Purification of an oxidation-sensitive enzyme, pI258 arsenate reductase from Staphylococcus aureus.
Arsenate reductase (ArsC) from Staphylococcus aureus pI258 is extremely sensitive to oxidative inactivation. The presence of oxidized ArsC forms was not that critical for NMR, but kinetics and crystallization required an extra reversed-phase purification to increase sample homogeneity. The salt ions observed in the X-ray electron density of ArsC were investigated. Carbonate was found to have the lowest dissociation constant for activation (K(a)=1.1 mM) and potassium was stabilizing ArsC (DeltaT(m)=+6.2 degrees C). Also due to the use of these salt ions, the final yield of the purification had improved with a factor of four, i.e. 73 mg/l culture.
Messens
Joris
J
Dienst Ultrastructuur, Vlaams Interuniversitair Instituut voor Biotechnologie (VIB), Vrije Universiteit Brussel, Paardenstraat 65, B-1640, Sint-Genesius-Rode, Belgium. jmessens@vub.ac.be
Martins
José C
JC
Zegers
Ingrid
I
Van Belle
Karolien
K
Brosens
Elke
E
Wyns
Lode
L
D053501:Arsenite Transporting ATPases; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D016623:Ion Pumps / Q000737:chemistry / Q000302:isolation & purification*; D007700:Kinetics; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013211:Staphylococcus aureus / Q000201:enzymology*
D016623:Ion Pumps; D009097:Multienzyme Complexes; D053501:Arsenite Transporting ATPases