Fe-P450(cam) in reaction with peroxy acetic acid as oxidizing agent have been characterized by EPR and Mossbauer spectroscopy. After 8 ms of reaction time the reaction mixture consists of approximately 90% of ferric low-spin iron with g-factors and hyperfine parameters of the starting material; the remaining approximately 10% are identified as a free radical (S' = 1/2) by its EPR and as an iron(IV) (S= 1) species by its Mossbauer signature. After 5 min of reaction time the intermediates have disappeared and the Mossbauer and EPR-spectra exhibit 100% of the starting material. We note that the spin-Hamiltonian analysis of the spectra of the 8 ms reactant clearly reveals that the two paramagnetic species, e.g. the ferryl (iron(IV)) species and the radical, are not exchanged coupled. This led to the conclusion that under the conditions used, peroxy acetic acid oxidized a tyrosine residue (probably Tyr-96) into a tyrosine radical (Tyr*-96), and the iron(III) center of substrate-free P450(cam) to iron(IV).  (+info)

Suitability of peracetic acid for sterilization of media for mycoplasma cultures. (2/77)

The utility of peracetic acid for sterilization of serum and yeast extract additions to mycoplasma medium was studied by culturing six Mycoplasma species. Culture media containing additions that had been sterilized with peracetic acid proved to be as good as filtered components. The use of 0.05 to 0.1% peracetic acid is recommended to sterilize the serum and yeast extract additions since savings in time and equipment can be accomplished.  (+info)

Cloning and biochemical characterization of Co(2+)-activated bromoperoxidase-esterase (perhydrolase) from Pseudomonas putida IF-3 strain. (3/77)

The gene encoding Co(2+)-activated bromoperoxidase (BPO)-esterase (EST), catalyzing the organic acid-assisted bromination of some organic compounds with H2O2 and Br(-) and quite specific hydrolysis of (R)-acetylthioisobutyric acid methyl ester, was cloned from the chromosomal DNA of the Pseudomonas putida IF-3 strain. The bpo-est gene comprises 831 bp and encoded a protein of 30181 Da. The enzyme was expressed at a high level in Escherichia coli and purified to homogeneity by ammonium sulfate fractionation and two-step column chromatographies. The recombinant enzyme required acetic acid, propionic acid, isobutyric acid or n-butyric acid in addition to H2O2 and Br(-) for the brominating reaction and was activated by Co(2+) ions. It catalyzed the bromination of styrene and indene to give the corresponding racemic bromohydrin. Although the enzyme did not release free peracetic acid in the reaction mixture, chemical reaction with peracetic acid could well explain such enzymatic reactions via a peracetic acid intermediate. The results indicated that the enzyme was a novel Co(2+)-activated organic acid-dependent BPO (perhydrolase)-EST, belonging to the non-metal haloperoxidase-hydrolase family.  (+info)

Biofilms and planktonic cells of Pseudomonas aeruginosa have similar resistance to killing by antimicrobials. (4/77)

Biofilms are considered to be highly resistant to antimicrobial agents. Strictly speaking, this is not the case-biofilms do not grow in the presence of antimicrobials any better than do planktonic cells. Biofilms are indeed highly resistant to killing by bactericidal antimicrobials, compared to logarithmic-phase planktonic cells, and therefore exhibit tolerance. It is assumed that biofilms are also significantly more tolerant than stationary-phase planktonic cells. A detailed comparative examination of tolerance of biofilms versus stationary- and logarithmic-phase planktonic cells with four different antimicrobial agents was performed in this study. Carbenicillin appeared to be completely ineffective against both stationary-phase cells and biofilms. Killing by this beta-lactam antibiotic depends on rapid growth, and this result confirms the notion of slow-growing biofilms resembling the stationary state. Ofloxacin is a fluoroquinolone antibiotic that kills nongrowing cells, and biofilms and stationary-phase cells were comparably tolerant to this antibiotic. The majority of cells in both populations were eradicated at low levels of ofloxacin, leaving a fraction of essentially invulnerable persisters. The bulk of the population in both biofilm and stationary-phase cultures was tolerant to tobramycin. At very high tobramycin concentrations, a fraction of persister cells became apparent in stationary-phase culture. Stationary-phase cells were more tolerant to the biocide peracetic acid than were biofilms. In general, stationary-phase cells were somewhat more tolerant than biofilms in all of the cases examined. We concluded that, at least for Pseudomonas aeruginosa, one of the model organisms for biofilm studies, the notion that biofilms have greater resistance than do planktonic cells is unwarranted. We further suggest that tolerance to antibiotics in stationary-phase or biofilm cultures is largely dependent on the presence of persister cells.  (+info)

Generation of controlled atmospheres for the determination of the irritant potency of peroxyacetic acid. (5/77)

Given the physical properties of peroxyacetic acid, which decomposes into acetic acid and hydrogen peroxide, the generation and analysis of controlled atmospheres used to test the irritant potency of this peracid in mice require specific developments. The sampling and analytical method was based on the simultaneous sampling on a titanyl sulphate-impregnated silica gel tube (allowing the determination of total peroxides, peroxyacetic acid and hydrogen peroxide) and in an impinger containing a methyl-p-tolyl sulphide solution (of which the analysis yields the concentration of total acids, peroxyacetic acid and acetic acid, and peroxyacetic alone). From these results the concentrations of the different products can be inferred without interference. A special device composed of inert materials was designed for the generation of the controlled atmosphere. Buffering the peroxyacetic solution at pH 7 with a phosphate buffer allowed the generation of peroxyacetic acid atmospheres with negligible concentrations of acetic acid and hydrogen peroxide.  (+info)

