Denaturation reactions

When the rate of an enzyme catalyzed reaction is studied as a function of temperature, it is found that the rate passes through a maximum. The existence of an optimum temperature can be explained by considering the effect of temperature on the catalytic reaction itself and on the enzyme denaturation reaction. In the low temperature range (around room temperature) there is little denaturation, and increasing the temperature increases the rate of the catalytic reaction in the usual manner. As the temperature rises, deactivation arising from protein denaturation becomes more and more important, so the observed overall rate eventually will begin to fall off. At temperatures in excess of 50 to 60 °C, most enzymes are completely denatured, and the observed rates are essentially zero. 

Brandts, J. F., Halverson, H. R., and Brennan, M. Consideration of the possibility that the slow step in protein denaturation reactions is due to cis-trans isomerism of proline residues. Biochemistry 14, 4953-4963 (1975). 

Systematic nmnerical calculations showed that the elution order, the peak sizes, their resolution, and their relative heights depend greatly on the gradient conditions and on the rate of the denaturation reaction [45]. hi contrast to the case of systems in which there is no reaction, reducing the gradient slope can actually re-... 

Using numerical solution, they showed that, in frontal analysis, denaturation results in multiple, unsymmetrical waves in the breakthrough curves, waves that can be mistakenly attributed to the presence of impurities. However, curves recorded at increasing flow rates permit the differentiation between the effects due to impurities and those due to denaturation. In isocratic elution, peaks of the native and the denatured forms cannot be fully separated because of the denaturation reaction. 

Given the following data, calculate Keq for the denaturation reaction of /J-lactoglobin at 25°C ... 

Enzyme immobilization is an attractive method to stabilize the enzyme against denaturating reaction conditions and to make the biocatalyst recyclable [34-37]. In combination with peroxidase-initiated polymerizations, this has been reported recently by Zhao et al. [38]. Incorporation of HRP into a hydrogel increased both storage and thermal stability of the enzyme also resistance against H202 was increased to some extent. Furthermore, the immobilized enzyme preparation could be reused at least four times, albeit with significant activity losses after each cycle. 

Other reactions of hemoglobin also permit a free radical interpretation, notably the coupled oxidation with ascorbic acid by molecular 02 which yields choleglobin, but further discussion requires a full kinetic analysis. Even though the denaturation reactions described above have not been examined kinetically it is worth emphasizing that their chief features can be explained by the formation of 02- as in the mechanism advanced for the autoxidation. The liberation of an activated O2 molecule is no longer required—02 is the active oxygen. 

Figure 12.4 In the presence of formaldehyde, the exocyclic nitrogen (encircled, upper left) on a nucleotide forms an Af-hydroxymethyl adduct (upper center). Further denaturing reactions of formalin-fixed nucleic acids during tissue processing lead to a compound ethoxymethyl adduct (lower right), fragments from depurination (lower left), cross-hnks to associated proteins (not shown), and hydrolysis of phosphodiester bonds (not shown).

It is known that the presence of phosphate ions slows a de-naturation reaction. Simpson and Kauzman (22) demonstrated in a study of the effects of various electrolytes on the rate of de-naturation of ovalbumin in urea that phosphate (HPO and H2P0t,) slowed the denaturation reaction. Thus, the removal of the pK far from the pK of the carboxylate anions in the hyaluronate studies examined here (J, 2, 3) may also be ascribed to the use of phosphate buffer. The following analysis proceeds, however, along thermodynamic lines, not kinetic. 

It is recognized (26, 27) that electrostatic theories of salting out of the kind developed by Debye and Kirkwood (23) do not account for the large differences between the effects of different salts, such as is evident in the preference of phosphate for amide group binding. Whether this preference and relative prevention of the denaturation reaction implicates an interaction with a changing structure of liquid water has yet to be determined (26, 28). 

Chymotrypsin has been covalently bound to water-soluble oxidized dextran through Schiff bases, and the stability of the modified enzyme investigated. The increase in stability observed was attributed to an increase in the enthalpy of activation of the denaturation reaction. 

Figures 5 and 6 show the course of the denaturation reaction in a healthy person and in a diseased person, respectively. The serum was first diluted 1 15 with normal saline (0.9% NaCl) solution, then an equal volume of a 0.25N potassium hydroxide solution was added, and at...

Figure 5. Course of nonfiltrate denaturation reaction in serum of healthy subject.

In principle the protein index expresses a relationship between the Brdidka denaturation reaction in Co solution and the Brdiika filtrate reaction in Co " solution. The authors of this index, Muller and Davis, recommend the following procedure ... 

Thermodynamic studies on the unfolding of a-lactalbumin caused by lithium chloride, lithium chlorate, and sodium chlorate have been reported. The characterization of the denatured states and the mechanism of the denaturation reactions are discussed and compared with previous results on the unfolding with organic denaturants. 

Since the reversibility of relatively few denaturation reactions has been demonstrated, it is not surprising that most denaturation studies have been concerned with the kinetics of denaturation. The most commonly used techniques are optical rotatory and precipitation methods. An example of the pH dependence of the rate of a denaturation reaction over a wide range of temperature and pH is shown in Fig. 59. The purpose of the final section of this chapter is to show how side-chain hydrogen bonding can lead to such pH-dependent denaturation kinetics (Laskowski and Scheraga, 1961). 

We studied the effects of total and partial deuteration on the kinetics of thermal denaturation of met-hemoglobin. The kinetics were shown to be first order with respect to protein concentration this was true both in H2O and in D2O within the entire range of temperatures examined. Deuterium oxide increased the stability of the native conformation of met-hemoglobin this effect increased progressively by increasing the amount of D2O in the solution. Extension of the experiments to the amplest possible temperature range (50-63°C) allowed the determination of the isotopic effect on the activation enthalpy and entropy of the denaturation reaction the isotopic effect resulted to be mainly entropic. 

At temperatures higher than 57°C the experimental points are well fitted by a single straight line and the denaturation reaction is first order with respect to time. At temperatures lower than 57°C deviations from the linear behavior are evident and the order of the reaction with respect to time is appreciably greater than unity. 

---