Fast atom bombardment/tandem mass spectrometry of physalaemin-like peptides.

Fragment ion spectra obtained from collision-induced decomposition of protonated molecular ions have been used to determine amino acid sequences of several physalaemin-like peptides which were recently purified from rabbit stomach. This technique was chosen because the peptides were available in microgram quantities and were anticipated to contain pyroglutamate as the blocked N-terminal residue. Such spectra of several synthetic analogs of the naturally occurring peptides were obtained and analyzed to confirm the veracity of this peptide sequencing strategy. In addition, methyl ester derivatives of these synthetic peptides provided a crucial test for the spectral interpretation via the mass shifts thus induced.

Physalaemin-like immunoreactive peptides from rabbit stomach.

Two physalaemin (PHY)-like immunoreactive peptides, designated PHLIPs, have been purified from extracts of rabbit stomach tissue. Fast atom bombardment/mass spectrometry (FAB/MS) indicated that the m/z values for the PHLIP protonated molecular ions were 867.419 and 796.4. FAB/tandem MS spectra, coupled with a knowledge of the amino acid composition and the aid of a computerized fragment-matching program, indicated the amino acid sequences to be: (formula; see text) The sequences of PHLIPs-7 and -8 were confirmed with synthetic peptides. The PHY-antiserum cross-reactivity of the PHLIPs reflects homology at amino acid residues 1, 3, 4 and 5 for the mammalian and amphibian residues.

Peptide sequencing program.

A computer program has been devised to automate rationalization of peptide fragmentation patterns. The program systematically generates all possible linear amino acid sequences which might be attributable to a peptide with a known amino acid composition. The generated sequences are then searched to find those that most closely match the spectrum of an unknown sequence.

Beta-epidermal growth factor is the des-asparaginyl form of the polypeptide.

Reversed-phase high performance liquid chromatography of mouse epidermal growth factor (EGF) yielded two major forms, alpha- and beta-EGF, and a minor component, gamma-EGF. All three forms exhibited receptor-binding activity. Analysis of native alpha- and beta-EGF by mass spectrometry and partial Edman degradation led us to propose that alpha-EGF has a primary structure equivalent to that originally reported for EGF and that beta-EGF is the des-asparaginyl form of the polypeptide. When the purified alpha- and beta-polypeptides were cultured with human embryonic palatal mesenchymal cells stimulation of cell proliferation was observed at concentrations as low as 0.01 ng/ml with maximal stimulation occurring at about 1 ng/ml. Essentially no difference was noted in the mitogenic potency of the two forms. This suggests that the NH2-terminal region of EGF is not critical for mitogenic activity.

Characteristics of an extraction and purification procedure for chlorinated dibenzo-p-dioxins and dibenzofurans in soil and liver.

Liver is extracted with chloroform-methanol to give essentially quantitative transfer of endogenous chlorinated dibenzo-p-dioxins (CDDs) and dibenzofurans (CDFs) into the organic phase. A new procedure involving LH-20 Sephadex is used to remove most of the lipids from the extract. Soil is extracted by a simple, rapid and economical procedure giving very high recoveries of CDDs and CDFs from sandy soil, various types of clay, and humus-rich loam. Subsequent cleanup on basic and acidic alumina complete the preparation for gas chromatography-mass spectrometric analysis. The use of propylene glycol as a "keeper" and of 2,3,7-trichlorodibenzo-p-dioxin as a carrier minimizes losses during evaporation of solvents and on glass surfaces. Interactions of 2,3,7,8-CDD with organic material in loam slightly reduce recovery but there is no indication of high affinity binding sites, the losses being apparently associated with simple distribution coefficients. Special precautions needed to avoid losses of CDFs on alumina chromatography are described, and the effect of "aging" spiked soil is discussed.

Dioxin in soil: bioavailability after ingestion by rats and guinea pigs.

Soil environmentally contaminated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was given by gavage to guinea pigs and rats. The development of a characteristic clinicopathologic syndrome in guinea pigs, the induction of aryl hydrocarbon hydroxylase in rats, and the presence of TCDD in the livers of both species show that TCDD in soil exhibits high biological availability after ingestion.

The oxidation of 2-aminofluorene by prostaglandin endoperoxide synthetase. Comparison with other peroxidases.

