RNase denaturation

Q-Sepharose was the separation medium chosen for preparative purification of plasmid pUC18.41 Cell proteins, fragments from RNase digestion of RNA, open coil plasmid and a denatured form of the plasmid were... 

Qi et al. (1998) have demonstrated that ribonuclease A exhibits behavior like that of cytochrome c. The burst phase observed on dilution of Gdm HCl-denatured RNase A is mimicked exactly by reduced RNase A. The latter, when carboxamidomethylated to prevent oxidation, has a CD at 222 nm that is nearly independent of temperature and indicative of extensive unfolding at zero denaturant. 

The RPA is a sensitive method for quantifying specific RNAs from a mixture of RNAs. This is achieved using a small-volume hybridization of an RNA probe to the RNA under study. Unhybridized probe and sample is then digested with RNAses and the protected probe fragment is visualized after denaturing gel electrophoresis. Commonly, the probe is radiolabeled for maximum sensitivity. Following is a method for RPA detection of R-luc-4 sites and F-luc mRNA. 

Cross-linking constrains the conformational flexibility of biopolymers and, as a rule, stabilizes their secondary, tertiary, and quaternary structures against the denaturing effects of high temperatures.29 We used differential scanning calorimetry (DSC) to compare the heat-induced conformational transitions of selected RNase A samples that were characterized in Figure 15.2. A brief introduction to DSC is provided in Section 15.15.1 for those readers unfamiliar with this biophysical method. Trace 1 in Figure 15.3a is the heat absorption... 

Figure 15.3 (a) Heat absorption in solutions of native RNase A (trace 1) and RNase A kept in 10% buffered formalin for 2 days (trace 2) and 6 days (trace 3) at pH 7.4 and 23°C. All samples were dialyzed against 75 mM potassium phosphate buffer (pH 7.4) prior to DSC. (b) Dependence of Td of the dialyzed RNase A samples on time of incubation in 10% buffered formalin at pH 7.4 and 23°C. (c) Heat absorption of solutions of formalin-treated RNase A fractions isolated by size-exclusion gel chromatography monomer (trace 1), dimmer (trace 2), and a mixture of oligomers with >5 cross-linked proteins (trace 3). Protein concentrations were 0.5 mg/mL. The thermal denaturation transition temperature (Td) is defined as the temperature of the maximum in the excess heat absorption trace associated with the protein s endothermic denaturation transition. See Rait et al.10 for details. 

These thermal analysis studies serve to establish a direct relationship between a heat-induced AR method and the reversal of formalin-induced intra- and intermolecular protein cross-links.10 2831 Further, while formalin-treatment provides thermal stability to RNase A, this stabilization is not sufficient to prevent thermally induced protein denaturation at temperatures (>100°C) typically used in heat-induced AR methods.32 34 The implications of this finding for the mechanism of AR will be discussed further in Section 15.6. 

In summary, formalin-treated does not significantly perturb the native structure of RNase A at room temperature. It also serves to stabilize the protein against the denaturing effects of heating as revealed by the increase in the denaturation temperature of the protein. However, formalin-treatment does not stabilize RNase A sufficiently to prevent the thermal denaturation of the protein at temperatures used in heat-induced AR methods as shown by both DSC and CD spectropolarimetry. This denaturation likely arrises from the heat-induced reversal of formaldehyde cross-links and adducts, as shown in Figure 15.4 of Section 15.4. Further, cooling formalin-treated RNase A that had been heated to 95°C for 10 min does not result in the restoration of the native structure of the protein, particularly in regard to protein tertiary structure. 

Figure 15.8 (a) Time course of the activity restoration of formalin-treated RNase A during incubation at 50°C (0-2h) and 65°C (2-4h) in TAE buffer, pH 7.0. (b) Time course of the activity restoration of formalin-treated RNase A during incubation at 65°C in TAE buffers of various pH values. All RNase A preparations were freed of excess formaldehyde by dialysis prior to the assay. The RNase A activity was determined with a colorimetric assay using cytidine 2,3,-cyclophosphate as the substrate as described by Crook et al.54 Note that the slopes of the curves decrease with incubation time at 65°C, which is near the denaturation temperature of native RNase A. This loss of activity is likely due to the competing effect of protein denaturation of the recovered RNaseA at this temperature. See Rait et al.10 for details. 

Chemical treatment with formaldehyde may not necessarily result in significant denaturation. A recent study of RNase A indicated that treatment with formaldehyde does not significantly alter secondary structure.21,22 Although formalin treatment may induce subtle changes in secondary structure, alpha helices and beta-pleated sheets are left essentially intact after formalin treatment. It is reasonable to assume, however, that boiling (as per antigen retrieval protocols) will significantly alter secondary structure. 

The quality of the RNA is the most important factor for the success of array analysis, and great care should be taken to ensure top quality. It is essential to work as quickly as possible during cell disruption to denature RNAses found in tissue cells before they can degrade the sample RNA. RNAses are also found on lab benches and on hands. Therefore, RNAse decontamination of the bench area used for isolation of the RNA is recommended. This is done using RNAseZap. Gloves should be changed frequently. 

Because the denaturation solution inactivates RNAses, mortar and pestle are cleaned with detergent only. Prechill the equipment with hquid nitrogen before usage. Once frozen in hquid nitrogen, the sample can also be stored in an airtight container at -80°C. 

