Thermal stability of low molecular weight urokinase during heat treatment. III. Effect of salts, sugars and Tween 80

Thomas M. Foster, Jonathon J. Dormish, Uday Narahari, Jeffrey D. Meyer, Michael Vrkljan, Jack Henkin, William R. Porter, Harold Staack, John F. Carpenter, Mark C. Manninga

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)193-201
Number of pages9
JournalInternational Journal of Pharmaceutics
Volume134
Issue number1-2
DOIs
StatePublished - May 28 1996

Keywords

  • Salts
  • Sugars
  • Thermal stability
  • Tween 80
  • Urokinase

ASJC Scopus subject areas

  • Pharmaceutical Science

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