Effects of Lyse-It on endonuclease fragmentation, function and activity.

Nucleases are enzymes that can degrade genomic DNA and RNA that decrease the accuracy of quantitative measures of those nucleic acids. Here, we study conventional heating, standard microwave irradiation, and Lyse-It, a microwave-based lysing technology, for the potential to fragment and inactivate DNA and RNA endonucleases. Lyse-It employs the use of highly focused microwave irradiation to the sample ultimately fragmenting and inactivating RNase A, RNase B, and DNase I. Nuclease size and fragmentation were determined visually and quantitatively by SDS polyacrylamide gel electrophoresis and the mini-gel Agilent 2100 Bioanalyzer system, with a weighted size calculated to depict the wide range of nuclease fragmentation. Enzyme activity assays were conducted, and the rates were calculated to determine the effect of various lysing conditions on each of the nucleases. The results shown in this paper clearly demonstrate that Lyse-It is a rapid and highly efficient way to degrade and inactivate nucleases so that nucleic acids can be retained for down-stream detection.

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.

Caged RNase: photoactivation of the enzyme from perfect off-state by site-specific incorporation of 2-nitrobenzyl moiety.

Photo-triggered activation of semisynthetic Ribonuclease S' from a perfect off-state was successfully carried out by incorporation of photo-labile caged moiety into a proximity to the active site.

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.

Formation of o-tyrosine and dityrosine in proteins during radiolytic and metal-catalyzed oxidation.

To evaluate their usefulness as chemical indicators of cumulative oxidative damage to proteins, we studied the kinetics and extent of formation of ortho-tyrosine (o-Tyr), dityrosine (DT), and dityrosine-like fluorescence (Ex = 317 nm, Em = 407 nm) in the model proteins RNase and lysozyme exposed to radiolytic and metal-catalyzed (H2O2/Cu2+) oxidation (MCO). Although there were protein-dependent differences, o-Tyr, DT, and fluorescence increased coordinately during oxidation of the proteins in both oxidation systems. The contribution of DT to total dityrosine-like fluorescence in oxidized proteins varied from 2-100%, depending on the protein, type of oxidation, and extent of oxidative damage. In proteins exposed to MCO, DT typically accounted for > 50% of the fluorescence at DT wavelengths. These studies indicate that o-Tyr and DT should be useful chemical markers of cumulative exposure of proteins to MCO in vitro and in vivo.

[The characteristics of the action of gamma irradiation on the secondary structure of ribonuclease in vitro and the effect of oxygen on this process]. / Osobennosti deistviia gamma-oblucheniia na vtorichnuiu strukturu ribonukleazy in vitro i vliianie kisloroda na étot protsess.

The change in the secondary structure of ribonuclease after 60Co gamma irradiation with a dose of 1,000 Gy in 0.2% aqueous solution was estimated using the circular dichroism method. The beta structures were significantly changed, while other types of the secondary structure (alpha-helix and beta-turns) changed insignificantly. The secondary structure injury was also affected by oxygen. The data are attributed to characteristics of the secondary structure of this enzyme.

[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.

Effect of near-UV (366 nm) on the activity of certain nucleic acid enzymes in Verticillium agaricinum.

Verticillium agaricinum when grown for 60 min under near-UV irradiation (366 nm) followed by 24 h in darkness produced maximal activity of a number of nucleic acid enzymes (DNase I, endonuclease, nuclease, RNase A, and RNase T1). Total protein and nucleic acid on the other hand showed a decrease under the same conditions. The nucleic acid enzymes which are involved in reversible reactions seem to favour nucleic acid degradation in this study.

[The effect of oxygen on denaturation and aggregation of enzyme macromolecules during gamma-irradiation]. / Vliianie kisloroda na denaturatsiiu i agregatsiiu makromolekul fermenta v protsesse gamma-oblucheniia.

The ribonuclease molecules irradiated in a solution in the presence of 18O2 were separated, by the method of gel-chromatography, into fractions of aggregated macromolecules, denatured monomers and the molecules which retained the original sizes. Oxygen was bound by the molecules of all fractions. The oxygen binding prevented the aggregation of macromolecules. The fractions of molecules of monomer forms, obtained after irradiation in the presence of oxygen, were more inactive than those obtained after irradiation in vacuum.

Radiolytic and enzymatic dimerization of tyrosyl residues in insulin, ribonuclease, papain and collagen.

Insulin, ribonuclease, papain and collagen solutions saturated with nitrogen, N2O or air were irradiated with doses of 10 to 640 Gy of gamma rays. Protein solutions were also oxidized enzymatically in a system of horse-radish peroxidase: hydrogen peroxide. Column chromatography (Sephadex G-75 or Sephacryl S-200) of treated protein solutions revealed that they contain protein molecular aggregates. Nitrogen saturation of solution before irradiation was most favourable for radiation-induced aggregation of proteins. Fluorescence analysis of protein solutions resulted in detection of dityrosyl structures in irradiated as well as in enzymatically oxidized proteins. Concentration of dityrosine in proteins studied was determined fluorimetrically in their hydrolysates separated on BioGel P-2 column. In irradiated proteins, dityrosine was present almost exclusively in their aggregated forms. In proteins oxidized enzymatically, dityrosine was also present in fractions containing apparently unchanged protein. Mechanisms which could account for differences in the yield of dityrosine formation in radiolysis and in enzymatic oxidation of proteins are suggested.

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.

Reactions of ethanol and formate radicals with ribonuclease A and bovine serum albumin in radiolysis.

Aqueous solutions of ribonuclease and bovine serum albumin were irradiated under N2O in the presence of ethanol or formate, which was partly 14C-labelled. The amount of bound ethanol and formate was measured after separation by gel filtration. Reactions of ethanol and formate radicals with proteins lead to covalent crosslinks between the organic solutes and the proteins as well as between the protein molecules. The amount of bound ethanol or formate depends on the structure of the protein and its degree of denaturation. Based on these results and known pulse radiolysis data a mechanism for the reaction of the organic radicals with proteins is proposed. Radiation-induced crosslinking of organic solutes to proteins can be used for studying protein structure in solution.

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.