- The reaction U?I may not be first order, butsecond order. A case
in point is aggregation of unfolded protein, which is rather common during
heating of not very dilute protein solutions. The extent of ‘‘denaturation’’
then is generally estimated from the increase in turbidity or from the
proportion of protein having become insoluble. Often, the overall reaction
order so observed is between one and two, and it may change in the course
of the reaction. - The change U?I may involvemore than one reaction, either
simultaneously or in sequence. For instance, a state I 1 may still allow slow
refolding (where one may think of the trans–cis equilibrium of peptide
bonds involving proline), whereas a state I 2 does not allow refolding. - An unfolded globular protein generally is very susceptible to
proteolytic cleavage, if a suitable protease is present in the active state. Such
a situation can readily produce inactivation curves like those in Figure
7.11b, where the fast inactivation between 45 and 55 8 C would be due to
proteolysis, until also the protease has attained the unfolded, i.e., inactive,
state. One example is the autodigestion at intermediate temperatures shown
by several proteolytic enzymes; an example is in Figure 7.11c. - The rates ofheating and coolingmay significantly affect the shape
of the inactivation curves, especially in the case just mentioned. Another
cause may beslow reactivation(refolding) as exemplified in Figure 7.10b; in
such cases, also the time elapsed between cooling and estimation of residual
activity plays a part.
FIGURE7.11 Heat inactivation of some enzymes, expressed as activity divided by
initial activity. (a) A bacterial protease as a function of heating time at various
heating temperatures ( 8 C, indicated). (b) Luciferase, same variables. (c) A bacterial
protease heated during 30 min as a function of heating temperature.