ESTIMATION OF MELTING CURVES FROM ENZYMATIC-ACTIVITY TEMPERATURE PROFILES

Authors
Citation
Dj. Hei et Ds. Clark, ESTIMATION OF MELTING CURVES FROM ENZYMATIC-ACTIVITY TEMPERATURE PROFILES, Biotechnology and bioengineering, 42(10), 1993, pp. 1245-1251
Citations number
27
Categorie Soggetti
Biothechnology & Applied Migrobiology
ISSN journal
00063592
Volume
42
Issue
10
Year of publication
1993
Pages
1245 - 1251
Database
ISI
SICI code
0006-3592(1993)42:10<1245:EOMCFE>2.0.ZU;2-E
Abstract
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.