Enzyme modification by polymers with solubilities that change in response to photoirradiation in organic media.

We have synthesized a hybrid subtilisin the solubility of which can be regulated by photoirradiation through coupling with a photoresponsive copolymer that carries spiropyran groups in its side chains. The copolymer was synthesized by polymerization of methacrylate, methacrylic acid, and spiropyran-carrying methacrylate. It was then covalently bonded to the amino groups of subtilisin Carlsberg via its carboxyl groups using a carbodiimide coupling agent. The hybrid subtilisin was perfectly soluble in toluene and efficiently catalyzed transesterification. After ultraviolet irradiation, the hybrid subtilisin precipitated and was easily and quantitatively recovered by centrifugation. Recovered hybrid subtilisin, resolubilized by visible light irradiation, retained its original transesterification activity even after several cycles of precipitation and solubilization.

[The possible role of the elements of protein secondary structure in adaptation to the action of ionizing radiation]. / O vozmozhnoi roli élementov vtorichnoi struktury belkov v adaptatsii k deistviiu ioniziruiushchei radiatsii.

Changes in the secondary structure of enzymes induced by gamma-rays 60Co at doses not exceeding one ionization per macromolecule were studied to elucidate a possible role of radiation-chemical processes in the evolution of proteins. The data on the comparative radioresistance of various types of secondary protein structures, alpha-helix, parallel and anti-parallel beta-structures, and beta-turn, were obtained by the method of circular dichroism. It was shown that beta-turns were resistant against radiation, alpha-helix was relatively stable, and beta-layer underwent significant changes. The importance of these structural types in the evolution of proteins is discussed. A special role of beta-turn as structural elements fixing the confirmation of macromolecules and therefore responsible for adaptation of the protein structure against a constant radiation background is proposed.

[Effect of an electromagnetic field on subtilisin from Bacillus subtilis 316 m]. / Vozdeistvie élektromagnitnogo polia na subtilizin Bacillus subtilis 316 m.

The influence of electromagnetic field on the subtilisin of Bacillus subtilis strain 316 M preparation has been studied. It is the increase 30-60% of the proteolytic activity during 60-100 min treatment was observed. The effect of activation is stable for a month at +4 degrees C. The increase of the proteolytic activity is connected with conformational changes of the enzyme molecule, with the decrease of pI from 11.4 to 9.2 in particular.

Photosensitization tissue model.

Photosensitization in a light scattering matrix was investigated with a tissue model consisting of polystyrene microsphere scattering particles, in the presence of a Photofrin II as a photosensitizing agent, and subtilisin Carlsberg as an enzyme target. The photodynamic rate constant for irradiation at 435 nm, 545 nm, and red light was measured at different microsphere concentrations. The reaction rate was almost independent of the microspheres, ruling out a significant effect of light scattering on the integrated photosensitization efficiency. Modeling with the one-dimensional diffusion approximation showed that increasing scatterer concentrations led to lower transmission and higher diffuse reflection, such that the fractional absorption was almost constant. The quantum efficiency of enzyme inactivation in the light-scattering systems was 0.0014 +/- 0.0003. In terms of incident dose, red and green light had approximately the same effectiveness and blue light was two-fold more efficient.

[Radiation inactivation of alpha-chymotrypsin in aqueous solutions. The nature of the dose-response relationship]. / Radiatsionnaia inaktivatsiia al'fa-khimotripsina v vodnykh rastvorakh. Kharakter zavisimosti éffekt--doza.

A study was made of possible reasons for deviations of the dose--response curves from the exponential function at low (less than or equal to 10(-6) M) and high (greater than or equal to 10(-4) M) initial concentrations of the enzyme. Factors influencing the degree of the deviation and type of the dependence of the radiation and chemical yield on the initial concentration of the enzyme are discussed. The data obtained are compared with those reported in the literature.

[Effect of the irradiation conditions on the radiation inactivation of subtilisin-72]. / Vliianie uslovii oblucheniia na radiatsionnuiu inaktivatsiiu subtilizina-72.

A comparative study was made of gamma-inactivation of subtilisin-72 solutions in 5 X 10(-3) M acetate buffer and 0.1 M NaCl in the presence and absence of Ca2+ ions. It was shown that the acetate buffer had a protective action, and the influence of Ca2+ ions depended on the ionic strength of the solution. In general, Ca2+ ions exerted a stabilizing effect irrespective of the subtilisin concentration in the acetate buffer, but this effect competed with the destabilizing influence of the ionic strength increased by Ca2+ ions.