THE THERMOSTABILITY OF NATURAL VARIANTS OF BACTERIAL PLASMINOGEN-ACTIVATOR STAPHYLOKINASE

Three natural variants (wild-type staphylokinase, [R36G, R 43H]staphyl okinase, and [G34S, R36G, R43H]staphylokinase) of the bacterial plasmi nogen-activator staphylokinase, a 136-amino-acid protein secreted by c ertain Staphylococcus aureus strains, have been characterized. These v ariants differ at amino acid positions 34, 36 and 43 only, and have a very similar plasminogen-activating capacity and conformation in solut ion, as revealed by fluorescence spectroscopy, dynamic light scatterin g and circular dichroism. However, the thermostability of these varian ts is significantly different. At 70 degrees C and 0.5 mg protein/ml, irreversible inactivation occurred with apparent half-life (t(1/2)) va lues 0.5 1 +/- 0.13, 0.81 +/- 0.20 and 3.7 +/- 0.7 h (mean +/- SEM) fo r wild-type staphylokinase. [R36G, R43H]stapaphykonana, and [G34S, R36 G, 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 temp eratures and enthalpies of unfolding. 0.08-0.34 mg/ml [G34S, R36G, R43 H]staphylokinase, however, did not aggregate at 70 degrees C but under went unfolding as revealed by a 20% increase in the Stokes' radius and a 30% decrease in circular dichroism. The unfolding was reversible up on cooling and was associated with full recovery of functional activit y. Thus, these natural variants of staphylokinase have a different sen sitivity to thermal inactivation, that is mediated by reversible unfol ding of the protein and concentration-dependent irreversible aggregati on. [G34S, R36G, R43H]staphylokinase, the most resistant natural varia nt, has a stability approaching the minimal requirements for pasteuriz ation, which would facilitate its development for clinical use.