Irreversible thermal inactivation, enzyme

Horse-radish peroxidase has been treated with acetic, butyric, valeric, enanthic, and succinic anhydrides of mono- and di-carboxylic acids, and with picrylsulphonic acid. ° The effects of these modifications on catalytic activity, rates of irreversible thermal inactivation, and absorption and c.d. spectra were studied. The degree of modification rather than the nature of the modifier was found to be responsible for the major effect on the macromolecular conformation and thermostability of the enzyme after modification. 

Zale, S.E. and Klibanov, A.M. (1983) On the role of reversible denaturation (unfolding) in the irreversible thermal inactivation of enzymes. Biotechnology and Bioengineering, 25, 2221-2230. 

Irreversible inactivation of L. mingrelica luciferase at 37°C. Thermal inactivation of the WT luciferase and its mutants was investigated at 37°C at various enzyme concentrations (10 6-10"8 mol/L). Two-exponential time-curves of thermoinactivation corresponding to the fast and the slow phases of inactivation were observed. The fast hi) and slow (k2) inactivation rate constants of the luciferase were shown to be dependent on the enzyme concentration. This phenomenon is typical for oligomeric enzyme when monomers are less stable than oligomers.4,5 At the concentration 10 6 mol/L, the rate constants kt and k2 of WT and mutant enzymes were similar. At 1 O 7 mol/L enzyme and lower, the ki and k2 values increased both for the WT and mutant luciferases, but constants ki and k2 of mutant forms were four to six times lower that those of the WT (Table 1). Thus, mutations significantly increased the stability of luciferase at both stages of inactivation. 

Most studies have concentrated on those conditions where reversible transitions can be demonstrated. However, at neutral pH the thermal transition temperature is high enough to introduce difficulties. Ribo-nuclease kept at 95° at pH 7 for 20 min is irreversibly denatured both in its spectral properties and enzymic activity (337). Tramer and Shugar showed that RNase inactivated at pH 7.8 by heating for 30 min has normalized all of its tyrosine residues as far as alkaline spectrophoto-metric titration is concerned. However, the magnitude of the acid difference spectrum is unaffected although the midpoint has shifted from pH 2 to 3. 

From an analytical perspective, the single most important physicochemical characteristic of riboflavin is its photosensitivity (80-82). Exposure of this vitamin to ultraviolet and visible light results in irreversible photoreduction to lumiflavin and lumichrome and loss of vitamin activity. In addition, the coenzymes are subject to hydrolysis by endogenous phosphatases that are present in a number of foods. Since these enzymes are generally inactivated by thermal processing, they are a concern only in the analysis of fresh products. 

The mechanism by which PRPP stabilizes APRT against heat inactivation is not yet clarified, but it would be reasonable to assume that PRPP is inducing a conformational change in the APRT molecule resulting in thermal stability, and that the increased heat stability of the enzyme in the UlS hemolysate is due to the increased level of PRPP in such cells. Indeed, both increased specific activity and thermal stability of the APRT in LNS hemolysates have been attributed to stabilization of the enzyme by the elevated concentration of PRPP accumulation in the HGPRT deficient erythrocytes (ll,12). In view of the fact however, that the increased stability of APRT is observed in dialyzed hemolysates, the postulated conformational change would have to be irreversible, unless the stabilizing PRPP is bound to the enzyme. 

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