M.A. Escudero

Cyclic Voltammetry, FTIRS, and DEMS Study of the Electrooxidation of Carbon Monoxide, Formic Acid, and Methanol on Cyanide-Modified Pt(111) Electrodes

We have used cyanide-modified Pt(111) electrodes, in combination with cyclic voltammetry (CV), Fourier transform infrared spectroscopy (FTIRS), and differential electrochemical mass spectrometry (DEMS), to investigate the oxidation of formic acid and methanol on Pt electrodes. Since CO is the poison intermediate formed during the oxidation of both formic acid and methanol, we have previously characterized the CO adlayer on cyanide-modified Pt(111) electrodes. Poison formation on cyanide-modified Pt(111) is nearly completely inhibited in the case of formic acid and methanol, the corresponding electro-oxidation reaction proceeding, hence, exclusively through the reactive intermediate pathway. These results suggest that, in the oxidation of formic acid and methanol, the formation of adsorbed CO would require the presence of at least three contiguous Pt atoms.

Phosphotriesterase activity identified in purified serum albumins

Abstract The phosphotriesterase in chicken serum that hydrolyses O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) was purified in three chromatographic steps. The activity copurified to apparent homogeneity with albumin monitoring by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS/PAGE) and by SDS-capillary electrophoresis in the purified fractions. Commercial chicken serum albumin was further purified and the phosphotriesterase activity remained associated with albumin. Capillary electrophoresis established a molecular weight of 59 ± 4 kDa for both purified proteins (chicken serum and commercial chicken serum albumin). The purified samples were assayed for hydrolytic activity against several carboxylesters, organophosphates and phosphoramidates. From carboxylesters, only p-nitrophenylbutyrate ( p-NPB) hydrolysing activity was found to copurify with the phosphotriesterase. The purified human, chicken, rabbit and bovine serum albumins and recombinant human serum albumin obtained from commercial sources hydrolysed HDCP and p-NPB. Serum albumin also hydrolysed O-butyl O-2,5-dichlorophenyl phosphoramidate, O-ethyl O-2,5-dichlorophenyl phosphoramidate and O-2,5-dichlorophenyl ethylphosphonoamidate but not other organophosphates and phosphoramidates.

An automatable microassay for phenyl valerate esterase activities sensitive to organophosphorus compounds

An automatable microassay method developed for phenyl valerate esterase (PVase) activity has been applied to determine the following activities in the soluble fraction of hen sciatic nerve: activity A (total PVase activity), activity B (paraoxon-resistant PVase activity), activity C (PVase activity resistant to 40 microM paraoxon and 250 microM mipafox) and neuropathy target esterase (NTE) activity (resistant to 40 microM paraoxon but sensitive to 250 microM mipafox), operationally defined as activity (B-C). This microassay is based on the technique described by Barril et al. (Toxicology. 1988. 49:107-114). The Automated Biomek 1000 Station was used, which guarantees both inter- and intra-assay reproducibility of the results, and shortens the total assay time. The technical problems involved when processing many samples were thus resolved and with same regards it can also apply manually and using a microplate reader. In the case of activity A, the sensitivity of the method allowed the detection of activity in 1 microgram of protein (0.15 mg fresh sciatic nerve tissue), and the response was linear for different concentrations of 0.15-1.7 mg fresh tissue. For B, C and NTE, sensitivity corresponded to 10 micrograms of protein (1.5 mg fresh tissue in the microassay), with a linear response in the range of 1.5-17 mg fresh tissue. The response was linear versus the time of enzyme-substrate reaction (30-150 min). As tissue concentration increased, the response became nonlinear at shorter time. The procedure may be used to measure other enzymatic activities that yield phenols and chlorophenols as reaction products.

Dichlorophenyl phosphoramidates as substrates for avian and mammalian liver phosphotriesterases: activity levels, calcium dependence and stereospecificity.

The present study shows the existence of both Ca2+-dependent and EDTA-resistant hydrolysing activities against HDCP and paraoxon in the particulate and soluble fractions of hen, rat and rabbit liver. HDCP was more extensively hydrolysed than paraoxon in both subcellular fractions and each of three individuals of the three animal species under study in spite of wide interindividual variations. However the ratio of HDCP versus paraoxon hydrolysing activity (HDCPase/paraoxonase), although within the same order of magnitude, cannot be considered as constant as it ranges one- to seven-fold between individuals of the same species. Also there is no constant ratio of Ca2+-dependent/EDTA-resistant activities. Rabbit liver showed the highest rates of Ca2+-dependent hydrolysis for both organophosphorus compounds whereas the hen paraoxonase activity was not inhibited by EDTA. The stereospecific hydrolysis of HDCP was mostly a Ca2+-dependent one, the S-HDCP isomer being hydrolysed faster than the R-HDCP one. The suggestion is made that HDCP could be conveniently used to measure PTE activity in the liver.

Separation of two forms of neuropathy target esterase in the soluble fraction of the hen sciatic nerve.

Neuropathy target esterase (NTE) activity is operatively defined in this paper as the phenyl valerate esterase activity resistant to 40 microM paraoxon but sensitive to 250 microM mipafox. Molecular exclusion column chromatography with Sephacryl S-300 of the soluble (S) fraction from chick sciatic nerve demonstrated two NTE activity peaks. The first eluted with the front, thus indicating a mol. wt. of over 700 kDa (peak Vo), while the second peak eluted with kd = 0.36, suggesting a mol. wt. of about 100 kDa. The curve of total phenyl valerate (PVase) activity inhibition with paraoxon (0.19-200 microM) shows that at a concentration of 40 microM the esterases highly sensitive to paraoxon are inhibited in the Vo and 100-kDa peaks. The NTE activity in these two peaks in turn represented 31% and 44% of the 40 microM paraoxon resistant activity, respectively. The mipafox inhibition curves (1.0-250 microM) revealed different sensitivities to mipafox, with I50 values (t = 30 min) of approximately 1.47 and 63 microM, for Vo and 100-kDa peaks respectively. Mipafox sensitivity of the Vo and 100-kDa peaks correlates with the two components, that had been deduced from the kinetic properties of the S-fraction.