Biomedical subjects

J L Corbin

Publications and source records attributed to J L Corbin.

14 recordsLinked to original sources

PubMed1990

A chemical preparation of pure reduced viologens for use as biomolecular reducing reagents.

A chemical method is reported for conveniently preparing a variety of pure, reduced low-potential viologens for use as biomolecular reductants. The free radical, semiquinone viologen form is prepared anaerobically in aqueous solution by coproportionation of the dihydroviologen with the fully oxidized viologen according to the following reaction, using methyl viologen (MV) as an example: MV + MVH2 = 2 MV.. By varying the substituents on the viologen nitrogen atoms, a series of viologens of varying charge and reduction potential is easily obtained. Applications involving the reduction of various metalloproteins are presented.

Animals↗

PubMed1989

An Improved Method for Analysis of Polyamines in Plant Tissue by Precolumn Derivatization with o-Phthalaldehyde and Separation by High Performance Liquid Chromatography.

An improved high-performance liquid-chromatographic method was developed for estimation of polyamines in crude plant extracts. Polyamines were derivatized with o-phthalaldehyde and mercaptoethanol (OPT). The fluorescent derivatives were eluted from a C(18) column with the dimethylcyclohexylamine-phosphate buffer derived by T. Skaaden and T. Greibrokk ([1982] J Chromatogr 247: 111-122) after treatment to remove impurities in the buffer. The method had a sensitivity of 1-2 picomoles and completely resolved nine polyamines (agmatine, spermine, nor-spermidine, spermidine, 3,5-homospermidine, 4,4-homospermidine, 1,3-diaminopropane, putrescine, and cadaverine) in 12 to 14 minutes. An optional ion-exchange step was used to remove less basic amines (including amino acids) and to concentrate the crude extracts. This method was compared with benzoyl chloride derivatization. Use of the benzoyl chloride method vastly under-estimated the amount of polyamine in some plant extracts, a problem not encountered with the OPT procedure. Additionally, the OPT procedure resolved two isomers of homospermidine found in Azolla caroliniana. These two isomers were not resolved with the benzoylation method. Overall, the OPT method described here requires preparation and analysis time similar to other current methods but provides greater sensitivity and selectivity.

Journal Article↗

PubMed1984

Liquid chromatographic-fluorescence determination of ammonia from nitrogenase reactions: a 2-min assay.

The analytical potential of the reaction of ammonia with o-phthalaldehyde mercaptoethanol reagent at pH 7 (an atypical fluorescence) has already been demonstrated. This, coupled with additional findings reported here, has led to an ammonia determination well suited to nitrogenase studies. As a result, large numbers of samples can be rapidly analyzed by high-pressure liquid chromatrography methods under mild conditions and without prior microdiffusion. Neither sodium dithionite (or other components of the usual nitrogenase assay), nor alternative substrates (cyanide, azide, methyl isonitrile), nor their products (methylamine, dimethylamine, hydrazine) interfere. High-pressure liquid chromatography showed that the fluorescent "product" of the o-phthalaldehyde mercaptoethanol reagent-ammonia reaction was, in fact, more than just a single compound. Despite this, once the proper solvent composition was found, high-pressure liquid chromatography with a small inexpensive C(18) "guard" column proved quite fast and reproducible for this measurement. Fluorescence response to ammonia was linear to at least 40 nmol/ml. A previous problem, long-term stability of the fluorescence, was solved by running the reactions in the dark. Background ammonia in the buffer could be substantially reduced by an analogous o-phthalaldehyde mercaptoethanol reagent reaction, using t-butyl mercaptan, and solvent extraction.

Journal Article↗

PubMed1983

Nitrogenase reactivity: methyl isocyanide as substrate and inhibitor.

