A nearly isosteric photosensitive amide-backbone substitution allows enzyme activity switching in ribonuclease s.

psi[CS-NH]4-RNase S, a site specific modified version of RNase S obtained by thioxylation (O/S exchange) at the Ala4-Ala5- peptide bond, was used to evaluate the impact of protein backbone photoswitching on bioactivity. psi[CS-NH](4)-RNase S was yielded by recombination of the S-protein and the respective chemically synthesized thioxylated S-peptide derivative. Comparison with RNase S revealed similar thermodynamic stability of the complex and an unperturbed enzymatic activity toward cytidine 2',3'-cyclic monophosphate (cCMP). Reversible photoisomerization with a highly increased cis/trans isomer ratio of the thioxopeptide bond of psi[CS-NH](4)-RNase S in the photostationary state occurred under UV irradiation conditions (254 nm). The slow thermal reisomerization (t(1/2) = 180 s) permitted us to determine the enzymatic activity of cis psi[CS-NH](4)-RNase S by measurement of initial rates of cCMP hydrolysis. Despite thermodynamic stability of cis psi[CS-NH](4)-RNase S, its enzymatic activity is completely abolished but recovers after reisomerization. We conclude that the thioxopeptide bond modified polypeptide backbone represents a versatile probe for site-directed photoswitching of proteins.

Broadband 10-300 GHz stimulus-response sensing for chemical and biological entities.

By illuminating the sample with a broadband 10-300 GHz stimulus and coherently detecting the response, we obtain reflection and transmission spectra of common powdered substances, and compare them as a starting point for distinguishing concealed threats in envelopes and on personnel. Because these samples are irregular and their dielectric properties cannot be modulated, however, the spectral information we obtain is largely qualitative. To show how to gain quantitative information on biological species at micro- and millimetre-wave frequencies, we introduce thermal modulation of a globular protein in solution, and show that changes in single-wavelength microwave reflections coincide with accepted visible absorption spectra, pointing the way towards gaining quantitative chemical and biological spectra from broadband terahertz systems.

[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.

[The effect of gamma irradiation on the structure and enzymatic activity of the nuclear membrane of the liver in pregnant rats and their embryos]. / Vliianie gamma-oblucheniia na strukturu i fermentativnuiu aktivnost' iadernoi obolochki pecheni beremennykh krys i ikh émbrionov.

Morphological and biochemical investigations of pregnant rats and embryo liver cell nuclei after in vivo irradiation in the doses of 1 and 2 Gr revealed their high radiosensitivity at all stages of gestation and embryonal development. At damaging effect of radiation, we managed to observe sharp accumulation of products of lipid peroxide oxidation and suppression of the activities of such enzymes as cytochrome-c-oxidase, NAD.N-cytochrome-c-reductase, ATPase and RNAase in liver nuclei of pregnant rats and embryos. The changes of such a kind are shown to intensify with the increasing of irradiation doses. The most profound inhibition of the activities of these enzymes in liver nuclei of embryos irradiated in utero was observed during the period of organogenesis (the 13th day of the development) and in fetal period of embryogenesis (the 17th day of the development), as well as at the 13th and 17th day of gestation. The morphological data also demonstrate the high level of cell nucleus sensitivity to the action of radiation during gestation and embryogenesis.

The influence of low temperature on the radiation sensitivity of enzymes.

When enzymes are exposed to ionizing radiation at low temperatures there is a progressive decrease in radiation sensitivity: considerably more enzymatic activity remains after the same dose of radiation at low temperature compared to room temperature. Detailed studies of five enzymes reveals the quantitative relationship between radiation sensitivity and temperature during exposure. Although 25 enzymes are shown to display this same relationship, recent reports have denied this effect in three enzymes. In this paper, we investigate two possible artifacts that could cause these discrepancies: 1) inaccurate determination of the temperature of the sample during irradiation, and 2) use of temperature-sensitive dosimeters to measure radiation dose. Procedures are described that carefully control these parameters. Thermoluminescent dosimeters are shown to be independent of temperature effects. These methods are used to investigate one of the enzymes, malate dehydrogenase, that has been reported to have a temperature-insensitive radiation inactivation. The radiation sensitivity of this enzyme is found to show the same temperature dependence as 24 other enzymes.

Contrasting oxygen-effects in the inactivation of ribonuclease A by N.3, (SCN).-2 and .OH radicals.

N.3 exhibits higher efficiency than .OH in the inactivation of RNase in de-acerated (neutral) aqueous solution. In O2-saturated solution the .OH-induced inactivation is enhanced, but N.3 and (SCN).-2 become remarkably inefficient. Our results suggest that semi-oxidized tyrosine, the predominant initial defect induced by N.3 and (SCN).-2 but not by .OH2 can be re-reduced upon reaction with O.-2 or cysteine.

