Renaturation process

Fig. 3.4 Temperature-dependent reconstitution of tetrameric K coli aspartase.29 A Reactivation of denatured aspartase. The enzyme denatured in 4 M guanidine-HCl was renatured at 4° C by dilution. After 14 min, the temperature of each preparation was shifted up as indicated in the figure. The temperature of each preparation was further shifted up to 30° C after 45 min. B HPLC analysis of intermediates in the renaturation process. Aspartase renatured at 4°C was incubated for 15 min at the indicated temperatures. An aliquot of each preparation was applied to a TSKgel G3000SWXL column (7.5 X 300 mm) and eluted with a flow rate of 0.5 ml/ min. The temperature of the sample in the sample loop, elution buffer and the column was maintained constant. (From Physiol Chem. Phys. Med. NMR, 21, 222 226 (1989)).

Figure 3. Time development of the renaturation process of add-denatured ooo-transferrin. Concentration of ovotransferrin in the denatured state (pH 3) was approximately 10 mg/ml. The sample was diluted 10 1 in Tris buffer at pH 7.8. Note comparison values of Dt for steady-state native (A), and renatured(O) samples (12).

The reversibility of the reductive scission of disulfide linkages has also been discussed in a previous section. Such a reversal is intimately related to the renaturation process because the thiol groups must accept their correct original partners in the formation of the disulfide linkage. In mixtures of different proteins or with other substances, the correct pairing may be prevented. 

Separated complementary strands of nucleic acids spontaneously reassociate to form a double helix when the temperature is lowered below This renaturation process is sometimes called annealing. The facility with which double helices can be melted and then reassociated is crucial for the biological functions of nucleic acids. Of course, inside cells, the double helix is not melted by the addition of heat. Instead, proteins called helicases use chemical energy (from ATP) to disrupt the structure of double-stranded nucleic acid molecules. 

A denatured protein may be brought back to its native form by in vitro folding. The folding process is often slow and yields can be poor. As each protein is unique, the in vitro folding conditions must be determined case by case often using specific co-solvents as additives. An example is the group of proteins where disulfide bonds must be re-established as part of the renaturation process. 

Recently, we found that when certain single-stranded polynucleotides such as poly(5 -cytidine monophosphate), denoted by poly(C), coexists in the renature process of P-(1 3)-glucans mentioned above, the polynucleotide and s-SPG form a macromolecular complex.Our subsequent studies show that this complexa-tion is a characteristic nature commonly observed for all P-(1 3)-glucans. As far as we know, this is the first study showing that a neutral polysaccharide can combine with polynucleotides. If s-SPG can enter a cancer cell and combine with... 

We examined whether the other glucans show the same change in the UV or CD spectra when they were treated in the same manner as schizophyllan (i.e., dissolved or dispersed in DMSO and exchanged the solvent for water in the presence of poly(C)). The results shown in Table 4.6 indicate that the complexation is only observed for p-(1 3)-glucans. Although the data are not shown, even when the natural triple helix of schizophyllan was mixed with poly(C), no appreciable change was observed for CD and UV. This shows that the renature process is indispensable for the complexation. 

Fig. 5 Projection of the CUR triple helix a in the x-z plane and b in the x-y plane, showing the hydrogen-bonding network constructed in the helix (hydrogen atoms are omitted for both structures) (Reprinted with permission from [64]). c Calculated SPG triple helix structures based on the crystal structure of CUR and d Schematic illustration of denature/renature processes...

Unlike the forgoing guest polymers, i.e., SWNT, PANI, PT, and PPE, all of which are soluble or dispersible either into water or into polar organic solvents, PMDS is soluble only in nonpolar organic solvents such as hexane. Accordingly, to prepare highly ordered PMDS/SPG composites with excellent reproducibility, establishment of a novel solubiUzation strategy is desired. A new biphasic procedure, on the basis of the renaturation process of s-SPG on the liquid/liquid interface, is thus exploited. 

Fig. 172. Schematic view of the renaturation process of gelatin. Reproduced from Biochemistry [ReC 460] by the courtesy of The American Chemical Society...

The renaturation process of globular proteins takes place within 10 and 10" seconds. Internal molecular rotations are known to be in the order of 10 seconds Taking into account only three energetically favorable conformational states for each amino acid, a relatively small protein of 150 residues can potentially adopt more than 10 conformations. A comparison of these figures illustrates in an impressive manner that the time available to a protein for the folding process is by far not sufficient to find the energetically most favorable conformation by a random search mechanism. 

Figure 2.8. Model proposed by Holzwarth and Prestridge (1977) to explain their electron microscopy observations on xanthan. The section of native xanthan is thought to denature into shorter strands because of breaks in one or other chain of the double helix. The renaturing process is a reverse procedure but imperfections arise during the reassembly, causing small hockles , as shown in the renatured molecule.

For various reasons, plasmid molecules are the preferred tools for genetic engineering. Plasmids can easily be amplified in bacteria. They are separated from the larger chromosomal bacterial DNA by a denaturation/renaturation process, where the chromosomal DNA forms an insoluble precipitate, because it renatures more slowly. Purified plasmids can be transferred into eukaryotic cells either in their natural, supercoiled form or as linearized molecules. 

The denaturation/renaturation process can be quantitatively monitored by a number of methods (12,13). The most commonly used ones include (i) binding to hydroxyapatite (only double strands bind in 0.12—0.14 M Na—P buffer), (ii) resistance (or sensitivity) to single strand-specific nucleases, and (iii) optical hyper-chromicity (the breakage of H-bonds and base unstacking result in a 30% increase in UV absorbance at 260 nm). 

The presence of trace of jS-mercaptoethanol accelerated the renaturation process of RNase and lysozyme (Haber and Anfinsen, 1962 Epstein et al, 1962 Epstein and Goldberger, 1963). In the case of RNase, j8-mercaptoethanol catalyzed the disulfide interchange in incorrectly reoxidized enzyme. The effect of )8-mercaptoethanol on the reactivation of lysozyme is shown in Fig. 5.13. It prevents aggregation of the protein and is required to obtain the maximal rate of reactivation of lysozyme, in contrast with RNase where dilution alone is able to overcome the rate limiting effect of aggregation. 

Fig. 5.28. Apparent irreversibility of denaturation-renaturation process for elastase at pH 5.4 measured by the return of antigenic activity ( ). by the return of enzymatic activity ( ) (a) elastase 4 fiM denatured in different concentrations of GuHCl (b) elastase 4 fiM previously denatured in 6 Af GuHCl and then incubated in different concentrations of GuHCl. For radial immunodiffusion measurements 0.12 nmoles of elastase were added to wells in gel. The 2 M trough at this pH value was found in all cases and by both methods (from Ghelis and Zilber. 1982).

A preferential pathway of folding was shown for elastase. The same folded but inactive intermediates being formed and trapped in critical concentration of denaturant,in the denaturation as well as in the renaturation process (Ghelis and Zilber, 1982 Ghelis, 1980). 

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