Ribonuclease preparation

This classic experiment, carried out by Christian Anfinsen in the 1950s, provided the first evidence that the amino acid sequence of a polypeptide chain contains all the information required to fold the chain into its native, three-dimensional structure. Later, similar results were obtained using chemically synthesized, catalyti-cally active ribonuclease. This eliminated the possibility that some minor contaminant in Anfinsen s purified ribonuclease preparation might have contributed to the renaturation of the enzyme, thus dispelling any remaining doubt that this enzyme folds spontaneously. 

Figure 1. Reverse-phase chromatographic maps of tryptic digests of reduced caiboxymethylated ribonuclease preparations (a) RNase A control (b) RNase S control and (c) RNase S treated for 1 min with C2N2. Modified and unmodified protein samples were analyzed by reverse-phase HPLC (Perkin-Elmer 250 Binary Pumping Model 235, Diode array Detector, Vydac C-18, 18TP54 Column) using the method of McWherter et al. (18). The sample digests were frozen and stored at -70°C until analyzed.

Solid phase peptide synthesis does not solve all purification problems however Even if every coupling step m the ribonuclease synthesis proceeded in 99% yield the product would be contaminated with many different peptides containing 123 ammo acids 122 ammo acids and so on Thus Memfield and Gutte s six weeks of synthesis was fol lowed by four months spent m purifying the final product The technique has since been refined to the point that yields at the 99% level and greater are achieved with current instrumentation and thousands of peptides and peptide analogs have been prepared by the solid phase method... 

The ROA spectra of partially unfolded denatured hen lysozyme and bovine ribonuclease A, prepared by reducing all the disulfide bonds and keeping the sample at low pH, together with the ROA spectra of the corresponding native proteins, are displayed in Figure 5. As pointed out in Section II,B, the short time scale of the Raman scattering event means that the ROA spectrum of a disordered system is a superposition of snapshot ROA spectra from all the distinct conformations present at equilibrium. Because of the reduced ROA intensities and large... 

Komiyama at al. have prepared two oligonuclear Zn(II) complexes (22 and 23) and tested their hydrolytic activity toward different diribonucleotides [45,46] (catalytic turnover was not demonstrated). The dimer and trimer structures of the active species were confirmed by measuring the hydrolytic activity as a function of Zn/L ratio, which show sharp maxima at the expected 2/1 and 3/1 ratios, respectively. The oligomer complexes have high ribonuclease activity (e.g. the hydrolysis of UpU is accelerated more than 4 and 5 orders of magnitude by 22 and 23, respectively), whereas the effect of the monomer complex 24 was not... 

A potent enzyme inhibitor (abbreviated DEP) that acts by ethoxyformylation of proteins, usually at histidine residues. DEP is an irreversible inhibitor of ribonuclease, and rinsing glassware with a 0.1% (weight/volume) DEP solution is recommended to render glassware nuclease-free. Aqueous solutions must be freshly prepared for maximal effectiveness, because DEP will hydrolyze in 6-12 hours at neutral pH. 

Of course, the goal of every synthetic organic chemist is to obtain crystalline products, and a few cases of crystalline synthetic proteins have been reported. These include the ribonuclease A synthesized in solution/35 which crystallized after chemical workup and affinity chromatography (see Section 5.1.6.2.2). Further examples include an HIV protease analogue/701 a ubiquitin analogue/59 and monellin/89 which were each prepared by solid-phase methods and purified by HPLC. 

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. 

Ribonuclease U2 may be used for the synthesis of guanylyl-(3 -5 )-nucleoside and for the addition of adenylyl residue to 5 terminal of oligonucleotides including no guanylyl residue. By the fractionation of RNase U2 digests of RNA, oligonucleotides of defined sequence with 3 -terminal adenylic acid can be prepared. 

The existence of a deoxyribonuclease in E. coli bound to an inhibitory RNA was first suggested by Kozloff (3< ) who found that the DNase activity of freshly prepared extracts could be markedly enhanced by pretreatment with ribonuclease. The enzyme was subsequently purified and freed of inhibitor (39). The purified enzyme termed endonuclease I could, in turn, be competitively inhibited by a variety of RNA s including transfer RNA, and Ri values as low as 10-8 M (nucleotide) have been observed (40). Examination of various purified RNA species and synthetic polyribonucleotides for their inhibitory activity has led... 

Walters and Loring (88) have purified a 3 -nucleotidase about 50-fold from mung bean sprouts (Phaseolus aureus Roxb.). The enzyme hydrolyzes 3 -AMP, 3 -GMP, 3 -CMP in decreasing order and also hydrolyzes the 3 -phosphate group of coenzyme A. (89), but it has no significant activity for 2 - or 5 -ribonucleotides. For 3 -GMP, 3 -AMP, 3 -UMP, and 3 -CMP, Km values are 0.67, 1.1, 7.7, and 15 mM, respectively. The enzyme preparation also contained acid stable ribonuclease activity (89). Both 3 -nucleotidase and acid ribonuclease were inactivated reversibly at pH 5.0 and by dialysis and this inactivation could be prevented by Zn2+. The two activities were similarly inactivated by heat at pH 5 and 7.5. Such data indicate that the two are metalloproteins— probably zinc metalloproteins. These similarities and other kinetic data provide evidence that the 3 -nucleotidase and ribonuclease activities reside in the same protein. 

In interesting recent studies, Poliak and co-workers (67, 68) have observed glucose-6-P phosphohydrolase, acid inorganic pyrophosphatase, and PPi-glucose phosphotransferase activities to be considerably higher in lipid-poor reticulosomes than in microsomal preparations. They hypothesized that reticulosomes, which they prepared by treatment of microsomal preparations from rat and chick livers with ribonuclease and deoxycholate (68), may be the precursors of endoplasmic reticulum, and... 

In view of the high stability of the enzyme most samples have been prepared by the procedure described by Kunitz (16) and modified by McDonald (17) to remove all traces of proteolytic activity. During this procedure the minced bovine pancreas is exposed to 0.25 N sulfuric acid, ammonium sulfate precipitation, 10 min at 95°-100° and pH 3, and, finally, reprecipitation. The product can be crystallized it was also shown later to contain a number of components all with ribonuclease activity. A practical summary of all details is given by Kunitz and McDonald (18). 

Martin and Porter (19) described a partition chromatographic procedure and first demonstrated the presence of at least one minor active component in the crystalline enzyme preparation. King and Craig (20) found a solvent system permitting effective countercurrent distribution of ribonuclease, ethanol water ammonium sulfate in the ratios 25.9 -57.6 16.5. The principal component of the Kunitz preparation behaved as an almost ideal solute with a partition ratio of 0.8. Albertsson has provided a liquid polymer countercurrent system based on dextrari and methyl cellulose (21). 

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