Denaturation process

The equilibrium constants determined by Brandts at several temperatures for the denaturation of chymotrypsinogen (see previous Example) can be used to calculate the free energy changes for the denaturation process. For example, the equilibrium constant at 54.5°C is 0.27, so... 

Denatonium benzoate is the bitterest-tasting substance known. It gets its name from denatured alcohol—alcohol that has been rendered unfit for drinking—and is often used in the denaturing process. Specially denatured alcohol 40, or SD-40, is ethanol that has been denatured by a tiny amount of denatonium benzoate. Denatonium benzoate is an ester of PABA, and is related to lidocaine, benzocaine, novocaine, and cocaine. 

The DNA structure involves two polyanionic phosphodiester strands linked together by hydrogen bonding of base pairs. The strands can be separated by a denaturation process (melting). The melting temperatnre increases with an increase in guanine (G)-cytosine (C) content, since this base pair possess three hydrogen bonds as compared to just two for the adenine (A)-thymine (T) pair. 

The study of reaction rates or kinetics of a particular denaturation process of a protein therapeutic can provide valuable information about the mechanism, i.e., the sequence of steps that occur in the transformation of the protein to chemically or conformationally denatured products. The kinetics tell something about the manner in which the rate is influenced by such factors as concentration, temperature, excipients, and the nature of the solvent as it pertains to properties of protein stability. The principal application of this information in the biopharmaceutical setting is to predict how long a given biologic will remain adequately stable. 

Calorimetric Deconvolution Models and the Reversibility or Irreversibility of Overall Denaturation Processes. The deconvolution procedures used to analyze the thermograms presented in this study are based on equilibrium models, even though the overall denaturation process seen over a cycle of heating to a temperature above T and then cooling to below is, depending on the pH, either completely or partially irreversible. There is ample precedent in the literature for the application of equilibrium models in such cases, however. Convincing evidence has been presented... 

What we observe, both at the pH value (4.80) chosen to be near the activity optimum for the enzyme and at the value (8.34) chosen to produce substantial pH-stress, is that in the presence of cellobiose the enzyme has marked higher T values, but the overall shape of the denaturation envelope is very similar to diat observed in the absence of the inhibitor. In addition, the overall AH° values in the presence of even quite high concentrations of inhibitor are very close to those observed at lower temperatures in the absence of inhibitor, rather than resembling the values that the linear regression of Figure 6 would seem to imply for denaturation processes at these elevated temperatures. 

The structural characteristics of a variety of food systems are complexly related to the physicochemical protein phenomena of aggregation, coagulation and/or gelation. These phenomena are physical manifestations of protein denaturation processes which are highly dependent upon the type and amount of protein, processing conditions, pH and Ionic environment. 

The value of 2(- 8gt can be calculated from standard values8 and a from the structure of the protein. A low value of mD N, compared with that calculated, indicates that the protein does not become highly unfolded on denaturation. The value of mD N thus provides a test for the degree of unfolding. A low value of mD N may also indicate that the denaturation process is occurring stepwise rather than in a single cooperative transition. 

An alternative interpretation of the data is that AA is proportional to the number of water molecules per protein participating in the adsorption and denaturation process and is related to water activity at the interface. 

Both the denaturation process in proteins and the melting transition (also referred to as the helix-to-coil transition) in nucleic acids have been modeled as a two-state transition, often referred to as the all-or-none or cooperative model. That is, the protein exists either in a completely folded or completely unfolded state, and the nucleic acid exists either as a fully ordered duplex or a fully dissociated monoplex. In both systems, the conformational flexibility, particularly in the high-temperature form, is great, so that numerous microstates associated with different conformers of the biopolymer are expected. However, the distinctions between the microstates are ignored and only the macrostates described earlier are considered. For small globular proteins and for some nucleic acid dissociation processes,11 the equilibrium between the two states can be represented as... 

