Enzyme activity was used as a probe for correct protein conformation after heat treatment (Lewendon et al., 1988). Therefore, the soluble fraction of crude cell extracts, prepared as described in section 4.4.4, was diluted in assay buffer (50 mM Tris.HCl pH 7.5, 100 mM NaCl, 0.1 mM EDTA) supplemented with bovine serum albumin (100 µg/ml). The latter prevented inactivation of CAT by adsorption to the wall of reaction vessels, which may occur in diluted enzyme preparations (Suelter and DeLuca, 1983). Unless stated otherwise, samples of 350 µl were subjected to a heat treatment in a water bath, immediately followed by chilling on ice. Temperature and duration of thermal inactivation are indicated for each experiment. Remaining activity in heat-treated samples was compared to the activity in samples stored at room temperature during the same period. It has to be emphasized that all conclusions concerning protein stability following from these experiments are restricted to the conditions of time and temperature applied in the specific experiment.
Protocol :
Voorbereiding stalen
Stabiliteitstest
Reagentia :
TNEB-buffer:
Reagentia voor CAT-activiteitstest :
Referentie 1 (Turner et al., 1992) :
Eiwit in concentraties van 10 µg/ml (0,2 U) verdunnen in voorverwarmde staalbuffer (50 mM Tris.HCl pH 7,8 ; 1 mM EDTA)
Aliquots van 120 µl ondergingen thermische behandeling
Variabel : temperatuur (40 tot 70°C) / incubatietijd (0 tot 15 min)
Na thermische behandeling : afkoeling van stalen op ijs gedurende 5 min
Bepalen van restactiviteit
Referentie 2 (Lewendon et al., 1988) :
Eiwit in concentraties van 4 µM in buffer (50 mM Tris.HCl pH 7,5 ; 0,1 mM EDTA ; 0,1 mM 2-mercaptoethanol ; 0,2 mM chlooramfenicol)
Incubatie in waterbad op 70°C
Staalname op gepaste tijdsintervallen (0 tot 60 min)