THERMAL-STABILITY OF LOW-MOLECULAR-WEIGHT UROKINASE DURING HEAT-TREATMENT .3. EFFECT OF SALTS, SUGARS AND TWEEN-80

A turbidimetric assay was used to determine the extent of thermally-in duced aggregation in low molecular weight urokinase (LMW-UK). Previous work has shown that, under 60 degrees C heat treatment, LMW-UK denatu res and the unfolded protein proceeds to form soluble aggregates. The effects of excipients on the extent of aggregation were examined. Both salts (ammonium sulfate and magnesium chloride) and sugars (sucrose, glucose, trehalose, raffinose) were found to be effective, concentrati on-dependent inhibitors of aggregation, although excessive salt concen trations did lead to salting out of the protein. Addition of Tween 80, a nonionic detergent: was ineffective. Overall, the effect of these a dditives on the stability of thermally-stressed LMW-UK can be understo od in terms of preferential exclusion of the solute from the surface o f the protein. These interactions affect the extent of denaturation, o r unfolding, of LMW-UK at 60 degrees C, thereby controlling the degree of aggregation. Purification and incubation experiments indicate that a thermally-unstable subpopulation of LMW-UK exists and is responsibl e for the majority of the aggregation observed.