Dj. Hei et Ds. Clark, ESTIMATION OF MELTING CURVES FROM ENZYMATIC-ACTIVITY TEMPERATURE PROFILES, Biotechnology and bioengineering, 42(10), 1993, pp. 1245-1251
Measuring the reversible thermal unfolding of enzymes is valuable for
quantifying the effects of environmental factors on the thermodynamic
stability of proteins. The thermal unfolding behavior of enzymes is ty
pically studied using calorimetry or optical techniques such as circul
ar dichroism, fluorescence, or light scattering. These techniques ofte
n have practical limitations and usually require the protein to be ele
ctrophoretically pure. An alternative technique for analyzing the ther
modynamic stability of enzymes is to estimate the melting curve from t
emperature-activity data. This technique does not require electrophore
tically pure enzyme, provided the sample does not have competing enzym
atic activities or proteins which can affect enzyme stability (e.g., p
roteases). Moreover, small amounts of contaminant proteins should not
affect the results as long as enzymatic assays are performed at low pr
otein concentrations where nonspecific protein-protein interactions ar
e negligible. To illustrate this technique, the melting curve for beta
-galactosidase from Escherichia coli in the presence of 1 mM EDTA, and
the shift caused by adding 1 MM Mg+2, were calculated from activity-t
emperature data. Melting temperatures predicted from activity-temperat
ure data compared closely with those obtained using other techniques.
Application of this analysis to multisubstrate enzymes is illustrated
by estimating the melting profiles for partially purified hydrogenases
from several thermophilic Methanococcii. Limitations and important co
nsiderations for estimating melting profiles from activity-temperature
data are discussed. (C) 1993 John Wile & Sons Inc.