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.

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.

Inactivation of bacteriophage, DNA, and ribonuclease by thermal hydrogen atoms.

T1 phage, BU-T1 phage, infectious DNA extracted from phage phiX 174, and chromatographically purified ribonuclease were exposed to thermal hydrogen atoms, and the loss of plaque-forming ability, infectivity, or enzymatic activity was determined after various exposure times. Atomic hydrogen was generated by two different methods: (1) by a high-frequency discharge in hydrogen gas and (2) by irradiating a foil of polyethyleneter-ephthalate with 2-MeV protons. With increasing exposure time the surviving fraction of all objects tested approaches a constant level. After subtracting this constant "indestructible" fraction in either system, all objects were inactivated according to exponential curves. Furthermore, no BU sensitization was found to occur in BU-T1 phage exposed to atomic hydrogen, whereas gamma irradiation of samples from the same batches revealed a BU effect of a factor of 2.2. These experiments demonstrate hydrogen atoms to be efficient in causing biological damage. Consequently the terminology of "direct" and "indirect" radiation effect may have to be redefined.

Crystal structure of monoclinic ribonuclease-S at 4 A resolution. The mode of binding of 4-thiouridylic acid and a fragment of folic acid, p-aminobenzoylglutamic acid.

A four-A electron density map was calculated for the monoclinic crystal of ribonuclease-S (RNase-S) based on two heavy-atom derivatives. Close geometrical similarity was found between the two crystallographically independent RNase-S molecules (called molecules ZA and ZB) in this crystal and that (called molecule Y) in the trigonal crystal. Using the rotational and translational parameters relating these three molecules, it was established that the crystallographic two-fold symmetry between the two molecules ZA in the monoclinic crystal was exactly identical to that between the two molecules Y in the trigonal crystal, suggesting the tendency of RNase-S molecules to associate in this way although the interaction is weak. The 4-A difference Fourier maps calculated for the monoclinic crystal established the following conclusions. (1) 4-Thiouridine-2'(3')-monophosphates binds to the B1 and R1 sites like other pyrimidine nucleoside-2'(3')-monophosphates as expected from previous spectrophotometric studies, but not to the B2 site even at the concentration of 20 mM. An attempt to visualize the photoproduct generated by irradiation of near-ultraviolet light in this complex failed. (2) p-Aminobenzoylglutamic acid, a fragment of folic acid, seems to bind to RNase-S with its benzene ring close to the B2 site and the alpha-carboxylate group close to the p1 site. The model is compatible with most of the chemical results obtained by Sawada et al. ((1977) Biochim. Biophys. Acta 479, 188-197).

Alkaline ribonuclease from rye germ cytosol.

1. Alkaline ribonuclease (pH optimum 7.6) was isolated from rye (Secale cereale L) germ cytosol and partially purified; the preparation was devoid of other nucleolytic activities. 2. The enzyme is a typical endonuclease hydrolysing all phosphodiester bonds in RNA, yielding ultimately purine and pyrimidine nucleoside 2',3'-cyclic phosphates and the corresponding 3'-phosphates. Upon extensive digestion of synthetic polyribonucleotides, pyrimidine, but not purine, nucleoside 3'-phosphates are formed. The enzyme does not hydrolyse synthetic purine cyclic nucleotides. 3. The enzyme does not depolymerize double-stranded complexes of poly(A) and poly(U). 4. Susceptibility to photooxidation and inhibition by 2-hydroxy-5-nitrobenzyl bromide and N-bromosuccinimide implies the involvement of tryptophan residue in the active centre of the enzyme.

Cross-sections for the inactivation of ribonuclease by slow heavy ions.

Average cross-sections for inactivation of dry ribonuclease by H, He and N projectiles with energies between 100 eV and 10 keV are presented. The trend of the damage cross-sections with particle energy is consistent with the trend of the molecular quasi-elastic scattering and ionization cross-sections. A tentative radiation damage model is proposed in which single hit damage attributed to direct physical interactions by the primary and secondary radiations is supplemented by chemical action of the elastically scattered knock-on atoms which inactivate additional targets with an efficiency of 50-100%. Further experiments at energies below 100 eV are required for positive confirmation of the chemical efficiency. Free electrons (sub-ionizing) are chemically relatively innocuous (less than 10% efficiency). Saturation effects are satisfactorily accounted for in this model. Inactivation penetration depths measured in the microcrystalline enzyme agree with the theoretical predictions if a residual range of about 8 mug cm-2 is included. The mean energy expenditure to produce inactivation of a ribonuclease molecule is found to vary from 30 eV for 100 eV protons to 220 eV He and N ions.

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.

Cross linking in the radiolysis of some enzymes and related proteins.

In non-covalently bound complexes of serveral serine proteases and of ribounclease with DNA the enzymes were protected against the effects of ionizing radiation. No scavenging by the nucleic acids was observed. Similarly, complexing trypsin with silica protected the enzyme from radiolytic destruction. Irradiation of solutions of serine proteases required about twice the D37 dose to produce about 10% polymerization: significantly lower relative doses were effective in causing polymerization in both lima bean protease inhibitor and in the octapeptidal hormone oxytocin. Several sulfhydryl enzymes which have been examined were very efficiently inactivated by ionizing radiation. There was, at the same time, apparent formation of novel intra-molecular -S-S- bonds.

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.

[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.

[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.

[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.

[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.