TY - JOUR
T1 - Thermal stability of low molecular weight urokinase during heat treatment. III. Effect of salts, sugars and Tween 80
AU - Foster, Thomas M.
AU - Dormish, Jonathon J.
AU - Narahari, Uday
AU - Meyer, Jeffrey D.
AU - Vrkljan, Michael
AU - Henkin, Jack
AU - Porter, William R.
AU - Staack, Harold
AU - Carpenter, John F.
AU - Manninga, Mark C.
PY - 1996/5/28
Y1 - 1996/5/28
N2 - A turbidimetric assay was used to determine the extent of thermally-induced aggregation in low molecular weight urokinase (LMW-UK). Previous work has shown that, under 60°C heat treatment, LMW-UK denatures 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, concentration-dependent inhibitors of aggregation, although excessive salt concentrations did lead to salting out of the protein. Addition of Tween 80, a nonionic detergent, was ineffective. Overall, the effect of these additives on the stability of thermally-stressed LMW-UK can be understood in terms of preferential exclusion of the solute from the surface of the protein. These interactions affect the extent of denaturation, or unfolding of LMW-UK at 60°C, thereby controlling the degree of aggregation. Purification and incubation experiments indicate that a thermally-unstable subpopulation of LMW-UK exists and is responsible for the majority of the aggregation observed.
AB - A turbidimetric assay was used to determine the extent of thermally-induced aggregation in low molecular weight urokinase (LMW-UK). Previous work has shown that, under 60°C heat treatment, LMW-UK denatures 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, concentration-dependent inhibitors of aggregation, although excessive salt concentrations did lead to salting out of the protein. Addition of Tween 80, a nonionic detergent, was ineffective. Overall, the effect of these additives on the stability of thermally-stressed LMW-UK can be understood in terms of preferential exclusion of the solute from the surface of the protein. These interactions affect the extent of denaturation, or unfolding of LMW-UK at 60°C, thereby controlling the degree of aggregation. Purification and incubation experiments indicate that a thermally-unstable subpopulation of LMW-UK exists and is responsible for the majority of the aggregation observed.
KW - Salts
KW - Sugars
KW - Thermal stability
KW - Tween 80
KW - Urokinase
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UR - http://www.scopus.com/inward/citedby.url?scp=0029933023&partnerID=8YFLogxK
U2 - 10.1016/0378-5173(95)04442-6
DO - 10.1016/0378-5173(95)04442-6
M3 - Article
AN - SCOPUS:0029933023
SN - 0378-5173
VL - 134
SP - 193
EP - 201
JO - International Journal of Pharmaceutics
JF - International Journal of Pharmaceutics
IS - 1-2
ER -