Search

A new formulation of the bireagent kit for serum uric acid assay was developed based on the effects of pH on enzyme stability. At 4 °C, half-lives of uricases from Bacillus fastidious and Arthrobacter globiforms were longer than 15 months at pH 9.2, but became shorter at pH below 8.0; half-lives of ascorbate oxidase and peroxidase were comparable at pH 6.5 and 7.0, but became much shorter at pH higher than 7.4. In the new formulation of the bireagent kit, Reagent A contained peroxidase, 4-aminoantipyrine, and ascorbate oxidase in 50 mM phosphate buffer at pH 6.5; Reagent B contained B. fastidious or A. globiforms uricase in 50 mM sodium borate buffer at pH 9.2; Reagents A and B were mixed at 4:1 to produce a final pH from 7.2 to 7.6 for developing a stable color. The new bireagent kit consumed smaller quantities of three enzymes for the same shelf life. With the new bireagent kit, there were linear responses of absorbance at 546 nm to uric acid up to 34 mM in reaction mixtures and a good correlation of uric acid levels in clinical sera with those by a commercial kit, but stronger resistance to ascorbate. Therefore, the new formulation was advantageous. [ABSTRACT FROM AUTHOR]

The feasibility for microplate-based screening of inhibitors of isozymes of cyclic nucleotide phosphodiesterase (PDE) was tested via the coupled action of a phosphatase on adenosine-5′-monophosphate and an improved malachite green assay of phosphate. Human full-length PDE4B2 and truncated mutant (152-528aa) were expressed in Escherichia coli via fusion to SUMO, which after purification through Ni-NTA column exhibited specific activities >0.017 U mg−1. In the presence of proteins <30 mg L−1, absorbance for 10 µΜ phosphate was measurable; a PDE isozyme of specific activity over 0.008 U mg−1 after reaction for 20 min thus suited for microplate-based screening of inhibitors. By using Biotek ELX 800 microplate reader, affinities of two forms of PEDE4B2 for cAMP, rolipram and papaverine varied over three magnitudes and were consistent with those by routine assay, respectively. Hence, the proposed method was promising for high-throughput-screening of inhibitors of phosphate-releasing enzymes bearing specific activities over 0.008 U mg−1. [ABSTRACT FROM AUTHOR]

Abstract: Homogeneous noncompetitive assay of a protein in biological samples based on Förster-resonance-energy-transfer (FRET) was proposed by using its tryptophan residues as intrinsic donors and its specific fluorescent ligand as the FRET acceptor that was defined as an analytical FRET probe. Conjugate of a suitable fluorophore, which should have an excitation peak around 340nm but an excitation valley around 280nm, with a moiety binding to a protein of interest gave an analytical FRET probe to the protein. To test this method, N-biotinyl-N′-(1-naphthyl)-ethylenediamine (BNEDA) was used as an analytical FRET probe for homogeneous noncompetitive assay of streptavidin (SAV). The occurrence of FRET between the bound BNEDA and tryptophan residues was supported by the modeled geometry of the complex. By excitation at 280nm, free BNEDA produced negligible fluorescence at 430nm, but the bound BNEDA produced much higher stable fluorescence at 430nm after 2min of binding reaction. The competitive binding between BNEDA and biotin gave the dissociation constant of (16±3)fM for BNEDA (n =3). By excitation at 280nm, fluorescence at 430nm of reaction mixtures containing 32.0nM BNEDA responded linearly to SAV subunit concentrations ranging from 0.40 to 30.0nM with the desirable resistance to common interferences in biological samples. Therefore, by using tryptophan residue(s) in a protein of interest as intrinsic donor(s) and its fluorescent ligand as the corresponding FRET acceptor, this homogeneous noncompetitive assay of the protein in biological samples was effective and advantageous. [Copyright &y& Elsevier]

Highlights: [•] 1-Anilinonaphthalene-8-sulphonate (ANS) emitted at 470nm in organic solvents. [•] The complex of ANS and albumin (ALB) was effectively excited at 280 and 350nm. [•] In 20.0mM phosphate at pH 7.0, ANS<0.65μM bound to ALB at 1:1 with K d of 0.13μM. [•] At 0.3μM ANS in the buffer, fluorescence at 470nm was proportional to ALB levels. [•] New method had stronger resistance to interference and higher sensitivity than classical methods. [Copyright &y& Elsevier]

Abstract: The classical malachite green (MLG) assay of phosphate, which added MLG after molybdate to the acidified reaction solutions of phosphate, tolerated interference from papaverine, sildenafil, and some similar hydrophobic amines. Resonance Rayleigh scattering signals, the alleviation of interference by poly(vinyl alcohol), and the precipitation of some yellow complexes supported that the irreversible aggregation of the complexes of a hydrophobic amine of interference and phosphomolybdate reduced the amounts of phosphomolybdate accessible to MLG and caused the interference. By adding MLG before molybdate to the acidified reaction solutions of phosphate, the complexes of phosphomolybdate and MLG were preferentially formed before the complexes of phosphomolybdate and such a hydrophobic amine effectively aggregated; thereby, an improved MLG assay of phosphate with the resistance to common hydrophobic amines was developed. Using the improved MLG assay of phosphate and a phosphatase to release phosphate from AMP, a spectrometric method successfully estimated the half-inhibition concentrations of papaverine on the recombinant human cyclic nucleotide phosphodiesterase (PDE) isozyme 4 and the mixture of PDE isozymes from rabbit brain. Therefore, the improved MLG assay of phosphate was a favorable and universal technique for developing spectrometric methods for characterizing and screening inhibitors of enzymes that release phosphate during their actions. [ABSTRACT FROM AUTHOR]