The thermostability of natural variants of bacterial plasminogen‐activator staphylokinase

Abstract

Three natural variants (wild‐type staphylokinase, [R36G, R43H]staphylokinase, and [G34S, R36G, R43H]staphylokinase) of the bacterial plasminogen‐activator staphylokinase, a 136‐amino‐acid protein secreted by certain Staphylococcus aureus strains, have been characterized. These variants differ at amino acid positions 34, 36 and 43 only, and have a very similar plasminogen‐activating capacity and conformation in solution, as revealed by fluorescence spectroscopy, dynamic light scattering and circular dichroism. However, the thermostability of these variants is significantly different. At 70°C and 0.5 mg protein/ml, irreversible inactivation occurred with apparent half‐life (t1/2) values 0.54 ± 0.13, 0.81 ± 0.20 and 3.7 ± 0.7 h (mean ± SEM) for wild‐type staphylokinase, [R36G, R43H]staphylokinase, and [G34S, R36G, R43H]staphylokinase, respectively, with corresponding values at 0.08 mg/ml of 5.3 ± 1.4 h and 11 ± 2.0 h for wild‐type staphylokinase and [R36G, R43H]staphylokinase, respectively. Dynamic light‐scattering measurements indicated that inactivation was associated with protein aggregation, which precluded accurate determination of transition temperatures and enthalpies of unfolding. 0.08–0.34 mg/ml [G34S, R36G, R43H]staphylokinase, however, did not aggregate at 70°C but underwent unfolding as revealed by a 20% increase in the Stokes’ radius and a 30% decrease in circular dichroism. The unfolding was reversible upon cooling and was associated with full recovery of functional activity. Thus, these natural variants of staphylokinase have a different sensitivity to thermal inactivation, that is mediated by reversible unfolding of the protein and concentration‐dependent irreversible aggregation. [G34S, R36G, R43H]staphylokinase, the most resistant natural variant, has a stability approaching the minimal requirements for pasteurization, which would facilitate its development for clinical use. Copyright © 1994, Wiley Blackwell. All rights reserved