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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.

The separate contributions of the primary aqueous radicals to the changes in activity, molecular configuration and amino acid composition of RNase, produced by γ-irradiation of dilute solutions, have been identified. The most effective radical for the inactivation of RNase is the H atom; approximately one out of four H atoms inactivates a RNase molecule, compared to 1 out of 15 ·OH or ${\\rm e}_{{\\rm eq}}^{-}$. Additional molecular products are formed on irradiation of the enzyme and when separated by gel filtration these have been related to denatured and aggregated forms of the protein molecule. Hydroxyl radicals are most effective in forming molecular aggregates while H atom attack leads predominantly to denaturation. The solvated electron is the least effective radical for producing changes in molecular configuration; both denatured and aggregated products are formed. In the presence of oxygen, the denatured product forms few, if any, aggregates, due presumably to peroxy formation and an intramolecular termination of the radical. Amino acid analyses of the denatured and aggregated products indicate a very specific attack by the radicals. Significant changes are seen only in the sulfur-containing cystine and methionine and the aromatic tyrosine residues after low doses of irradiation. At higher doses there is, in addition, some effect on the aromatic phenylalanine residues.

The alkaline RNAse level of mucosal scrapings from rat small intestine was increased after 700 R of whole-body X-irradiation, when expressed as total intestinal content or relative to DNA or protein levels; this increase occurred as early as 3 hours after irradiation. Increases also occurred after local irradiation of the head. Both the inhibition of neuroendocrine reactions in mature rats by the administration of drugs, and the use of infant rats in which these reactions do not occur, suppressed the alkaline RNAse increase after irradiation, thus indicating that neuroendocrine reactions may be the cause of the enzyme increase. The application of ACTH, hydrocortisone, or general stressing agents did not produce any increase in the enzyme level, but the radiomimetic chemicals Chlorambucil and Myleran did cause increases similar to those induced by radiation.

The effect of free radicals derived from thiocyanate, bromide, and carbonate ions on the activity of ribonuclease have been studied in neutral and alkaline solutions. Thiocyanate ions and, to a lesser extent, carbonate ions, protect ribonuclease against radiation-induced inactivation, although the presence of bromide ions has little effect. The absolute rate constants for the reactions of these radical anions with ribonuclease have been measured as a function of pH and salt concentration, and the transient absorption spectra of the reaction products have been determined. The results suggest that damage to a histidine residue, or residues, leads directly to loss of activity and support previous conclusions that exposed tyrosine residues in the enzyme are not essential for activity.

The effect of γ-irradiation on the lysosomal enzymes (acid phosphatase, DNase, RNase, β-galactosidase, β-glucuronidase, muramidase, and arylsulfatase) in the absence and in the presence of cysteine or 4-amino-1-naphthol (4A1N) was studied. Inactivation of these enzymes in the absence of the radioprotectors, cysteine and 4A1N, followed first-order kinetics. Muramidase was the most radiosensitive of all enzymes, having a $D_{37}$ value of 4 krads, whereas acid phosphatase was the most resistant, having a $D_{37}$ of 290 krads. Cysteine (0.1 mM) exerted no radioprotective effect on β-glucuronidase, the $D_{37}$ decreasing by 4 krads, whereas β-galactosidase was slightly protected, the $D_{37}$ increasing by 3 krads. The following enzymes were markedly protected: arylsulfatase, $D_{37}$ increased by 8 krads; muramidase, increased by 9 krads; DNase, increased by 13 krads; acid phosphatase, increased by 360 krads. Cysteine exerted some radio-protection on RNase only, with doses ranging from 5 to 10 krads. 4-Amino-1-naphthol (0.1 mM) exerted no protective effect on arylsulfatase; the $D_{37}$ decreased by 12 krads. The remaining enzymes were protected: the $D_{37}$ of acid phosphatase increased by 400 krads; β-glucuronidase, 193 krads; β-galactosidase, 148 krads; DNase, 40 krads; muramidase, 22 krads; RNase, 4 krads.

Exposure of lyophilized native ribonuclease or lysozyme in vacuo to about 30 Mrads causes aggregation of the protein molecules. There is no evidence of extensive degradation of these globular molecules. Guanidine-HCl (4 M) does not disassemble the aggregated material. If the S-carboxymethylated form of ribonuclease or lysozyme (that is, a form devoid of disulfide linkages) is exposed to the gamma ray, there is also no indication of marked degradation of the polypeptide chain, and again the molecules are prone to aggregate. On the other hand, lathyritic collagen exhibits a definite lowering of molecular weight, under the same conditions. Equivalent irradiation in an hydrogen sulfide atomosphere rather than in vacuo causes a diminished aggregation of the S-carboxymethylated ribonuclease and less degradation of the collagen.

The production of denatured and aggregated products from crystalline RNase was studied following irradiation by 60 Co γ-rays in vacuo, in oxygen and H2 S, and in the presence of the paramagnetic ion, Cu++. The yields of these products were determined by gel filtration of the irradiated enzyme on G-75 Sephadex. The results showed that aggregation occurred through relatively slow free radical reactions which were eliminated or altered by free radical scavengers such as O2 and H2 S. Conversely, the yield of denatured products was enhanced from 1.2/100 eV (in vacuo) to 2.4/100 eV by these gases. The yield of both aggregated and denatured molecules decreased in the presence of Cu++ ions, suggesting that these ions reduce the effective radiation dose. Detailed reaction schemes were proposed to account for the rates of formation of the products, and the yields deduced from these schemes were in reasonable agreement with the yields for inactivation of the enzyme under the same conditions. Two peaks, which eluted in the region of the aggregates, were identified as dimers and trimers of RNase. The trimer was observed even at low radiation doses.

The mechanism of formation of secondary radicals in x-irradiated proteins has been studied by electron spin resonance spectroscopy. Ribonuclease, thiolated gelatin, and egg albumin were irradiated at 77°K, alone, as well as in the presence of oxygen or adenine. The spectra were recorded immediately after irradiation and after heat-treatment to predetermined temperatures. The presence of high concentrations of adenine during irradiation prevented the formation of sulfur radicals in ribonuclease and doublet-type radicals in thiolated gelatin. The presence of oxygen did not alter the radical yield or the qualitative spectra observed at 77°K. Oxygen, like adenine, prevented the formation of the typical secondary radicals on heat-treatment. Qualitative spectra were observed which were ascribed to transient HO2 radicals, presumed to be formed by decomposition of organic peroxide radicals. The results are interpreted to mean that in proteins irradiated in vacuum the secondary radicals formed on heat-treatment arise largely by intermolecular reactions mediated by diffusible radicals, primarily H atoms, which can be scavenged by a second component like adenine.