Acid denaturation nuclease

Staphylococcal nuclease is a small a + ft protein of 149 amino acids that is stable to denaturation by only +5.5 kcal/mol (Anfinsen et al.,... 

Fig. 4. Scatterplots of the Nh residual dipolar couplings from three different denatured states of staphylococcal nuclease plotted against each other. (A) Wild-type, full-length nuclease in 4 M urea (y-axis) versus A131A in buffer (x-axis). (B) Wild-type, full-length nuclease denatured by acid (25 mM citrate, pH 3.0) (y-axis) versus A131A in buffer (x-axis).

Most studies have concentrated on those conditions where reversible transitions can be demonstrated. However, at neutral pH the thermal transition temperature is high enough to introduce difficulties. Ribo-nuclease kept at 95° at pH 7 for 20 min is irreversibly denatured both in its spectral properties and enzymic activity (337). Tramer and Shugar showed that RNase inactivated at pH 7.8 by heating for 30 min has normalized all of its tyrosine residues as far as alkaline spectrophoto-metric titration is concerned. However, the magnitude of the acid difference spectrum is unaffected although the midpoint has shifted from pH 2 to 3. 

The isolation of bacterial DNA described in this experiment, patterned after the work of Marmur (1961), accomplishes these objectives. Bacterial cells are disrupted by initial treatment with the enzyme, egg-white lysozyme, which hydrolyzes the peptidoglycan that makes up the structural skeleton of the bacterial cell wall. The resultant cell walls are unable to withstand osmotic shock. Thus, the bacteria lyse in the hypotonic environment. The detergent, sodium dodecyl sulfate, (SDS, sodium do-decyl sulfate) then completes lysis by disrupting residual bacterial membranes. SDS also reduces harmful enzymatic activities (nucleases) by its ability to denature proteins. The chelating agents, citrate and EDTA (ethylenediamine tetraacetic acid), also inhibit nucleases by removing divalent cations required for nuclease activity. 

DNase I stock solutions are stored at -2O C (1 mg/ml) in 5- xl aliquots (each aliquot is used once). Variation in the activity of DNase I preparations is often observed and the exact amount needed to introduce the desired number of nicks should be determined for each enzyme batch. Sometimes template switches occur which will result in snap-back structures (zero-binding nucleic acid), which remain S, nuclease-resistant upon denaturation. Rigby et al. (1977) suggested that this effect was due to a differential loss of 5 - 3 exonuclease activity upon storage leading to a displacement of the nicked strand and a template switch from the complementary... 

For digestion of unhybridized sequences, the tube(s) is briefly centrifuged and 10 vols. of SI buffer and SI nuclease are added (Section 12.3.2.2) and incubated for 1 h at room temperature. Protected nucleic acid is precipitated and run on a denaturing polyacrylamide gel as described in the previous section. The amount of RNA can be determined by densitometry, in comparison to known amounts of RNA in the controls. 

RNase A is a small, monomeric enzyme of 124 amino acid residues. The catalytic core is unusually stable to heat and denaturants. RNase A is an endoribo-nuclease specific for pyrimidine bases. The enzyme cleaves phosphodiester bonds of RNA at the 3 side of C and U residues, resulting in mono- and oligoribonucleo-tide products with 3 -P and 5 -OH termini (Scheme 3.2) ... 

Nuclease activity is assayed by measuring the release of acid-soluble products from heat-denatured DNA (e.g., calf thymus). A typical assay mixture (0.5 ml) contains 30 mM NaAc (pH 4.6), 50 mM NaCl, 1 mM zinc acetate, 5% (v/v) glycerol, 0.5 mg/ml heat-denatured DNA, and enzyme. The assay mixture is incubated for 10 min at 37°C. 

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