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 inorg anic pentavalent arsenate, was conjugated to Sepharose 4B via its para-amin o group to form an As(V)-Sepharose matrix. The cellular proteins from KB ce lls 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 mas s of 58 kDa was shown on a sodium dodecyl sulfate-polyacrylamide gel. By im munoblotting, amino acid sequencing and enzymatic analysis, the sodium arse nate-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 pu rified 35.4-fold with a specific activity of 153.15 U/mg protein in the pre sence of 6 mM fructose-1,6-biphosphate. Although PK was eluted from an As(V )-Sepharose column with sodium arsenate, PK activity was apparently inhibit ed by the used eluent system, but not by p-arsanilic acid, indicating a spe cific interaction of As(V) to PK. In summary, our results indicate that As( V)-Sepharose can serve as a simple and efficient chromatographic support fo r PK purification from KB cells. (C) 2000 Elsevier Science B.V. All rights reserved.