Figure 16.7 The differential power output obtained from a DSC containing a dilute buffered solution of lysozyme in the sample cell and the buffer in the reference cell during the denaturation process. Adapted from P. L. Privalov, Adv. Prot. Chem., 33, 179 (1979).

Calorimetric studies have established several general features of protein denaturation.12 13 14 For small globular proteins, the denaturation process is well represented by a two-state process,... 

Double-helical DNA in solution can undergo strand separation or dena-turation as a consequence of extremes of pH, heat, or exposure to chemicals such as urea or amides. Decrease in viscosity, increase in absorbance at 260 nm (hyperchromic effect), decrease in buoyant density, or negative optical rotation indicates denaturation of DNA. The denaturation process disrupts only noncovalent interactions between the two strands of DNA. Since G-C base pairs are held together by three hydrogen bonds in contrast to two for an A-T base pair, A-T rich DNA is easily denatured compared to G-C rich DNA (Figure 6.3). Electron microscopy can detect these A-T-rich regions in a DNA molecule since they form bubblelike structures. Hence the temperature of melting (Tm) of DNA increases in a linear fashion with... 

Cie la, K., Roos, Y., Gluszewski, W. 2000. Denaturation process in gamma irradiated proteins studied by differential scanning calorimetry. Radiat Phys Chem 58 233-243. 

Most of the DNA in nature has the double helical secondary structure. The hydrogen bonds between the base pairs provide the stability of the double helix. Under certain conditions the hydrogen bonds are broken. During the replication process itself, this happens and parts of the double helix unfold. Under other conditions, the whole molecule unfolds, becomes single stranded, and assumes a random coil conformation. This can happen in denaturation processes aided by heat, extreme acidic or basic conditions, etc. Such a transformation is often referred to as helix-to-coil transition. There are a number of techniques that can monitor such a transition. One of the most sensitive is the measurement of viscosity of DNA solutions. 

What can you conclude about the denaturation process of /3-galactosidase ... 

Figure 4.17 displays -galactosidase activity in the presence of 7 mM of guanidine chloride as a function of time. Analysis of the decay curve yields two decay times equal to 2.9 and 14.5 min. These times reveal mainly that the denaturation process of a protein does not occur in one simple step. The structure and dynamics of the protein should play an important role in the accessibility of the guanidine chloride to the amino acids and so to the unfolding process. 

The intrinsic pKas of the proteins depend on the local environment. They are further influenced by ionic strength, dielectric constant, and temperature. Mobility estimates should account for the effective mass-to-charge ratio and molecular shape contributions. The denaturation processes produce sets of... 

The dependence of the protein conformation on the charge state distribution of its ions produced by ESI can be applied to study protein conformation. This also allows the denaturation process to be followed over this time and sometimes possible intermediates... 

The first reaction to be discussed is denaturation. Denaturation is involved in most structure forming processes although gels may form from already denatured proteins. It is important to control the denaturation process in order to obtain structures with the desired textural properties. Unfortunately most studies on protein denaturation have been made by biochemists mainly interested in the native structure. One definition given... 

The denaturation process starts at the point where the curves begin to deviate from the baseline. This temperature is, however, difficult to identify in a reproducable way especially for the second peak, since it is not clear whether the peaks overlap or not. Instead the intercept of the extrapolated slope of the peak and the baseline, was taken as a measure of the denaturation temperature (T ). The temperature at the peak maximum (T ) was also used for relative comparisons. From Figure 3 it can Be seen that the highest denaturation temperature was obtained at pH 5 close to the isoelectric point. Only one peak was observed at low (2-3) and high (10) pH. It is obvious that the protein system is partially denatured at these pH s due to the high net charge favoring chain-solvent interactions. 

The highest denaturation temperature of the second peak is found at pH 4.0 - 4.5. It is further seen that the second peak is bigger and more well defined at pH <4.5. The area under the peaks is proportional to enthalpy involved in the denaturation process. 

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