Thermal Inactivation. Thermal inactivation of the purified
enzymes was performed in capillary tubes (1 mm i.d., 200 íL total
volume). Samples were heated in a circulating water bath (model 20B,
Julabo, Allentown, PA) to the indicated temperatures (between 50 and
100 °C depending on the enzyme) for the times specified. The comeup
time for the capillary tubes was determined to be <10 s (6). Heated
samples were cooled immediately in ice-water, and the residual
enzymatic activity was evaluated as described previously. Rate constants
were calculated for POD inactivation in samples treated at specified
temperatures and times. The rate constant for inactivation (k) was
obtained from the slope of a plot of the natural log of residual activity
versus time of heat exposure. The values of activation energy for
inactivation of the enzymes were calculated from the slope of the
straight lines obtained using Arrhenius plots of the logarithm of the
first-order rate constant for inactivation (log k) versus the reciprocal
of absolute temperature (1/T).
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