Structure and time-dependent behavior of acetylated and non-acetylated forms of a molluscan metallothionein.

Cadmium-induced metallothionein in a mollusc, the oyster Crassostrea virginica, occurs in both blocked and unblocked forms (Roesijadi, G., Kielland, S.L. and Klerks, P. (1989) Arch. Biochem. Biophys. 273, 403-413). The block, which is the sole difference in the structure of the two proteins, was identified as an acetyl group with use of tandem mass spectrometry. The blocked and unblocked proteins carried N-acetylserine and serine, respectively, at the N-terminus and were designated CvNAcMT and CvMT. Only CvNAcMT was detected under basal conditions. Both forms were induced by Cd. Pulse-labeling with [35S]cyteine at specified times during exposure showed that the rate of CvNAcMT synthesis in gills increased rapidly, initially exceeding that of CvMT, then declined to the rate attained by CvMT. Turnover rates for Cd-induced CvMT and CvNAcMT were similar to each other. They appeared to be faster when measured in the absence of Cd in the external medium (k = 0.18 and 0.16/day, respectively), than in its presence (k = 0.03 and 0.06/day, respectively).

High-performance liquid chromatography-time-of-flight mass spectrometry and its application to peptide analyses.

High-performance liquid chromatography (HPLC) has been successfully interfaced on-line with liquid secondary-ion time-of-flight mass spectrometry, utilizing a continuous-flow interface. Time-of-flight mass spectrometry (TOF-MS) is a low-resolution, high-mass-range technique, compatible with extremely rapid data acquisition rates. Thus a TOF-MS system is extremely well suited for coupling with HPLC. This paper describes the interface used to couple the HPLC and TOF-MS as well as the basic operating principles of such a system. Using both standard and packed-capillary reversed-phase HPLC columns, the HPLC-TOF-MS system has been successfully used to separate and detect peptides, providing molecular weight information for the peptide analytes. Experimental data, including chromatograms (UV, reconstructed ion and selected ion) and mass spectra, are presented to demonstrate the ability of the HPLC-liquid secondary-ion TOF-MS system to resolve chromatographically analytes as well as to resolve mass spectrometrically analytes which are unresolved on the chromatographic column.

Assessment of glycosylation-site heterogeneity using plasma desorption mass spectrometry.

Plasma desorption mass spectrometry (PD-MS) was used to assess the molecular weight heterogeneity of glycopeptides (6-12 amino acids) from each of the three N-linked glycosylation sites of bovine fetuin (R.G. Spiro (1962) J. Biol. Chem. 237, 382-388). The glycopeptides were purified by a combination of anion exchange chromatography and reverse-phase HPLC. Since no detectable fragmentation was observed in the PD-MS of these asialoglycopeptides, the observation of multiple molecular ions could be attributed to either carbohydrate or peptide heterogeneity. Assignment of molecular ions, within 3 to 5 amu of the theoretical mass, of glycopeptides from each glycosylation site was made from amino acid composition, peptide sequence around the glycosylation sites, and previously reported triantennary oligosaccharide structures (B. Nilsson, N.E. Nordén, and S. Svensson (1979) J. Biol. Chem. 254, 4545-4553). Ion groups differing in mass by one N-acetyllactosamine unit were observed in glycopeptides from the Asn-Asp and Asn-Cys sites, localizing these previously observed biantennary oligosaccharide structures (R.R. Townsend, M.R. Hardy, T.C. Wong, and Y.C. Lee (1986) Biochemistry 25, 5716-5725; S. Takasaki and A. Kobata (1986) Biochemistry 25, 5709-5715) to these two sites. The presence of biantennary oligosaccharides at the Asn-Asp sites could be substantiated using 1H NMR but were not detected in the Asn-Cys glycopeptides. PD-MS was also implemented in the purification protocol for these glycopeptides and proved to be useful in assessing purity of chromatographic fractions which were mixtures of glycopeptides displaying both carbohydrate and peptide heterogeneity. A preparation scheme was developed to obtain molecular ions of desialylated glycopeptides by PD-MS.

Derivatives of dicyclopentadiene in ground water.

Dicyclopentadiene, a waste product of manufacturing at the Rocky Mountain Arsenal near Denver Colorado, has been detected in ground water at this facility. Ground water extracts were analysed by gas chromatography-mass spectrometry (GC/MS) to determine if derivatives of dicyclopentadiene were present in addition to dicyclopentadiene and other chemical wastes. The derivatives thus identified were characterized by GC high resolution MS, deuterium exchange for active hydrogen in the chemical ionization source of the mass spectrometer, and GC/MS following on-column base catalyzed deuteration of enolizable hydrogen. Two ketone derivatives of dicyclopentadiene were identified by comparison with synthetic standards. Ground water derivatives of dicyclopentadiene were compared with mammalian enzymatic metabolites produced in vitro by incubation with immobilized rabbit liver cytochromes P-450. The metabolites were analysed by GC/MS and by GC/MS following derivatization and deuterium labelling. Although not found in ground water samples, the two metabolites were identified as monoepoxides of dicyclopentadiene by comparison with compounds synthesized in the laboratory.

Reaction of 1-O-acyl glucuronides with 4-(p-nitrobenzyl)pyridine.

In this investigation, 31 1-O-acyl glucuronides were synthesized and 25 of these were shown to react with 4-(p-nitrobenzyl)pyridine (NBP), a standard chemical nucleophil, on thin layer chromatography plates. A quantitative NBP assay was developed based on existing methods, and the rates of reaction of three acyl glucuronides, clofibric 1-O-acyl glucuronide, indomethacin 1-O-acyl glucuronide, and flufenamic 1-O-acyl glucuronide, were determined. These rates ranged from 0.436 min-1 to 1.08 min-1. Chlorambucil, a powerful alkylating agent, reacted with NBP 127 times faster than the most reactive of the three glucuronides assayed. The half-lives of these three 1-O-acyl glucuronides, determined at pH 2.0, 4.0, 6.0, 7.4, and 10.0 in aqueous phosphate solution, ranged from greater than 1000 hr at pH 2 to less than 1 min at pH 10.0. Determination of the rates of reaction of 1-O-acyl glucuronides with NBP and the rates of hydrolysis as a function of pH further characterize these compounds as activated phase II metabolites.

