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.

Improving radiation-damage substructures for RIP.

Specific radiation damage can be used to solve macromolecular structures using the radiation-damage-induced phasing (RIP) method. The method has been investigated for six disulfide-containing test structures (elastase, insulin, lysozyme, ribonuclease A, trypsin and thaumatin) using data sets that were collected on a third-generation synchrotron undulator beamline with a highly attenuated beam. Each crystal was exposed to the unattenuated X-ray beam between the collection of a 'before' and an 'after' data set. The X-ray 'burn'-induced intensity differences ranged from 5 to 15%, depending on the protein investigated. X-ray-susceptible substructures were determined using the integrated direct and Patterson methods in SHELXD. The best substructures were found by downscaling the 'after' data set in SHELXC by a scale factor K, with optimal values ranging from 0.96 to 0.99. The initial substructures were improved through iteration with SHELXE by the addition of negatively occupied sites as well as a large number of relatively weak sites. The final substructures ranged from 40 to more than 300 sites, with strongest peaks as high as 57sigma. All structures except one could be solved: it was not possible to find the initial substructure for ribonuclease A, however, SHELXE iteration starting with the known five most susceptible sites gave excellent maps. Downscaling proved to be necessary for the solution of elastase, lysozyme and thaumatin and reduced the number of SHELXE iterations in the other cases. The combination of downscaling and substructure iteration provides important benefits for the phasing of macromolecular structures using radiation damage.

Isolation and analysis of dityrosine from enzyme-catalyzed oxidation of tyrosine and X-irradiated peptide and proteins.

Dityrosine (DT) was isolated in a single-step by reversed-phase HPLC in 25% yield from enzyme-catalyzed oxidation of N-acetyl tyrosine followed by deacetylation. The isolated product was characterized by 1H NMR. A three-step chromatographic procedure was reported to facilitate the preparation of DT from the enzyme-catalyzed oxidation of tyrosine in 26% yield of theoretical maximum. Upon irradiation at 284 nm in acidic and 315 nm alkaline conditions, DT exhibits strong fluorescence at 400 nm-range. However, when excited at 300 nm-range, contribution of similar fluorescence by Trp oxidation and other protein modifications cannot be overruled. In order to identify the formation of DT unequivocally, Tyr was subjected to X-irradiation under nitrogen at pH 4 and labeled with dansyl chloride. HPLC conditions were devised to resolve dansylated DT from dansylated standard amino acids. Radiation-induced DT was identified by cochromatography with a dansylated, authentic sample of DT isolated and characterized from enzyme-catalyzed oxidation of Tyr. The formation of DT in the irradiated samples, determined by the integrated peak area, increased with dose (0-600 Gy). HPLC analysis of dansylated hydrolysate of the major product from an irradiated tripeptide (Tyr Gly Gly) detected Gly and DT (2:0.5). Extension of the model study to irradiated BSA and RNase A also showed DT as the major oxidation product of Tyr under the experimental conditions. Fluorescence signal of dansylated DT was linear from 0.5 pmol to 1.5 nmol (correlation coefficient 0.999, n = 3). The detection limit 0.5 pmol per 5 microliters injection hydrolysate corresponds to one molecule of DT per 300 molecules of BSA (BSA at 1 mg/ml). DT can be used as a marker for assessing oxidative damage of proteins. Most standard amino acid analysis techniques are limited to detect normal residues of proteins. The assay reported in the present study has potential for low-level detection of DT unequivocally and may be useful for monitoring oxidative stress-related physiological and pathological processes.

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.

Structural investigation of radiation-induced aggregates of ribonuclease.

Following irradiation of bovine pancreatic ribonuclease in aqueous solution with 60Co gamma-rays protein aggregates are formed. The nature of the bonds linking these radiation-induced aggregates together has been investigated by chromatographic and electrophoretic methods. Thin-layer gel filtration and polyacrylamide gel electrophoresis, both in the presence of sodium dodecyl sulphate, demonstrated the existence of covalent crosslinks between the aggregates. However, non-covalent crosslinking also plays a role in the radiolysis of ribonuclease. Thin-layer gel filtration with and without 6 M urea and 2 per cent beta-mercaptoethanol added to the gel, revealed that only part of the covalent bonds between the aggregates consisted of disulphide linkages. By separation of the reduced aggregates by thin-layer gel filtration and electrophoresis, both with SDS, this finding was substantiated. Densitometric measurements indicated for example that the percentage of covalently linked dimers held together by disulphide bridges amounted to about 40-45 per cent, whereas the remaining 55-60 per cent of the dimers must be linked by other covalent bonds. The existence of covalent crosslinks other than disulphide bonds was also confirmed by isoelectric focusing. By this method definite differences were established between the proteolytic hydrolysates of the reduced aggregates and the reduced monomer of gamma-irradiated ribonuclease.