Sensory irritation of acetic acid, hydrogen peroxide, peroxyacetic acid and their mixture in mice. (6/77)

The expiratory bradypnoea indicative of upper airway irritation in mice was evaluated during a period of 60 min of oronasal exposure to acetic acid, hydrogen peroxide and peroxyacetic acid vapours. The airborne concentration resulting in a 50% decrease in the respiratory rate of mice (RD50) was calculated for each chemical. The concentration-response curves of acetic acid, hydrogen peroxide and peroxyacetic acid had similar slopes. The results did however show that the three chemicals had different irritant potencies. The RD50 values of acetic acid, hydrogen peroxide and peroxyacetic acid were 227, 113 and 5.4 p.p.m. respectively. Moreover, a mixture containing 53% acetic acid, 11% hydrogen peroxide and 36% peroxyacetic acid had an RD50 of 10.6 ppm, 3.8 ppm being peroxyacetic acid, which is 1.4 times lower than the theoretical value estimated from the fractional concentrations and the respective RD50s of the individual components. On the basis of a TLV-STEL (threshold limit value for short-term exposure limit) equal to 0.1 RD50, the TLV-STELs for acetic acid, hydrogen peroxide and peroxyacetic acid should not exceed 20, 10 and 0.5 p.p.m. respectively. On the basis of a TLV-TWA (time-weighted average) equal to 0.03 RD50, the TLV-TWAs for these same chemicals should not exceed 5, 3 and 0.2 p.p.m. respectively. Finally, these values and existing TLVs in Europe and the USA are compared.  (+info)

Role of Acinetobacter calcoaceticus 3,4-dihydrocoumarin hydrolase in oxidative stress defence against peroxoacids. (7/77)

The physiological role of a bifunctional enzyme, 3,4-dihydrocoumarin hydrolase (DCH), which is capable of both hydrolysis of ester bonds and organic acid-assisted bromination of organic compounds, was investigated. Purified DCH from Acinetobacter calcoaceticus F46 catalysed dose- and time-dependent degradation of peracetic acid. The gene (dch) was cloned from the chromosomal DNA of the bacterium. The dch ORF was 831 bp long, corresponding to a protein of 272 amino acid residues, and the deduced amino acid sequence showed high similarity to those of bacterial serine esterases and perhydrolases. The dch gene was disrupted by homologous recombination on the A. calcoaceticus genome. The dch disruptant strain was more sensitive to growth inhibition by peracetic acid than the parent strain. On the other hand, the recombinant Escherichia coli cells expressing dch were more resistant to peracetic acid. A putative catalase gene was found immediately downstream of dch, and Northern blot hybridization analysis revealed that they are transcribed as part of a polycistronic mRNA. These results suggested that in vivo DCH detoxifies peroxoacids in conjunction with the catalase, i.e. peroxoacids are first hydrolysed to the corresponding acids and hydrogen peroxide by DCH, and then the resulting hydrogen peroxide is degraded by the catalase.  (+info)

Role of radical formation at tyrosine 193 in the allene oxide synthase domain of a lipoxygenase-AOS fusion protein from coral. (8/77)

Coral allene oxide synthase (cAOS), a fusion protein with 8R-lipoxygenase in Plexaura homomalla, is a hemoprotein with sequence similarity to catalases. cAOS reacts rapidly with the oxidant peracetic acid to form heme compound I and intermediate II. Concomitantly, an electron paramagnetic resonance (EPR) signal with tyrosyl radical-like features, centered at a g-value of 2.004-2.005, is formed. The radical is identified as tyrosyl by changes in EPR spectra when deuterated tyrosine is incorporated in cAOS. The radical location in cAOS is determined by mutagenesis of Y193 and Y209. Upon oxidation, native cAOS and mutant Y209F exhibit the same radical spectrum, but no significant tyrosine radical forms in mutant Y193H, implicating Y193 as the radical site in native cAOS. Estimates of the side chain torsion angles for the radical at Y193, based on the beta-proton isotropic EPR hyperfine splitting, A(iso), are theta(1) = 21 to 30 degrees and theta(2) = -99 to -90 degrees. The results show that cAOS can cleave nonsubstrate hydroperoxides by a heterolytic path, although a homolytic course is likely taken in converting the normal substrate, 8R-hydroperoxyeicosatetraenoic acid (8R-HpETE), to product. Coral AOS achieves specificity for the allene oxide formed by selection of the homolytic pathway normally, while it inactivates by the heterolytic path with nonoptimal substrates. Accordingly, with the nonoptimal substrate, 13R-hydroperoxyoctadecadienoic acid (13R-HpODE), mutant Y193H is inactivated after turning over significantly fewer substrate molecules than required to inactivate native cAOS or the Y209F mutant because it cannot absorb oxidizing equivalents by forming a radical at Y193.  (+info)