Activated phase II metabolites: comparison of alkylation by 1-O-acyl glucuronides and acyl sulfates.

1-O-acyl glucuronides are reactive Phase II metabolites which can alkylate chemical nucleophiles. Industrial sulfate ester mixed anhydrides have been reported to be active acylating agents. This study was undertaken in order to establish that sulfate ester mixed anhydrides of clinically useful drugs could be synthesized, purified, and characterized as reactive chemical species. Their ability to alkylate 4-(p-nitrobenzyl)pyridine (NBP) and their stability in aqueous solution was compared with 1-O-acyl glucuronide conjugates of the same drugs. Synthesis of the 1-O-acyl glucuronides of the hypolipidemic agent, clofibric acid, and the nonsteroidal antiinflammatory drugs flufenamic acid and indomethacin were catalyzed by immobilized microsomal rabbit liver UDP-glucuronyltransferase. Potassium salts of the sulfate ester mixed anhydrides of these drugs were synthesized chemically by temperature-controlled reaction with chlorosulfonic acid in anhydrous pyridine. Half-lives at pH 2.0, 6.0, 7.4, and 10.0 were determined for each compound. The reactivity of the acyl glucuronides and sulfate ester mixed anhydrides towards the standard chemical nucleophile, 4-(p-nitrobenzyl pyridine (NBP), was measured using a spectrophotometric assay at several substrate concentrations. Acyl sulfate ester mixed anhydrides were shown to be 3-20 times more reactive towards NBP than their corresponding 1-O-acyl glucuronides. For both glucuronides and sulfate esters, relative reactivity towards NBP was: clofibric acid greater than indomethacin greater than flufenamic acid. This behavior paralleled the hydrolytic instability of the compounds.

Determination of the sialylation pattern of human fibrinogen glycopeptides with fast atom bombardment.

Sialylated biantennary glycopeptides from human fibrinogen were analyzed with fast atom bombardment mass spectrometry. The mass spectrometric conditions used in the positive mode showed predominantly molecular ions with no fragment ions due to the loss of sialic acid. Standard mixtures of glycopeptides with zero, one, and two sialic acid residues revealed a linear relationship between ion abundance and molar fraction. The desorption efficiency varied according to the number of sialic acid residues in these biantennary glycopeptides. The relative abundance of different molecular ion species differing only in amino acid content was in agreement with chemical analysis. Sensitivity, precision, and requirements for sample preparation were assessed. Both assignment of molecular weights and quantification of individual glycopeptide species from human fibrinogen were obtained. The glycopeptides from human fibrinogen were found to consist of a mixture of equal amounts of monosialylated and disialylated species with no asialoglycopeptides.

Mass spectrometric studies of a modified active-site tetrapeptide from delta 5-3-ketosteroid isomerase of Pseudomonas testosteroni.

Examination of the product of affinity labeling of delta 5-3-ketosteroid isomerase (EC 5.3.3.1) of Pseudomonas testosteroni by the suicide substrate [7-3H]5,10-secoestr-5-yne-3,10,17-trione has demonstrated that the steroid becomes bound by an acid- and base-labile linkage to an active-site peptide representing residues 55-58 (H2N-Tyr-Ala-Asn-Ser-CO2H) of the primary structure (Penning, T. M., and Talalay, P. (1981) J. Biol. Chem. 256, 6851-6858). Upon release of the steroid by mild acid hydrolysis, the peptide is converted into a more basic structure while retaining its amino acid composition (as determined by conventional means). These findings were rationalized by postulating that the steroid is bound as an imido ester via the amide group of asparagine 57 and that the polypeptide backbone participates (via attack by nitrogen or oxygen) in the hydrolysis of this ester with the formation of a cyclic amidine or basic oxazine. By comparing the properties of the isolated tetrapeptide, from which the steroid has been removed, with those of synthetic H2N-Tyr-Ala-Asn-Ser-CO2H and H2N-Tyr-Ala-Asp-Ser-CO2H by electron impact and fast atom bombardment mass spectrometry, we now have evidence for the presence of an oxazine (5,6-dihydro-6-iminio-4H-1,3-oxazine) in the modified peptide. Our results draw attention to the hitherto unsuspected degree of nucleophilicity of the amide group of the side chain of asparagine and the participation of this group in the formation of an imido ester.

Metabolism of clofazimine in leprosy patients.

We have identified two metabolites of clofazimine (B663; Lamprene; 3-(p-chloroanilino)-10-(p-chlorophenyl)-2,10-dihydro-2-isopropyliminophenazine) in our initial investigation of its metabolism in leprosy patients. Based on mass, ultraviolet, and visible spectrometry, we characterized an unconjugated (metabolite I, 3-(p-hydroxyanilino)-10-(p-chlorophenyl)-2,10-dihydro-2-isopropyliminophenazine ) and a conjugated (metabolite II, 3-(beta-D-glucopyranosiduronic acid)-10-(p-chlorophenyl)-2,10-dihydro-2-isopropyliminophenazine) metabolite from the urine of patients. Both metabolites were red in color, similar to clofazimine; however, both were considerably more polar than the parent drug. We suggest that metabolite I was formed by a hydrolytic dehalogenation reaction, and metabolite II by hydrolytic deamination followed by glucuronidation.