We have examined the interaction of methyl isocyanide with the purified component proteins of Azotobacter vinelandii nitrogenase (Av1 and Av2). CH3NC was shown to be a potent reversible inhibitor (Ki = 158 microM) of total electron flow, apparently uncoupling magnesium adenosine 5'-triphosphate hydrolysis from electron transfer to substrate. CH3NC is a substrate (Km = 0.688 mM at Av2/Av1 = 8), and extrapolation of the data indicates that at high enough CH3NC concentration, H2 evolution can be eliminated. The products are methane plus methylamine (six electrons) and dimethylamine (four electrons). There is an excess (relative to methane) of methylamine formed, which may arise by hydrolysis of a two-electron intermediate. A rapid high-performance liquid chromatography/fluorescence method was developed for methylamine determination. The products C2H4 and C2H6 appear to be formed via a reduction followed by an insertion mechanism. CH3NC appears to be reduced at an enzyme state more oxidized than the one responsible for H2 evolution or N2 reduction. Other substrates (C2H2 greater than N2 congruent to azide greater than N2O) all both relieve CH3NC inhibition and inhibit CH3NC reduction. Both effects occur in the same relative order, implying productive (substrate) and nonproductive (inhibitor) modes of binding of CH3NC to the same site.

Adenosine Triphosphate↗

PubMed1982

Nitrogenase reactivity: cyanide as substrate and inhibitor.

We have examined the reduction of cyanide by using the purified component proteins of nitrogenase (Av1 and Av2). The previously reported self-inhibition phenomenon was found to be an artifact. One of the two species present in cyanide solutions, CN-, was shown to be a potent reversible inhibitor (Ki = 27 microM) of total electron flow, apparently uncoupling MgATP hydrolysis and electron transfer. There appears to be no differential effect of CN- on the specific activities of Av1 and Av2 nor is there any apparent irreversible physical damage to Av2. CN- inhibition is completely reversed by low levels of CO, implying a common binding site. Azide partially relieves the inhibitory effect, but other substrates and inhibitors (N2, C2H2, N2O, H2) have no effect. The other species present in cyanide solutions, HCN, was shown to be the substrate (Km = 4.5 mM at Av2/Av1 = 8), and extrapolation of the data indicates that at high enough HCN concentration H2 evolution can be eliminated. The products are methane plus ammonia (six electrons), and methylamine (four electrons). There is an excess (relative to methane) of ammonia formed, which, according to electron balance studies, may arise from a two-electron intermediate. Both nitrous oxide and acetylene (but not N2) influence the distribution of cyanide reduction products, implying simultaneous binding. HCN appears to bind to and be reduced at an enzyme state more oxidized than the one responsible for either H2 evolution or N2 reduction.

Adenosine Triphosphate↗

PubMed1982

Iron-molybdenum cofactor from nitrogenase. Modified extraction methods as probes for composition.

Five modifications of the preparative procedure for isolating iron-molybdenum cofactor (FeMoco) from the molybdenum-iron (MoFe) protein of Azotobacter vinelandii nitrogenase have been developed. This variety of isolation methods has established that no single component of the original isolation protocol, i.e. Tris, Cl-, citrate, HPO4(2-), N,N-dimethylformamide, and N-methylformamide, is essential for the effective isolation and/or structural stability of FeMoco, although any of them may act as ligands to FeMoco when present. The acid-bse status (effective pH) of the extracting solvent is a key adjustable parameter in the isolation procedure. The new procedures produced FeMoco with yields, metal analysis, charge, EPR spectrum, and specific activity (after reconstituting crude extracts from A. vinelandii UW45 mutant cells) essentially identical with FeMoco isolated by the original procedure. After purification, FeMoco apparently contains molybdenum, iron, and sulfide in a 1:7:4 ratio with N-methylformamide as a ligand but no amino acid residues, common sugars, coenzyme A, or lipoic acid. Reaction with o-phenanthroline allows quantitation of both adventitious and FeMoco-associated iron. Correlations of total activity after UW45 reconstitution with molybdenum, total iron, and o-phenanthroline-resistant iron contents show that only the last gives a consistent relationship of 35 +/- 5 nmol of C2H4/min/ng atom of Fe. Both o-phenanthroline and EDTA interact with FeMoco to abolish its EPR signal in reactions reversible by additions of Fe2+ or Zn2+, respectively. These and related reactions point against the presence of an endogenous organic component in FeMoco and toward the presence of exogenous ligands and imply a relatively labile coordination sphere whose nature may be determinable by a systematic investigation.

Amino Acids↗

PubMed1978