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Biomedical subjects

R C Kelly

Publications and source records attributed to R C Kelly.

At least 37 records · Page 2Linked to original sources
PubMed1984

Improved recovery and stability of ethanol in automated headspace analysis.

Solutions are reported here for two problems in automated headspace gas chromatographic analysis for ethanol. The first is the catalytic oxidation of ethanol by oxyhemoglobin, a process that is limited only by the amount of oxygen in the sealed vessel. This reaction is prevented by the addition of sodium dithionite. The other problem, the lack of a suitable salting-out agent, led to the selection of ammonium sulfate, which improved recovery of ethanol as well as enhancing method precision. A procedure incorporating these improvements is described.

Chromatography, Gas↗
PubMed1982

Identification of phencyclidine and its analogues at low concentrations in urine by selected ion monitoring.

This paper presents retention behavior and mass spectra of eluting peaks for twelve phenyl and thienyl analogues of phencyclidine (PCP) as well as PCP itself. An on-column decomposition product common to all thienyl analogues is described. Finally, a practical analytical procedure is given for the detection and identification of these compounds in low (under 5 ng/mL) concentrations in urine.

Humans↗
PubMed1979

Toxicological determination of benzodiazepines in serum: methods and concentrations associated with high-dose intravenous therapy with diazepam.

This paper describes a rapid and simple procedure for the determination of benzodiazepines in biological samples. Five common benzodiazepines (diazepam, oxazepam, clorazepate, flurazepam, and chlordiazepoxide) and/or their major metabolites are extracted from a buffered serum sample at pH 9.2 by a mixture of toluene, hexane, and isoamyl alcohol. The phases are separated by centrifugation, and a small aliquot of the organic layer is injected into a gas chromatograph equipped with an electron-capture detector. The drugs are identified and quantitated by comparison with standards simultaneously processed similarly. A sample can be analyzed in about 30 min. The technique is illustrated by its application to sequential samples from a patient being treated for delirium tremens with large intravenous doses of diazepam. Serum diazepam, N-desmethyldiazepam, and oxazepam concentrations several times higher than usually encountered therapeutically were observed.

Adult↗
PubMed1978

Preparation and spectral properties of 12-hydroxyl eicosatetraenoic acid (HETE).

A convenient method for the biosynthesis of 12L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE) from archidonic acid with human platelets was developed, and milligram quantities were made for biological and spectroscopic characterization. This compound has only weak activity in two of five biological prostaglandin assays and no activity in the other three. The 13C and 1H NMR spectra of HETE were obtained and the signal due to each carbon atom was assigned.

Arachidonic Acids↗
PubMed1977

Solvent suppression of the decomposition of phenobarbital during on-column methylation with trimethylanilinium hydroxide.

Gas-liquid chromatographic (GLC) analysis of phenobarbital by on-column methylation with trimethylanilinium hydroxide gives rise to a major decomposition product, N-methyl-2-phenylbutyramide, in addition to the methylated barbiturate, N,N'-dimethylphenobarbital. This reaction occurs nearly exclusively in the solution phase in the injection port of the gas chromatography. A mechanism for the decomposition reaction consistent with the available information is presented. This decomposition is shown to be inhibited by certain solvents and this effect forms the basis of a new analytical technique for the simultaneous GLC analysis of phenobarbital, primidone, and diphenylhydantoin.

Aniline Compounds↗
PubMed1976

DNA "melting" proteins. II. Effects of bacteriophage T4 gene 32-protein binding on the conformation and stability of nucleic acid structures.

Bacteriophage T4-coded gene 32-protein is an essential component of the T4 replication and recombination systems. Alberts and co-workers (Alberts, B.M., Amodio, F.J., Jenkins, M., Gutmann, E.D., and Ferris, F.L. (1968) Cold Spring Harbor Symp. Quant. Biol. 33, 289-305) have shown that the major physiological activity of the protein involves preferential and cooperative binding to single-stranded DNA. In this paper, the physiochemical parameters characterizing this "melting" protein system are quantitatively determined. Boundary sedimentation velocity experiments are used to measure the interaction of gene 32-protein with native DNA. The binding is shown to be non-cooperative and involves an overlapping site size (nh) of approximately 10 nucleotide residues (or approximately 5 nucleotide pairs). In analogy with the ribonuclease results (Jensen, D.E., and von Hippel, P.H. (1976) J. Biol. Chem. 251, 7198-7214), the logarithm of the association constant (Kh) is found to be linerarly related to log [Na+]. The binding of gene 32-protein to denatured (single-stranded) DNA involves appreciable distortion of the polynucleotide backbone from the unliganded conformation; binding totally unstacks the bases of both ribose- and deoxyribose-containing polynucleotides at 10 degrees, and results in a hyperchromic change exceeding that which can be induced by heating. This hyperchromism induced in poly(dA) on binding gene 32-protein under low salt (tight binding) conditions is used to determine a value of nc (the single-stranded DNA site size) of approximately 6.7 nucldotide residues per protein. In addition, gene 32-protein binding to single-stranded polynucleotide induces an unusual circular dichroic spectrum characterized principally by a marked decrease in the magnitude of the positive CD band centered at approximately 265 nm. This spectral change is attributed to significant uncoupling of the transition moments of the vicinal bases of the single-stranded polynucleotide on gene 32-protein binding, in accord with the ultraviolet hyperchromism observed. Binding of gene 32-protein to double helical DNA has virtually no effect on the spectral properties of this conformation...

