Ribonucleases separation

What holds the ribosomes together to form polysomes From Rich s original preparation, one would suspect it to be messenger RNA. Ribonuclease separates polysomes that are effective in protein synthesis into ineffective smaller fragments. Furthermore, actinomycin, which blocks the synthesis of messenger RNA, also breaks the polyribosomes into smaller units in the organs of animals injected with the antimetabolite. 

Ghatorae, A. S., Guerra, M. J., BeU, G. and Hailing, P. J. (1994) Immiscible organic solvent inactivation of urease, chymotrypsin, lipase, and ribonuclease separation of dissolved solvent and interfacial effects. Biotechnol. Bioeng., 44, 1355-1361. 

FIGURE l.l Hydrophobic interaction and reversed-phase chromatography (HIC-RPC). Two-dimensional separation of proteins and alkylbenzenes in consecutive HIC and RPC modes. Column 100 X 8 mm i.d. HIC mobile phase, gradient decreasing from 1.7 to 0 mol/liter ammonium sulfate in 0.02 mol/liter phosphate buffer solution (pH 7) in 15 min. RPC mobile phase, 0.02 mol/liter phosphate buffer solution (pH 7) acetonitrile (65 35 vol/vol) flow rate, I ml/min UV detection 254 nm. Peaks (I) cytochrome c, (2) ribonuclease A, (3) conalbumin, (4) lysozyme, (5) soybean trypsin inhibitor, (6) benzene, (7) toluene, (8) ethylbenzene, (9) propylbenzene, (10) butylbenzene, and (II) amylbenzene. [Reprinted from J. M. J. Frechet (1996). Pore-size specific modification as an approach to a separation media for single-column, two-dimensional HPLC, Am. Lab. 28, 18, p. 31. Copyright 1996 by International Scientific Communications, Inc.. Shelton, CT.]... 

On the other hand, the HIP value for ribonuclease was practically independent of the length of the polyether ligate. These stationary phases were also employed for the separation of oligophenylalanines containing up to 4 residues by isocratic elution with 0.5 mol/1 phosphate buffer, pH 6.3. The retention increments of the Phe residues did not depend on the ligate length, too, and were 0.82 and 0.89 for the stationary phases composed of PEOs (1500 and 4000, respectively). 

Figure 6 Separation of basic proteins on an untreated fused silica capillary with diaminopropane as buffer additive. Capillary 75 cm (55 cm to detector) x 50 p i.d. Buffer pHs are as noted on the figure with 30 to 60 mM DAP as an additive 200 to 240 V/cm peak identification 1 = lysozyme, 2 = cytochrome, 3 = ribonuclease, 4 = a-chymotrypsin 5 = trypsinogen, 6 = r-huIL-4. (From Bullock, J. A. and Yuan, L.-C., /. Microcol. Sep., 3, 241, 1991. With permission.)...

Final alcohol precipitation not only allows for removal of the phenol and any remaining non-covalently bound hydrocarbon but also concentrates the DNA. Ribonuclease treatment removes any contaminating RNA. Additional purification by cesium chloride centrifugation (35) is also often performed. This is particularly suited to small quantities of DNA. Hydroxyapatite chromatography is also effective in separating RNA, proteins, and DNA (36.37). 

Oligonucleotides have also been separated by ion-exchange chromatography of yeast ribonucleic acid treated either with acid216 or with ribonuclease.209 Alkaline hydrolysis of the fission products obtained with the latter gives rise to pyrimidine nucleoside 3-phosphates and mixtures of purine nucleoside 2- and 3-phosphates. Bone phosphomonoesterase196 followed by alkaline hydrolysis gives pyrimidine nucleosides and purine... 

Fig. 20. Test of stability of weak cation exchange monolithic column (ISCO). Conditions column, 50 X4.6 mm i.d., mobile phase gradient of sodium chloride in 0.01 mol/1 sodium phosphate buffer (pH 7.6) from 0.1 to 0.5 mol/1 in 4.5 min and to 1 mol/1 in 6.5 min, overall gradient time 11 min, flow rate 10 ml/min. Peaks Ribonuclease (1), cytochrome c (2), lysozyme (3). The two separations shown in this figure were achieved 503 runs apart...

Fig. 21. Separation of cytochrome (peak 1), ribonuclease, (peak 2), carbonic anhydrase (peak 3), lysozyme (peak 4), and chymotrypsinogen (peak 5) by hydrophobic interaction chromatography on a molded poly(acrylamide-co-butylmethacrylate-co-N,AT,-methylenebisacry-lamide) monolithic column. (Reprinted with permission from [ 135]. Copyright 1998 Elsevier). Conditions column, 50 x8 mm i.d., 10% butyl methacrylate,mobile phase gradient from 1.5 to 0.1 mol/1 ammonium sulfate in 0.01 mol/l sodium phosphate buffer (pH 7) in 3 min, gradient time 3.3 min, flow rate 3 ml/min...

Figure 3.23 Selectivity of phenyl and alkyl bonded stationary phase materials for protein separation. Column A, TSK gel phenyl-5PW RP, 75 mm x 4.6 mm i.d. B, TSK gel TMS 250, 75 mm x 4.6 mm i.d. eluent, 60 min linear gradient elution from 5% of 0.05% trifluoroacetic acid in 5%> aqueous acetonitrile to 80% of 0.05% trifluoroacetic acid in 80% aqueous acetonitrile flow rate, lml min-1 detection, UV 220 nm. Peaks 1, ribonuclease 2, insulin-, 3, cytochrome c 4, lysozyme-, 5, transferrin-, 6, bovine serum albumin-, 1, myoglobin-, and 8, ovalbumin.

The ease that certain protein mixtures can be separated using reverse micelle extraction was clearly demonstrated by Goklen and Hatton [46], Goklen [31], and Jarudilokkul et al. [25], who investigated a series of binary and ternary protein mixtures. In two cases, they were able to quantitatively extract cytochrome c and lysozyme from a ternary mixture of these proteins with ribonuclease A. Woll and Hatton [24] investigated the separation of a mixture of ribonuclease A and concanavalin A, and showed that the system behaved ideally and that there was no interaction between the proteins. 

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