We have examined the prostaglandin endoperoxide synthetase-dependent metabolism of the arylamine carcinogen 2-aminofluorene (2-AF). Ram seminal vesicle microsomes fortified with arachidonic acid metabolize 2-AF to products covalently bound to microsomal macromolecules, water-soluble metabolites, and two organic extractable metabolites. The organic extractable metabolites were identified by co-chromatography, uv-visible spectrophotometry, and mass spectrometry as 2-nitrofluorene and 2,2'-azobisfluorene (azofluorene). Hydrogen peroxide also supports 2-AF metabolism to the same products, suggesting that the hydroperoxidase activity of prostaglandin endoperoxide synthetase is responsible for the co-oxidation. The highly reactive oxygenated metabolites of 2-AF, N-hydroxy-2-AF, and 2-nitrosofluorene, are metabolized by prostaglandin endoperoxide synthetase to one major product, 2-nitrofluorene. The metabolism of 2-AF, N-hydroxy-2-AF, and 2-nitrosofluorene is extremely rapid, reaching completion in less than 30 s. The horseradish peroxidase/H2O2 system also metabolites 2-AF to 2-nitrofluorene and azofluorene. The chloroperoxidase/H2O2 system, however, yields primarily 2-nitrosofluorene from 2-AF. These results suggest that 2-AF is oxidized to an electrophilic intermediate(s) by prostaglandin endoperoxide synthetase, which either binds covalently to tissue macromolecules or is further rapidly oxidized to 2-nitrofluorene and azofluorene. Furthermore, this reaction probably proceeds through a free radical mechanism similar to that of horseradish peroxidase.

Mass spectrometry of di-(2-ethylhexyl)phthalate metabolites.

The electron impact, methane positive chemical ionization, oxygen negative chemical ionization and collision induced dissociation mass analyzed ion kinetic energy spectra of nine metabolites of di-(2-ethylhexyl)phthalate are presented. The collision induced dissociation mass analyzed ion kinetic energy spectra of selected fragment ions were useful for structural information.

Adduct formation between the carcinogen N-acetoxy-2-acetylaminofluorene and synthetic polydeoxyribonucleotides.

The chemical carcinogen N-acetoxy-2-acetylaminofluorene (NA-AAF) was reacted with poly(dG-dC) - poly(dG-dC); poly dG - poly dC; poly(dA-dT) - poly (dA-dT); and poly dA - poly dT under a variety of conditions. Poly (dG-homo GC polymer and 10--20 more reactive the A + T polymers. Lowering the ionic strength increased the extent of reaction, while pH change (8.9 vs. 5.5) had only a small effect. If ionic strength was adjusted so that the two guanine-containing polymers showed equal thermal stability (as judged by Tm) then the alternating copolymer was 7 times as reactive as the homopolymer. In aggreement with previous investigators, the major product was found to be 8-(N-2-fluorenylacetamido) deoxyguanosine.

Effect of mercuric and silver ions on cesium sulfate equilibrium buoyant densities of synthetic polydeoxyribonucleotides.

The effect of Hg2+ and Ag+ on the buoyant density (rho) of four synthetic DNA polymers, poly[d(A-T)]; poly(dA) - poly(dT); oikt[d(G-C)]; and poly(dG) - poly(dC), was investigated. The buoyant density of poly[d(A-T)] in Cs2SO4 increased dramatically after complexing with Hg2+, but little change in the buoyant density of other polymers resulted except at very high molar ratios of Hg2+/DNA-P (rf). Hg2+ raised the thermal transition temperature (Tm) of alternating polymers and lowered the Tm of homopolymers. Measurements in the preparative ultracentrifuge indicated that lowered Tm correlated with Hg2+-induced strand separation of one homopolymer [poly(dA) - poly(dT)], but strand separation was not observed with another homopolymer [poly(dG) - poly(dC)] complexed with Hg2+. When Ag+ was mixed with the polymers, the buoyant density of poly(dG) - poly(dC) increased most markedly. A substantial increase in the buoyant density of poly[d(A-T)] and a small increase in the buoyant density of poly[d(G-C)] were also observed. The Tm changes induced by Ag+ were not related in any obvious way to buoyant density changes. These findings indicate that nucleotide sequence as well as overall base composition is of importance in understanding the buoyant density changes induced by metal ions. Although these data do not allow construction of a detailed molecular model of polymer-metal ion interactions, they may be used to explain much of the behavior of naturally occurring DNA sequences, such as heterochromatic satellite sequences and 5 S and rRNA sequences, in Hg2+/Cs2SO4 and Ag+/Cs2SO4 gradients.