The thermostability of natural variants of bacterial plasminogen-activator staphylokinase.
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 degrees 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 degrees 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.
Institute for Molecular Biotechnology, Jena, Germany.
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A
Gase
E
E
Birch-Hirschfeld
K H
KH
Gührs
M
M
Hartmann
S
S
Vetterman
G
G
Damaschun
H
H
Damaschun
K
K
Gast
R
R
Misselwitz
D
D
Zirwer
D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers; D004795:Enzyme Stability; D008027:Light; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010960:Plasminogen Activators / Q000378:metabolism*; D011489:Protein Denaturation; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013211:Staphylococcus aureus / Q000201:enzymology*; D013696:Temperature; D013816:Thermodynamics
D017931:DNA Primers; D010960:Plasminogen Activators; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus