Robert L. Blakeley

Ellman's reagent: 5,5'-dithiobis(2-nitrobenzoic acid)--a reexamination.

Accurate determination of thiol groups by means of Ellmans reagent [5,5′-dithiobis(2-nitrobenzoic acid), DTNB] has been limited by uncertainty about the molar absorption coefficient of the dianion of the product, 2-nitro-5-thiobenzoic acid (TNB). A procedure is described for the purification of TNB by reduction of commercial DTNB followed by gel chromatography and crystallization. Pure DTNB is prepared by reoxidation of purified TNB followed by gel chromatography and crystallization. The molar absorption coefficient of the dianion of TNB is 14,150 at 412 nm in dilute aqueous salt solutions. This value was confirmed independently by reduction of purified DTNB with cysteine. Titration of sulphydryl groups with DTNB can be done at pH 7.27 where the thiol group of TNB is 99.8% in the intensely-colored conjugate base form while the hydroxide-promoted hydrolysis of DTNB is minimal.

Jack bean urease: the first nickel enzyme

The article defines the development of our present understanding of the urease molecule. It describes physical and chemical properties of the enzyme, the interaction of inhibitors with the active site Ni(II) ion(s), substrate specificity and differential effects of pH on catalytic efficiency. A mechanism consistent with these data is presented. In addition, a simple model is described in which Ni(II) ion promotes nucleophilic attack of ethanol and water on a urea.

An interactive web-based Pseudomonas aeruginosa genome database: discovery of new genes, pathways and structures

Using the complete genome sequence of Pseudomonas: aeruginosa PAO1, sequenced by the Pseudomonas: Genome Project (ftp://ftp.pseudomonas. com/data/pacontigs.121599), a genome database (http://pseudomonas. bit.uq.edu.au/) has been developed containing information on more than 95% of all ORFs in Pseudomonas: aeruginosa. The database is searchable by a variety of means, including gene name, position, keyword, sequence similarity and Pfam domain. Automated and manual annotation, nucleotide and peptide sequences, Pfam and SMART domains (where available), Medline and GenBank links and a scrollable, graphical representation of the surrounding genomic landscape are available for each ORF. Using the database has revealed, among other things, that P. aeruginosa contains four chemotaxis systems, two novel general secretion pathways, at least three loci encoding F17-like thin fimbriae, six novel filamentous haemagglutinin-like genes, a number of unusual composite genetic loci related to vgr/RHS: elements in Escherichia coli, a number of fix-like genes encoding a micro-oxic respiration system, novel biosynthetic pathways and 38 genes containing domains of unknown function (DUF1/DUF2). It is anticipated that this database will be a useful bioinformatic tool for the Pseudomonas: community that will continue to evolve.

Jack Bean Urease VI. Determination of thiol and disulfide content: Reversible inactivation of the enzyme by the blocking of the unique cysteine residue

The total thiol content of highly pure jack bean urease (urea amidohydrolase, EC 3.5.1.5) has been determined by titration with 5,5′-dithiobis(2-nitrobenzoic acid) in the presence of 6 M guanidinium chloride. Urease contains 15 thioi groups per 96.6-kDa subunit. Coupled with amino acid analysis data, this result establishes that urease contains one cystine disulfide bond per 96.6-kDa subunit. Slow loss of enzymatic activity in the presence of β-mercaptoethanol and oxygen is due to the formation of a mixed disulfide which involves the unique active-site cysteine residue. Enzymatic activity can be fully restored by treatment of inactive urease with 0.1 M dithiothreitol at pH 7.3. Urease is quite stable when stored in 0.05 M sulfite/0.02 M phosphate buffer (pH 7.2, 1 mM in EDTA).

