Single-particle study of protein assembly.

A study of protein assembly in solution with single-particle imaging and reconstruction techniques using cryoelectron microscopy is reported. The human glutamine synthetase enzyme, important in brain metabolism, and previously assumed to be assembled into a homogeneous quaternary structure, is found to be heterogeneous, with three oligomeric states that co-exist at room temperature. This result corrects an old structural and kinetic model determined by ensemble averaging techniques that assumed a homogeneous system. Unexpectedly fast protein dissociation kinetics results from a stabilized transition state.

A rapid, sensitive HPLC method for the determination of ganciclovir in human plasma and serum.

A method for ganciclovir determination in human serum and plasma has been developed and validated. The method has a lower limit of quantification (LLOQ) adequate for sensitive pharmacokinetic studies ( < or = 0.05 microg/ml), has run times of < or = 15 min, and uses aliquot volumes adequate for pediatric studies (0.25 ml). In the method, proteinaceous material in serum or plasma is precipitated by trichloroacetic acid. An aliquot of the supernatant is analyzed by HPLC; automated column switching removes late-eluting materials that might interfere with the analyte peak in subsequent runs. Detection and quantification of ganciclovir is by fluorescence (lambda(ex) = 278 nm; lambda(em) = 380 nm). The method has a validated range of 0.0400-4.00 microg/ml and an LLOQ of 0.0400 microg/ml. All intra- and inter-assay % C.V. values were < 8%; all recoveries (accuracy) were within 7% of nominal values. No interference was observed by mycophenolic acid or its glucuronide metabolite, by AZT, salicylic acid, acetaminophen, ibuprofen, naproxen prednisone, acyclovir, or cyclosporine. Ganciclovir is very stable in the samples and the extract during storage and sample processing. Both serum and plasma methods have been validated for use and have been successfully used to analyze samples from clinical studies.

Determination of femtomole/milliliter concentrations of enprostil acid in human plasma using high-performance liquid chromatography-laser-induced fluorescence detection.

This paper describes the use of multiple-column high-performance liquid chromatography (HPLC) combined with laser-induced fluorescence for the determination of femtomole/milliliter concentrations of enprostil acid, a prostaglandin analogue, in human plasma. The drug is isolated from plasma by phenyl solid-phase extraction and fluorescently labeled at its carboxyl functional group with a large excess of 2-bromoacetyl-6-methoxynaphthalene. A multi-column method using both normal- and reversed-phase chromatography is necessary to separate the labeled drug from the unreacted reagent. Post-column dilution of the mobile phase with water after the reversed-phase chromatography allows on-line concentration of the labeled analyte onto a guard column prior to the microbore HPLC. A loop guard column device provides a simple way to inject up to 1.0 ml of sample solution onto a microbore column without significantly reducing the column efficiency. A 325-nm He-Cd laser is used to excite the labeled drug, and fluorescence emission is monitored at 450 nm. Using this system, we are able to derivatize, detect, and quantify 5 pg of the prostaglandin analogue in 1.0 ml of plasma.

High-performance liquid chromatographic determination of the 1,4-diketo and 1,4-diketo-2,3-dihydroxy metabolites of lonapalene in rat urine.

Individual high-performance liquid chromatographic (HPLC) methods have been developed for the determination of two major metabolites of lonapalene in rat urine. The highly unstable and polar 1,4-diketo-2,3-dihydroxy metabolite (II) is extracted from urine by two extraction columns (phenyl followed by silica), further purified by means of HPLC with a fully end-capped C18 HPLC column and quantified by an ultraviolet detector at 280 nm. Ascorbic acid is used as an antioxidant during extraction and overnight injection of II. Urine samples for total II (free plus conjugated) determination are incubated with arylsulfatase and beta-glucuronadase prior to extraction. The 1,4-diketo metabolite (III) is extracted from urine with a C18 extraction column, further purified with a C18 HPLC column, and quantified by an ultraviolet detector at 260 nm. The detection limit for both metabolites is 100 ng/ml of urine (signal-to-noise = 2.5). The methods were used to analyze urine samples from a long-term toxicology study of lonapalene in rats and to determine the linearity of dose-concentration relationships for both metabolites.