Remove excess PBS and immediately apply 50 pi double-FAM LNA (see Note 2) probe solution and gently shield with cover glass (see Note 3). The probe solution is prepared as follows denature LNA probe and dilute the probe in Exiqon ISH buffer. For example, for 2 ml hybridization mix containing 20 nM double-FAM-labeled miR-21 LNA probe (from 25 pM probe stock), transfer 4 pi into the bottom of a 2 ml nonstick RNase-free tube and place the tube at 90°C for 4 min. Spin down shortly using a tabletop centrifuge, and immediately... 

The rate of hydrolysis of DNA, RNA, and polynucleotides can be measured by a sensitive spectrophotometric assay which is based on the hyperchromicity that occurs upon hydrolysis of these substrates (S). The enzyme has a 7-fold greater affinity for denatured DNA than for RNA (8). No inhibitory products accumulate during the course of the reaction. The pH optimum for RNase and DNase activities is between 9 and 10, depending on the Ca2+ concentration. At higher pH values less Ca2+ is required. The inhibitory effect of high Ca2+ observed consistently by many investigators is more pronounced at higher pH values (S). 

A curious observation by Podder and Tinoco (47) that G-(2, 5 )-G bond was synthesized by RNase Tx from G-cyclic-p led Egami and Inoue (unpublished) to reinvestigate the phosphotransferase activity at various temperatures. At 100°, unlike at 36°, G-(2, 5 )-G3 -p is split to produce G3 -P, and G-(3, 5 )-G3. p is attacked in quite a different way at 100° than it is at 36°. This may result from the altered action of partly heat-denatured RNase Tx. Native RNase Tx is specific, however, to the internucleotide 3, 5 -phosphodiester bonds at normal temperature. 

Fio. 7. Schematic diagram of RNase-S system. The single bond is cleaved converting RNase-A to RNase-S. Ribonuclease-S dissociates reversibly to S-peptide-f-S-protein. The latter can recombine with denatured forms of RNase-A where the tail is loosened from the rest of the molecule. Reproduced from Richards (91a). 

The modification of carboxyl groups has been carried out (1) by esterification with dry methanol and HC1, (2) by esterification with aliphatic diazo compounds, (3) by the formation of adducts with carbodi-imides, or (4) by the formation of amides through activation with carbodiimides. Both complete and, apparently specific, partial modification of the 11 free carboxyl groups have been obtained. In general, the first method suffers from the denaturing medium, the second from incomplete reaction, and the third from the uncertain nature of the products. The fourth procedure is perhaps subject to the least question. There are a total of 11 free carboxyl groups in native RNase-A l (Val), 5/ (Asp), 5y(Glu). A summary of the derivatives is given in Table V. 

The sulfur atom of methionine residues may be modified by formation of sulfonium salts or by oxidation to sulfoxides or the sulfone. The cyanosulfonium salt is not particularly useful for chemical modification studies because of the tendency for cyclization and chain cleavage (129). This fact, of course, makes it very useful in sequence work. Normally, the methionine residues of RNase can only be modified after denaturation of the protein, i.e., in acid pH, urea, detergents, etc. On treatment with iodoacetate or hydrogen peroxide, derivatives with more than one sulfonium or sulfoxide group did not form active enzymes on removal of the denaturing agent (130) [see, however, Jori et al. (131)]. There was an indication of some active monosubstituted derivatives (130, 132). 

Iodination of PIR (147) showed 1 residue buried, Tyr 25, and all others iodinated at least to the monoiodotyrosyl form. Pepsin-inactivated RNase also has only one abnormal tyrosyl by titration which is thus assumed to be 25. Iodination of RNase-S is very similar to RNase-A in the early stages (lift). Extensive iodination leads to dissociation of the protein and peptide components. Direct iodination of S-protein indicated that all 6 tyrosyl residues were accessible, in this sense comparable to urea-denatured RNase-A. Substantial structural changes must be involved for both S-protein and PIR if Tyr 97, in particular, is to become susceptible to attack (see Section IV,B,3). 

Cyanuric fluoride has been used to modify tyrosine residues, substituting the phenolic hydroxyl group. A maximum of 3 residues in RNase was found to react at pH 10.9 and 25° (148a). However, some mystery surrounds this number, as with other estimates of accessibility, since alkaline-denatured material where all tyrosine residues are available still showed the reaction of only 3 residues with cyanuric fluoride. However, similar observations have been made on iodination in 8 Af urea (11 )- At pH 9.3, Takenaka et al. (149) found that only 2 residues reacted and that 115 was not one of them. Two more reacted after alkali denaturation. Two were resistant under all conditions tested. No enzymic activity data were reported. 

The investigations of W. H. Stein and Moore and their colleagues were first reported in 1959 157). The inactivation of RNase by iodo-acetate was studied. A maximum in the rate of activity loss was noted at pH 5.5. Reaction with a methionine residue was found at pH 2.8 at pH 8.5-10 lysine residues were modified, but at pH 5.5-6.0 only histidine appeared to be involved. The specific reaction required the structure of the native enzyme. Reaction with histidine was not observed under a variety of denaturing conditions 158). Iodoacetamide did not cause activity loss, or only very slow loss, or alkylate His 119 in the native enzyme at pH 5.5. The negative charge on the carboxyl group of the iodoacetate ion was apparently essential. 

S-Protein was used to show that l-CM-His-119-RNase-A still had the NHa-terminal tail closely associated with the rest of the molecule as no activity was regenerated on mixing. Prior denaturation of the derivative produced full activity when mixed with S-protein. 

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