Renaturation temperature

At low concentrations of the denatured collagen peptides (less than 0.1 mg/ml) renaturation is a first-order process with respect to protein concentration (39). This suggests that refolding is entirely an intramolecular process resulting in formation of single-stranded structures folded back upon themselves. If the renaturation temperature is substantially below the Tu for chain unfolding, the renaturation will occur rapidly with many nucleation sites, and maximization of noncovalent... 

To answer the question whether the ds-transisomerization of the bridged polypeptides with a Ala-Gly-Pro sequence represents the rate-determining step, the following experiment was carried out The polypeptide with a chain length n = 8 was denaturated in a rapid reaction with a temperature jump from 9.2 to 30 °C and subjected to renatura-tion at 9.2 °C after an incubation time of 25 s. In a second and a third experiment, the incubation in the coiled state was prolonged respectively to 75 and 125 s. It could be observed that the amplitude of the rapid phase depends on the time that lapses between the denaturation and renaturation (Fig. 32). 

The study of temperature effects on the reactivation of an enzyme that has been completely unfolded allows one to distinguish between reactivation (referring to kinetic analysis exclusively) and renaturation, the latter of which would reflect both the refolding transition and the formation of misfolded or aggregated byproducts. 

For the in vitro selection process, the RNA pool containing 10 different sequences and structural motifs is generated by an in vitro transcription reaction. Folding of the RNA molecules is induced by heat denaturation and renaturation at room temperature (26). 

The first step of a PCR involves DNA denaturation at 90-95 °C, in a buffered, neutral, aqueous solution containing DNA polymerase, the four deoxynucleotide triphosphates and Mg++, in the presence of a large excess of the two primers (Fig. 27). In the second step, the temperature of the reaction is lowered to about 10 °C below the melting temperature of the primers and the primers (which are considerably smaller than the DNA) are allowed to hybridize to their complementary sequence on the DNA template molecule. This temperature is still too high for the DNA to fully renature. The temperature is then raised to 72 °C, the optimal temperature for extension of the primers by the DNA polymerase, which catalyses the addition of nucleotide triphosphates to extend the sequence in each direction from the... 

The rate of urea denaturation was inhibited by a variety of anions known to bind to the enzyme in decreasing order of effectiveness, pyrophosphate, 2 -CMP, phosphate, citrate, tartrate, and sulfate (353). This inhibition was greater at pH 5.6 than 7.3. The binding constants were the same as those estimated by inhibition of the enzymic reaction. As with the pH and temperature effects, the anions had no demonstrable effect on the rate of renaturation. 

Effect of temperature on the relative absorbance of native, renatured, and denatured DNA. When native DNA is heated in aqueous solution, its absorbance does not change until a temperature of about 80°C is reached, after which the absorbance rises sharply, by about 40%... 

Steps in denaturation and renaturation of a DNA duplex. In step l the temperature is raised to the point where the two strands of the duplex separate. If denatured DNA is slowly cooled, the events depicted as steps 2 and 3 follow. In step 2 a second-order reaction occurs in which two complementary strands of DNA must collide and form interstrand hydrogen bonds over a limited region. Step 3 is a first-order reaction in which additional hydrogen bonds form between the complementary strands that are partially hydrogen-bonded (zippering). Once complementary strands are partially bonded, the zippering reaction occurs rapidly. In the overall process, step 2 is rate-limiting. 

For renaturation, the purified RNA is dissolved in selection buffer after denaturing PAGE and precipitation (Section 7.3.1.4). Then this solution is heated to 70 °C for 5 min and subsequently cooled to the selection temperature within approximately 15 min. Alternatively, the RNA can be renatured, for example, by heating to other temperatures (65-95 °C) and immediately incubating it on ice. Mg2+ can be added either before or after heating. Because RNA can be degraded if heated in the presence of Mg2+, the heating should not be too excessive if the buffer contains Mg2+ ions. 

The RNA pellet was dissolved in the selection buffer, heated at 70 °C for 10 min. Then, Mg2+ was added and the RNA was renatured by allowing the mixture to cool to room temperature within 30 min. 

A wide and 1000-1500 A long (Fig. 11D), in which striat-ions are not so clear as in the renatured and 60°C-set preparations. X-ray studies of the oriented curdlan gels showed that the heat treatment at temperatures above 120°C(autoclaving) caused a... 

Double-stranded DNA denatures into single strands as the temperature rises but renatures into a double-stranded structure as the temperature falls. Any two single-stranded nucleic acid molecules can form double-stranded structures (hybridize) provided that they have sufficient complementary nucleotide sequence to make the resulting hybrid stable under the reaction conditions. 

Cloned, pH stability, Temperature stability [°C], Organic solvent stability, Oxidation stability, General stability, Storage stability, Renatured, and Links to other databases and references. 

Fig. 7. Microcalorimetric recording of the heat effect on cooling and subsequent heating of metmyoglobin solution at pH 3.83. The low temperature peaks correspond to heat release on cold denaturaton and heat absorption on subsequent renaturation of protein. The shift of these peaks in temperature is caused by slow kinetics of unfolding and folding of myoglobin structure at low temperature (for details, see Privalov et al 1986).

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)).

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