Isolation and identification of 6-desmethylnaproxen sulfate as a new metabolite of naproxen in human plasma.

A new metabolite of naproxen, 6-desmethylnaproxen sulfate (6-DMNS), has been identified in plasma from normal and uremic subjects after oral administration of a single 500 mg dose of naproxen. Tentative identification was achieved by the finding of an increase in the concentration of 6-DMN upon incubation of the plasma with arylsulfatase from Helix pomatia or from Aerobacter aerogenes. More definitive identification was established through demonstration that the HPLC retention time of the conjugate is identical to that of an authentic reference sample of 6-DMNS. Unequivocal identification was accomplished by means of LC-MS after the metabolite was isolated from the plasma by protein precipitation with acetonitrile and further purified by anion-exchange and reversed phase HPLC. Plasma profiles of 6-DMNS for normal and uremic subjects, obtained by a procedure involving differential enzymatic hydrolysis using arylsulfatase from H. pomatia, revealed that 6-DMNS was present in plasma from all subjects but in relatively high concentrations only in subjects with impaired renal function and that the extent of the conjugation is related directly to the severity of the dysfunction. No evidence was found for the presence of glucuronide or sulfate conjugates of naproxen in plasma.

Purification and characterization of endo-xylanases from Aspergillus niger. I. Two isozymes active on xylan backbones near branch points.

Two endo-xylanases (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) were purified to homogeneity from a crude Aspergillus niger pentosanase preparation by Ultrogel AcA 54 gel permeation chromatography, SP-Sephadex C-25 cation exchange chromatography at pH 4.5, Sephadex G-50 gel permeation chromatography, and a second SP-Sephadex C-25 step, this one at pH 5.8. The two xylanases hydrolyzed soluble xylan more rapidly than insoluble branched xylan, but attacked each substance to an equal extent. Their low activity on a linear xylooligosaccharide mixture and absence of activity on insoluble xylan freed of branches suggest that the xylanases require a branch point nearby for significant attack. No xylose or L-arabinose was produced, the major products of low molecular weight being tri- and pentasaccharides and smaller amounts of di-, tetra-, and hexasaccharides. There was low activity on untreated and crystalline cellulose and on carboxymethylcellulose and no activity on other polysaccharides tested. These two xylanases had molecular weights of ca. 1.3 x 10(4) and similar amino acid profiles, high in acidic and low in sulfur-containing residues. Isoelectric points were 8.6 for I and 9.0 for II. Optimum pH values for activity were 6.0 and 5.5, respectively. In a 20-min assay at pH 5.5, each was most active at 45 degrees C, with activation energies up to 40 degrees C of 30.4 and 38.8 kJ/ mol, respectively. Optimum pH levels for stability were 5.0 and 6.0, with half-lives at 60 degrees C and those pHs of 20 and 75 min, respectively.

Determination of acetylmethadol and metabolites by use of high-performance liquid chromatography.

A method is described for the simultaneous determination of 1, alpha-acetylmethadol (LAAM) and five active metabolites--noracetylmethadol, dinoracetylmethadol, methadol, normethadol, and dinormethadol--in biofluids by high-performance liquid chromatography using a normal-phase column and a UV detector at 218 nm. The compounds are recovered from biofluids by a multistep liquid--liquid extraction. The mobile phase is methanol--acetonitrile (70:30, v/v) containing 0.015% ammonium hydroxide as the modifier. Retention times can be varied by adjusting the composition of the mobile phase to maximize peak height for quantitation using l-propranolol as the internal standard or peak separation for the collection of fractions. Using a UV detector the lower limit of sensitivity is 10 ng/ml of biofluid. Using fraction collection of radiolabeled drug and metabolites followed by liquid scintillation counting the lower limit of sensitivity is 1.0 ng/ml. Commonly used or abused narcotics including morphine, heroin, meperidine, methadone and propoxyphene do not interfere with the analysis. The method has been applied to plasma and urine samples from humans, sheep and rats. Extracts of urine from patients receiving maintenance treatment with LAAM contain LAAM and each of the five active metabolites.