[Comparative stability assessment of laccases from the basidiomycetes Coriolus hirsutus and Coriolus zonatus in the presence of effectors]. / Sravnitel'noe izuchenie stabil'nosti lakkaz iz bazidiomitsetov Coriolus hirsutus i Coriolus zonatus v prisutstvii éffectorov.

Stability characteristics of the laccases of the basidiomycetes Coriolus hirsutus and Coriolus zonatus were measured comparatively at temperatures 25 and 40 degrees C in the presence of various effectors (proteins, salts, polyalcohols, polyacids, and polyelectrolytes). Stabilization effects of cations on the laccases from C. hirsutus and C. zonatus decreased in the descending series Cu2+ > Mg2+ > Ca2+ and Ca2+ > Mg2+ > Mn2+, respectively. Tween 20 caused insignificant stabilization of the two enzymes. The C. zonatus laccase was also insignificantly stabilized as a result of treatment with bovine serum albumin. The enzymatic activity of the laccase preparations from C. hirsutus and C. zonatus was conserved virtually completely after vacuum drying (84 and 93%, respectively). The most effective stabilizer of the C. hirsutus laccase was found to be dextran (17 kD). Dry preparations treated with this agent conserved up to 95% of the enzymatic activity. The most effective stabilizer of the C. zonatus was polyacrylic acid (102% of the initial activity).

Flow-injection glucose determination with long-wavelength luminescent oxygen probes.

A flow-injection method for the determination of glucose in serum is presented. It is based on the enzymatic measurement of oxygen consumption detected via oxygen quenching of the luminescence of certain metalloporphyrins. Phosphorescent water-soluble Pt2+ and Pd(2+)-porphyrins have been characterized by luminescence spectroscopy and decay-time measurements in various buffers, and found to be suitable for oxygen detection in biological systems. A new method for the flow-injection analysis of glucose has been developed based on the use of a column of immobilized glucose oxidase and the indicators Pt(2+)-coproporphyrin III and Pd(2+)-coproporphyrin I. The system has been optimized for glucose determination in aqueous samples and in whole serum with the 0.5-200 mM glucose range. Twenty assays can be performed in an hour, and the system has potential for commercial development with biotechnological and medical applications.

Fibre-optic oxygen sensor based on phosphorescence quenching.

A fibre-optic oxygen sensor is described which is based on an oxygen-sensitive luminescent film made from platinum octaethylporphyrin and polystyrene. The luminescence and quenching characteristics of such films were studied for their use in a fibre-optic oxygen biosensor. A prototype oxygen sensor was made from this material, and was tested in aqueous solutions and in the gaseous phase at physiological oxygen concentrations. Measurements of luminescence intensity and decay time were employed to determine oxygen concentration from luminescence quenching. The main working characteristics of this prototype oxygen sensor were studied.

Photodestruction in vitro of tumour cells sensitized by porphyrins and their conjugates with specific antibodies.

The photodynamic action of a number of carboxylic porphyrins and their covalent conjugates with specific monoclonal IgM antibodies on a human lung adenocarcinoma cell line was studied. All the conjugates showed strong phototoxicity which did not correspond with the phototoxicity of the free porphyrins. Absolute quantum yields of singlet oxygen photogeneration by the porphyrins and their conjugates were obtained and compared with the phototoxicity of the compounds.

[Chromatography of water-soluble porphyrins and their conjugates with proteins]. / Khromatografiia vodorastvorimykh porfirinov i ikh kon''iugatov s belkami.

The chromatographic behaviour of tetra- and octacarboxylic porphyrins and tetra-(p-sulphophenyl)-porphin was being studied by HPLC using Protein Pak and TSK-type gel permeation columns. The mobility of the porphyrins on the carriers depended on the ionic strength of the eluating buffer Tris-HCl. The conditions for separation of the porphyrins and their protein conjugates were optimized. The separation was performed under mild conditions with minimum denaturation of conjugates. The purification technique can be used to separate porphyrin conjugates with various proteins. The technique can be also used in conventional column chromatography on Toyopearl supports.

[Fluorescent immunoanalysis. Synthesis, spectro-fluorescent and immunologic properties of conjugates of coproporphyrin I with antibodies]. / Fluorestsentni immunoanaliz. Sintez, spektral'no-fluorestsentnye i immunologicheskie svoistva kon''iugatov koproporfirina I s antitelami

The synthesis of protein conjugates with the new high-efficient fluorescent labile coproporphyrin-I was optimized. A number of conjugates of monoclonal antibodies with different coproporphyrin-I content were synthesized, and their spectral properties were studied in water and micellar solutions, i.e. adsorption, excitation and emission spectra, fluorescence quantum yields, fluorescence pH-dependences. The binding constants of coproporphyrin-I and its protein conjugates with serum albumin were determined. The antibodies labelled with coproporphyrin-I retain the functional activity and photochemically stable in water solutions. The sensitivity of fluorometric detection of coproporphyrin-I and its conjugates with proteins is more than 10 times greater than in case of FITC.

[Fluorescent immunoassay of thyroxine in blood serum]. / Fluorestsentnyi immunoanaliz tiroksina v syvorotke krovi.

A method of solid-phase fluoroimmunoassay of thyroxine (T4) on polystyrene 96-microtiter plates using coproporphyrin III-labeled antibodies was developed; 0.02 ml of blood serum without preliminary treatment was required. Thimerosal was added to lessen the T4-binding effect of the serum on the results of T4-binding effect of the serum on the results of the immunoassay. A calibration curve was obtained to determine T4 covering the entire physiological range of concentrations (from 3 to 21 micrograms of thyroxine in 100 ml of the serum). A variation coefficient within this range did not exceed 15%. For 14 unknown blood serum samples of patients the coefficient of correlation with the results obtained using a commercial kit for T4 determination (Abbott, USA) was 0.960. The time of the assay was 2-2.5 h. This method is simple and easy for use on a large scale.