Purification of Staphylococcus aureus beta-lactamases by using sequential cation-exchange and affinity chromatography.
Boronic acids are active-site inhibitors of serine beta-lactamases, and a phenylboronic acid-agarose affinity column has been used to purify beta-lactamase from crude cell extracts of several bacterial species. We applied phenylboronic acid-agarose chromatography to the purification of Staphylococcus aureus beta-lactamase. Two factors interfered with the success of the previously described single-step chromatographic protocol. First, staphylococcal beta-lactamase exhibited non-active-site-mediated adsorption to the agarose used as a support for the meta-aminophenylborate ligand, preventing the recovery of beta-lactamase from the column. Second, the staphylococcal beta-lactamases exhibited low affinity for meta-aminophenylborate with inhibition constants (Kis) ranging from 8.0 x 10(-3) to 20.0 x 10(-3) M. These problems were resolved by modifying the buffers utilized during chromatography and increasing the dimensions of the affinity column, and a two-stage procedure consisting of cation-exchange chromatography followed by affinity chromatography was used to purify each of the four variants of staphylococcal beta-lactamase. The mean specific activities of the purified type A, B, C, and D beta-lactamases were 44.6, 12.2, 10.6, and 30.8 mumol of nitrocefin hydrolyzed per min/mg of protein, respectively. Dimer formation, presumably from intramolecular cysteine-cysteine cross-linking, was observed with the type D beta-lactamase but not with the type A, B, or C enzyme.
Kernodle
D S
DS
Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Zygmunt
D J
DJ
McGraw
P A
PA
Chipley
J R
JR
D001897:Boronic Acids; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D007202:Indicators and Reagents; D012685:Sepharose; D013211:Staphylococcus aureus / Q000201:enzymology*; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000302:isolation & purification*
D001897:Boronic Acids; D003470:Culture Media; D007202:Indicators and Reagents; D065093:beta-Lactamase Inhibitors; D012685:Sepharose; D001618:beta-Lactamases; C010686:benzeneboronic acid