Binding Sites↗
PubMed1976

DNA "melting" proteins. III. Fluorescence "mapping" of the nucleic acid binding site of bacteriophage T4 gene 32-protein.

The intrinsic tryptophan fluorescence of bacteriophage T4-coded gene 32-protein is found to be partially quenched on binding a variety of mono-, oligo-, and polynucleotides. This phenomenon is exploited to partially "map" the nucleic acid binding site of the protein. The intrinsic fluorescence spectrum of the protein peaks at about 347 nm, compared to 359 nm for the fully solvated model fluorophore, N-acetyl-L-tryptophanamide. Nucleotide binding, or collisional quenching by iodide ion, reduces the intensity of the fluorescence, with little or no peak shift. Small ligands, ranging in size from ribose- and deoxyribose-phosphate to tetranucleotides, quench the fluorescence by 2 to 6%; larger ligands quench from 20 to 35% of the intrinsic protein fluorescence. Iodide quenching experiments subjected to Stern-Vollmer analysis suggest that the binding of short nucleotide-containing ligands brings about a conformational change in the protein, fully exposing a tryptophan side chain to the solvent environment. The fluorescence of this tryptophan is fully quenched by the binding of d(Ap)2, but is largely unaffected by the binding of d(ApA) or d(pA)2, indicating both that this (tryptophan) "reporter" residue is located in the nucleic acid binding site and that binding is polar, i.e. polynucleotide chains of only one orientation are complexed. Long oligonucleotides fully quench the fluorescence of this binding site tryptophan. At high salt concentration (2 M NaCl), gene 32-protein forms self-limited dimers (Carroll, R.B., Neet, K.E., and Goldthwait, D.A. (1972) Proc. Natl, Acad. Sci. U.S.A. 69, 2741-2744; (1975) J. Mol. Biol. 91, 275-291). These dimers, in either high salt or in low salt after cross-linking, fail to bind nucleotides, suggesting that dimer formation partially occludes the nucleic acid binding site and thus that these dimers are probably not involved as intermediates in cooperative protein binding to the DNA. On the other hand, dimerization apparently results in a conformational change which fully exposes the "reporter" tryptophan to iodide quenching. These results are used to formulate a model of some of the nucleic acid-protein and protein-protein interactions involved in the cooperative binding of gene 32-protein to single-stranded DNA.

Binding Sites↗
PubMed1976

DNA "melting" proteins. IV. Fluorescence measurements of binding parameters for bacteriophage T4 gene 32-protein to mono-, oligo-, and polynucleotides.

The quenching of the intrinsic tryptophan fluorescence of T4-coded gene 32-protein on binding to nucleotide ligands, which was described in the preceding paper, is here exploited to measure thermodynamic parameters of the single-stranded nucleic acid-gene 32-protein interaction. It is shown that binding of small ligands follows a single site binding isotherm, with association constants increasing from approximately 20 M-1 for phosphate, to approximately 10(3) M for ribose or deoxyribose 5'-phosphate, to approximately 10(4) M-1 for mononucleotides, and to approximately 10(5) M-1 for dinucleoside monophosphates (all in 0.1 M Na+). The measured binding constants appear to be about the same for homologous ribose- and deoxyribose-containing ligands and to be independent of oligonucleotide base sequence and composition. Furthermore, beyond the dinucleotide level and up to octanucleotides, the increase in binding constant with increasing chain length is only about that expected from the statistical factor resulting from the increased number of ways a longer oligonucleotide can form a protein complex. This suggests that the basic binding unit involved in gene 32-protein associations with single-stranded nucleic acids can be approximated by a dinucleoside monophosphate. Oligonucleotides long enough to accomodate two or more protein monomers are characterized by much larger association constants, indicating that binding is cooperative in protein concentration. A cooperativity parameter (omegac) of approximately 10(3) is estimated from these data, in good agreement with that deduced from the application of ligand-perturbed helix in equilibrium coil transition calculations. Values of association constants (Kcomegac) of approximately 10(8) M-1 (in 0.1 M Na+) and site size (nc) of approximately 5 (+/-1) nucleotide residues/protein monomer are determined by the fluorescence titration technique for the cooperative binding of gene 32-protein to both poly(dA) and poly(rA); these values are also in agreement with those measured by Jensen et al. (Jensen, D.E. Kelly, R.C., and von Hippel, P.H. (1976) J. Biol. Chem. 251, 7215-7228). Possible in vivo consequences and correlations of these findings with proposed roles for gene 32-protein in replication and recombination are discussed.

Binding Sites↗