Inactivation RNases

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. 

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). 

Ribonuclease Pepsin-inactivated RNase has 1 anomalous Tyr Oxidized RNase has none anomalous Bigelow (1960)... 

The extreme sensitivity of RNA to the ubiquitous inter- and intracellular nudeases (for example on the skin of the investigator) makes special precautions necessary for effective RNA preparations. The use of disposable containers is recommended, or glassware that has been soaked in dilute hydrochloric acid and rinsed with autoclaved distilled water. Disposable gloves must be worn in all procedures where RNA is handled, or is likely to come into contact with RNA, such as solutions, chemicals, glassware, spatulas etc. Buffers for RNA work should be prepared from reagents reserved for this purpose, and stored separately. Buffers can be treated with 0.2 % (v/v) diethylpyrocarbonate (care - this is carcinogenic) and autodaved to inactivate RNases, or at least those with adive site histidines. Since most nucleases require Mg2 for activity, the addition of EDTA in mM concentrations to solutions is also recommended. 

Pretreatment of affixed specimens Endogenous enzyme inactivation, RNase treatment, permeabilization, and protease treatment are common pretreatments that increase signal-to-noise ratio and enhance access of the probe to the target. 

Despite the information about RNase H and the demonstration that many oligonucleotides may activate RNase H in lysate and purified enzyme assays, relatively little is yet known about the role of structural features in RNA targets inactivating RNase H( 146-148). In fact, direct proof that RNase H activation is the mechanism of action of oligonucleotides in cells has until very recently been lacking. 

RNA is relatively an unstable molecule and significantly more susceptible to degradation than DNA, mainly because of the presence of ribonudeases (RNases), which represent different enzymes that break down RNA molecules. RNases are very stable, do not require cofactors, are effective in very small quantities and are difficult to inactivate. RNase contamination can come from human skin and dust partides, which can carry bacteria and molds. They may be present as well in cell lysates as in the surrounding environment, consequently the isolation and analysis of RNA requires highly specialized techniques. 

Sterile, disposable polypropylene tubes are recommended for working with RNA. These tubes are generally RNase-free and do not require pretreatment to inactivate RNases. In ordertodestroy RNases, non-disposableplastic-warecan be rinsed withO.l M NaOH, 1 mM EDTA, followed by RNase-free water. Alternatively, chloroform-resistant plastic-ware can be rinsed with chloroform, which is sufficient to inactivate RNases. 

RNA Extraction from Blood The following method is a cheap, simple and fast method to isolate RNA from blood, based on the strongly denaturing solution RNAzol, which inactivates RNase within a few seconds and stabilizes DNA. 

In case of doubt, autoclave the water for 15 min at 120°C (or 15 lb/ sq.in. on liquid cycle). This will eliminate microbial contamination but will not sufficiently inactivate RNases. Water can be rendered RNase free by treatment with diethyl pyrocarbonate (DEPC) at 0.1%. Residual DEPC, which is reactive with amines (and is a suspected carcinogen), can either be left overnight at room temperature to decompose to ethanol and carbon dioxide or preferably be destroyed by autoclaving. 

RNAseZap inactivates RNA-degrading RNAses, which are found on surfaces in the laboratory. 

RIBOFLAVINASE RIBOFLAVIN KINASE RIBOFLAVIN SYNTHASE RIBOFLAVINASE RIBOFLAVIN KINASE RIBOFLAVIN SYNTHASE RIBONUCLEASE (or RNase) PSEUDOROTATION DIETHYL PYROCARBONATE RIBONUCLEASE F RIBONUCLEASE II RIBONUCLEASE III Ribonuclease inactivation,... 

Ribonuclease A (RNase A) is dissolved at 1 mg/ml in water, and stored at -20°C. To inactivate the possible contaminated DNase completely, RNase A solution is sometimes boiled once when prepared. 

The single trytophan residue in the native RNase Tt seems to be embedded in the interior of the molecule and cannot be modified by specific reagents. The modification of the residue in 8M urea was carried out with 2-hydroxyl-5-nitrobenzyl bromide by Takahashi (64a) and with A-bromosuccinimide by Kawashima and Ando (65). In both cases complete inactivation occurred when the tryptophan residue was completely modified. However, RNase T, modified with 2-hydroxyl-5-nitrobenzyl bromide in 4 M urea retained about 30% of the original activity (66). Thus, it may be concluded that the tryptophan residue does not participate directly in the catalytic process, since it is situated quite close to the essential Glu 58, it may indirectly participate in the building up of the active conformation (11). 

An enzyme similar to the 3 -nucleotidase of mung bean has been isolated from germinating wheat seedlings and purified 800-fold (90). The preparation possessed DNase, RNase, and 3 -nucleotidase activities. These three activities were similar in pH optima, requirements for Zn2+ and sulfhydryl compounds, stability to storage, temperature inactivation... 

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. 

When RNase-S was treated with iodoacetate at pH 6, both inactivation and histidine modification occurred 164). The modified histidine was in S-protein and was assumed to be His 119 since the sole product on analysis was 1-CM-His. In the absence of S-peptide only methionine modification occurred in S-protein. The loss of potential activity probably resulted from the reaction of the second of the two modifiable Met residues. The location of these residues in the sequence was not established. 

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 probable general structure of the dimers was established in elegant experiments by Fruchter and Crestfield 381) involving alkylation with iodoacetate. The two isomeric dimers referred to above behave identically in these reactions. The two active sites in the dimers behave just like that of the monomer. Histidines 12 and 119 both react, but the reactions are mutually exclusive. The proposed structure is outlined in Fig. 19. The tail of one monomer combines with the body of the other and vice versa. The His 12 and 119 pairs are now on separate molecules. When the dimers, fully inactivated by reaction with iodoacetate, are dissociated by heating at neutral pH, the following monomers would be expected native RNase (active), CM-His-12-RNase (inactive) CM-His-119-RNase (inactive), and di-CM-His-12-His-119-RNase (inactive). These were, in fact, found. About 2b% activity reappeared from the inactive dimer. Equally important the di-CM compound was found. This material... 

Residual RNA in a DNA preparation can be removed by treatment with ribonuclease (RNase). RNase A, which is free of DNase, is available commercially, or the contaminant DNase in the crude RNase A solution can be heat inactivated by heating RNase A solution (10 mg/mL in 10 mM Tris-Cl, pH 7.5, 15 mMNaCl) at 100°C for 15 minutes [4], DNA solution in TE at a concentration of at least 100 pg/mL is treated with RNase to a final concentration of 1 pg/mL followed by incubation at 37°C for 1 hour [3], RNase... 

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