The phosphotriesterase from Pseudomonas diminuta hydrolyzes a wide variety of organophosphate insecticides and acetylcholinesterase inhibitors. The rate of hydrolysis depends on the substrate and can range from 6000 s ؊1 for paraoxon to 0.03 s ؊1 for the slower substrates such as diethylphenylphosphate. Increases in the reactivity of phosphotriesterase toward the slower substrates were attempted by the placement of a potential proton donor group at the active site. Distances from active site residues in the wild type protein to a bound substrate analog were measured, and ؊1 . These studies demonstrated for the first time that it is possible to significantly enhance the ability of the native phosphotriesterase to hydrolyze phosphorus-fluorine bonds at rates that rival the hydrolysis of paraoxon.The phosphotriesterase (PTE) 1 from Pseudomonas diminuta has a rather broad substrate specificity. This protein has been shown to catalyze the hydrolysis of the insecticides parathion, paraoxon, coumaphos, methylparathion, and diazonin, among others (1). The enzyme also detoxifies the potent acetylcholinesterase nerve agents sarin, soman, and diisopropylfluorophosphate (2). The enzymatic reaction mechanism has been shown to proceed via an S N 2-like process whereby an activated water molecule attacks the phosphoryl center resulting in the displacement of the leaving group with an inversion of stereochemical configuration (3). The active site of this enzyme is comprised primarily of a binuclear metal cluster that contains Zn 2ϩ but can also accommodate Co Ϫ1 s Ϫ1 , respectively (4). However, an extensive structure reactivity analysis for the hydrolysis of paraoxon analogues has clearly demonstrated that the magnitude of these kinetic constants is very much dependent on the pK a of the leaving group phenol (7). Moreover, the rate-limiting step changes from diffusion of the enzyme-substrate or enzyme-product complex to hydrolytic cleavage of the P-O bond when the pK a of the phenol exceeds ϳ7. The Brnsted value ( lg ) for this series of compounds with pK a values exceeding 7 is approximately Ϫ2. Therefore, the rate for the enzymatic hydrolysis of paraoxon with a p-nitrophenolate leaving group (pK a ϭ ϳ7) is nearly a million times faster than is diethylphenylphosphate with a phenolate leaving group (pK a ϳ 10). The very substantial Brnsted value for this reaction indicates two important factors about the enzymatic machinery at the active site of this protein. First, the transition state for the hydrolysis of paraoxon analogues is very late, suggesting that the P-O bond is nearly fully broken. Second, the enzyme does not appear to be contributing to the activation of the leaving group through Lewis acid catalysis, via complexation with the binuclear metal center, or by general acid catalysis through protonation of the phenolic oxygen from a nearby acidic residue. In addition, it would appear that the environment of the leaving group binding site is quite hydrophobic.
A new program for lipoprotein characterization is outlined where capillary electrophoresis (CE) plays a central role in the analysis of intact lipoprotein serum components and the apoprotein domains. The first characterization step involves separation and particle density analysis of very low-, low-, and high-density lipoprotein fractions (VLDL, LDL, HDL) by ultracentrifugation and image analysis. VLDL, HDL, and LDL fractions are analyzed by capillary electrophoresis. Sodium dodecyl sulfate (SDS) at low concentrations in the background electrolyte used in the CE analysis is incorporated into the lipoprotein particle without appreciable delipidation, as determined by ultracentrifuge particle density analysis. Increasing the concentration of SDS results in extensive delipidation, resulting in the release of apoproteins (apo) which are detected as components of the electropherogram. Apo B-100 is detected in the delipidated VLDL and LDL fractions along with micelles of the lipids. Micelles from LDL delipidation have uniform charge densities. Apo A-I and A-II are detected in the HDL fraction. A new method for lipoprotein delipidation is introduced where the lipoprotein fraction is adsorbed on a reversed-phase hydrophobic cartridge. Delipidation and recovery of the apoprotein fractions is made by serial elutions with acetonitrile. CE of the lipid-free apoprotein mixture shows the presence of apoC-I,II,III and apoE in the VLDL fraction, and apoA-I,II apoC-I and apoE in the HDL fraction. Electrospray ionization mass spectrometry analysis gives the isoform distribution for each apoprotein. The identification of the apoproteins in the electropherograms is the first step in developing a CE-based quantitation method for measuring serum levels of these apoproteins and their distribution between the lipoprotein fractions. The assay described in this paper is being used as a level 2 and 3 cardiac risk profile analysis for individuals with normal lipid profiles who have a documented or family history of cardiovascular disease.
New isoforms of apolipoprotein (apo)C-I and apoC-III have been detected in delipidated fractions from very low density lipoprotein (VLDL) using matrix-assisted laser desorption (MALDI) and electrospray ionization (ESI) mass spectrometry (MS). The cleavage sites of truncated apoC-III isoforms have also been identified. The VLDL fractions were isolated by fixed-angle single-spin ultracentrifugation using a self-generating sucrose density gradient and delipidated using a newly developed C18 solid phase extraction protocol. Fifteen apoC isoforms and apoE were identified in the MALDI spectra and the existence of the more abundant species was verified by ESI-MS. The relative intensities of the apoCs are closely correlated in three normolipidemic subjects. A fourth subject with type V hyperlipidemia exhibited an elevated apoC-III level and a suppressed level of the newly discovered truncated apoC-I isoform. ApoC-II was found to be particularly sensitive to in vitro oxidation. The dynamic range and specificity of the MALDI assay shows that the complete apoC isoform profile and apoE phenotype can be obtained in a single measurement from the delipidated VLDL fraction.-Bondarenko,
Group separation of Maya crude oil was achieved by the saturates-aromatics-resins-asphaltenes (SARA) method. The sulfur-containing compounds in the saturate and aromatic fractions were then separated using a ligand exchange chromatography method based on organosulfur affinity for Cu 2+ and Pd 2+ , respectively. The separation into group types is effective as a prelude to the structural characterization of crude oil fractions using elemental analysis, Fourier transform infrared, and both 1 H and 13 C nuclear magnetic resonance spectroscopy. In addition, gel permeation chromatography was compared with atmospheric pressure chemical ionization/mass spectrometry (APCI/MS) in order to determine whether the APCI/MS method could provide a rapid means for the determination of the average molar mass.
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