Arsanilic acid–Sepharose chromatography of pyruvate kinase from KB cells

In the present study, arsanical-based affinity chromatography for pyruvate kinase (PK) isolation was explored. p-Arsanilic acid (4-aminophenyl arsonic acid), which contains an arsonic acid moiety structurally similar to inorganic pentavalent arsenate, was conjugated to Sepharose 4B via its para-amino group to form an As(V)–Sepharose matrix. The cellular proteins from KB cells bound to arsonic acid moieties were eluted by 50 mM sodium arsenate in Tris–HCl buffer (50 mM, pH 7.6). A single protein band with a molecular mass of 58 kDa was shown on a sodium dodecyl sulfate–polyacrylamide gel. By immunoblotting, amino acid sequencing and enzymatic analysis, the sodium arsenate-eluted 58-kDa protein was demonstrated to be a human PK (type M2). By using this one-step As(V)–Sepharose chromatography, PK from KB cells was purified 35.4-fold with a specific activity of 153.15 U/mg protein in the presence of 6 mM fructose-1,6-biphosphate. Although PK was eluted from an As(V)–Sepharose column with sodium arsenate, PK activity was apparently inhibited by the used eluent system, but not by p-arsanilic acid, indicating a specific interaction of As(V) to PK. In summary, our results indicate that As(V)–Sepharose can serve as a simple and efficient chromatographic support for PK purification from KB cells.