[26] Appendix: An accurate gravimetric determination of the concentration of pure proteins and the specific activity of enzymes

Publisher Summary In studies of subunit structure, equivalent weight and mechanism of action of enzymes, an accurate measure of the weight-concentration of the highly purified protein in solution is required. Concentration is usually measured spectrophotometrically, utilizing chromophores associated with the protein itself or chromophores produced in the Folin–Lowry or biuret procedures. In any spectrophotometric assay of protein concentration, it is necessary to know the relationship between the weight-concentration of the protein and the absorbance measured. The chapter describes an absolute gravimetric method for determining the weight-concentration of a protein solution. This method is useful in standardizing the measurement of concentration of a pure protein by any spectrophotometric method. One of the most important physical characteristics of an enzyme is its specific activity. Because of the convenience of measuring the concentration of protein by means of its A 280 , it is suggested that, where possible, the specific activity of an enzyme be expressed in terms of the empirical A 280 rather than in terms of milligram per milliliter or milligram of protein.

Development of polymerase chain reaction and fluorescent in situ hybridisation techniques for the detection of a bacterial strain that degrades the cyanobacterial toxin microcystin LR

Polymerase chain reaction (PCR) and fluorescent in situ hybridisation (FISH) techniques were developed for the detection of a Sphingomonas bacterium (strain MJ-PV), previously demonstrated to degrade the cyanobacterial toxin microcystin LR. A PCR amplification protocol using the primer set Sph-f1008/Sph-r1243 demonstrated specific amplification of the target 16S ribosomal DNA (rDNA) of strain MJ-PV. A 16S ribosomal RNA (rRNA) targeted probe, Sph-r1264, labelled with a rhodamine fluorescent dye was successfully used in whole-cell FISH for the detection of MJ-PV in seeded controls. DNA primers and a PCR protocol were developed for the specific amplification of a gene, mlrA, which codes for the enzyme MlrA, responsible for hydrolysis of the cyanobacterial toxin microcystin LR. A survey using 16S rDNA and mlrA primers on extracted DNA from environmental samples of a lake that suffers regular toxic cyanobacterial blooms demonstrated no amplified products indicative of the presence of MJ-PV or mlrA. Although not detecting the MJ-PV strain in the tested environmental samples, these developed methods are useful to study the distribution of strain MJ-PV demonstrated to degrade mycrocystin LR in seeded bioremediation trails, as well as the distribution and the regulation of mlrA shown to be involved in mycrocystin LR degradation.

VARIABLE STOICHIOMETRY IN THE DECOMPOSITION OF AROMATIC DISULFIDES IN ALKALINE SOLUTION. ON THE PROPERTIES OF 3-CARBOXYLATE-4-NITROBENZENE SULFENATE ION

Abstract The alkaline hydrolysis of low concentrations of the aromatic disulfide 5,5′-dithiobis(2-nitrobenzoic acid) in 3.0 M NaOH, quantitatively forms 3-carboxylate-4-nitrobenzenesulfenate ion as expected for a simple displacement of the thiophenoxide ion by hydroxide ion. In the absence of residual disulfide, the sulfenate ion is stable, apart from slow oxidation to the corresponding sulfinate ion. The red sulfenate ion has an absorption maximum at 492 ± 2 nm with a molar absorption coefficient (∊) of 10,600 M−1 cm−1 at 490 nm. At high initial concentrations of disulfide or at lower concentrations of hydroxide ion, appreciable amounts of the sulfenate ion react with unreacted disulfide to form a transient sulfinic acid thiolester intermediate which decomposes to form the corresponding sulfinate ion and the thiophenoxide ion. This work constitutes the first unambiguous description of the mechanism and variable stoichiometry of the alkaline decomposition of an aromatic disulfide.

Mechanism of the conversion of a nitrobenzene into a 4-nitrosophenol in 15·8 M potassium hydroxide

Quantitative conversion of 2-nitrobenzoate ion into 2-nitroso-5-hydroxybenzoate ion occurs in 15·8 M potassium hydroxide; kinetic and spectral studies are consistent with a mechanism which involves rate-limiting formation of a Meisenheimer adduct with hydroxide ion followed by proton abstraction.