Further investigation on the radiation induced inactivation of ribonuclease and the radioprotective effect of some selenium-containing compounds.

Steady state inactivation data on dilute aqueous solutions of RNase show that all water radicals, e-aq, OH, and H are responsible for the inactivation, but the most efficient radical is H atom, only about 4 of them being required for one inactivating event. The data are, therefore, more in agreement with the conclusions of Mee et al. (1972). In the transient absorption spectra of pulse irradiated ribonuclease different components derived by the individual radicals are observed. Organic and inorganic selenium-containing compounds offer a great protection of the enzyme activity, in agreement with the data obtained in other chemical and biological systems. In particular the effects of two new secondary radicals (CNSe)-2 and SeO-3 are in good accord with the known structure of ribonuclease.

Photochemical inactivation of enzymes.

The mechanisms of enzyme inactivation by ultraviolet light and visible light in the presence of sensitizing dyes are reviewed. Recent flash photolysis studies on amino acids and enzymes are summarized in terms of proposed models relating the initial photochemical reactions to permanent chemical and biological damage. The generation and reactions of singlet oxygen are discussed in connection with photodynamic processes. The photochemical results are compared with ionizing radiations, particularly pulse radiolytic methods employing radical anions as selective probes. The interrelationships between the various modes of enzyme inactivation are discussed, as well as the new information to be learned about the structure and functions of the native enzymes from selective radiation-induced alterations.

[Kinetic equation for the process of accumulation of paramagnetic centers in proteins exposed to UV-radiation]. / Kineticheskoe uravnenie protsessa nakopleniia paramagnitnykh tsentrov v proteinakh pri deistvii UF-sveta

The process of accumulation of paramagnetic centres in UV-irradiated solutions of simple proteins at 77 degrees K has been studied. A kinetic equation describing the accumulation of radicals in protein is obtained. Experimentally obtained curves of radical accumulation coincide with the theoretical ones.

[Mechanism of the formation of paramagnetic centers in proteins exposed to UV-radiation]. / Mekhanizm obrazovaniia paramagnitnykh tsentrov v belkakh pri deistvii UF-sveta

It is shown that the process of formation of paramagnetic centres in UV-irradiated at 77degreesK protein solutions in a biquantum one. The protein molecule in triplet state is an intermediate product.

[Accumulation of paramagnetic centers in protein solutions exposed to UV-radiation]. / Issledovanie nakopleniia paramagnitnykh tsentrov v rastvorakh belkov pri deistvii UF-sveta

It is shown that concentration of paramagnetic centres (PC) in UV-irradiated protein solutions at 77degreesK approximates the limiting value. The limiting number of PC (n) per one molecule is in direct proportion to that of aromatic amino acid residues in it n(sigma)=2+0,1 sigma. The formation of PC slopps because all the energy absorbed by aromatic amino acid residues is transfered to the radicals formed.

[Action of Co60 gamma radiation on the nucleic acid concentration and ribonuclease activity in the splenic tissues of inbred and hybrid mice]. / Deistvie gamma-izluchenii Co60 na kontsentratsiiu nukleinovykh kislot i aktivnost' ribonukleaz v tkaniakh selezenki inbrednykh i gibridnykh myshei

The spleen tissue radiation injury expressed in the organ weight loss, nucleic acid concentration decrease and ribonuclease activity increase was observed to a greater extent in mice of the AKR line and to a less extent in those of C57BL line; C57BL X AKR hybrids occupied an intermediate position. It shows that animal radiosensitivity is probably determined by the genotype.

Circular dichroism study of the conformation of ultraviolet-irradiated ribonuclease A.

RNAase A irradiated by ultraviolet light at 254 nm shows a linear dependence between loss of activity and destruction of cystine. At least one of the cystine modified forms in irradiated RNAase is catalytically active. Circular dichroism spectra of irradiated RNAase show a marked decrease in ellipticity between 210 nm and 230 nm, an increased ellipticity between 230 nm and 240 nm, and a blue shift of the 210-nm minimum toward 205 nm. These circular dichroism changes indicate a pariial disorganization of the native secondary and tertiary changes with irradiation. The temperature dependency of the circular dichroism shows the irradiated enzyme to be conformationally less stable to thermal perturbation than native RNAase. Differences in the polypeptide conformations of unirradiated RNAase denatured by heat and sodium dodecylsulfate, and irradiated RNAase treated with heat and sodium dodecylsulfate are discussed.