Thermal stability of low molecular weight urokinase during heat treatment. I. Effects of protein concentration, pH and ionic strength.

Exposure of low molecular weight urokinase (LMW-UK) to prolonged heating (60 degrees C, 10 hours) is used to inactivate possible viral contaminants. This process leads to a significant loss of active enzyme. Amidolytic activity was monitored following heat treatment in order to establish the conditions for maintaining the optimal stability of LMW-UK. The effects of pH, ionic strength, protein concentration, and various ionic additives were examined. While LMW-UK is stable across a wide pH range (pH 2-11), heating LMW-UK in aqueous solution leads to complete loss of activity except between pH 4 and 7.5. The mechanism of inactivation was delineated using activity assays as well as turbimetric and spectroscopic methods. Thermal inactivation occurs via aggregation of unfolded LMW-UK, followed by subsequent precipitation. Threshold effects upon the thermally-induced aggregation of LMW-UK were observed.