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.
Huang
R N
RN
Institute of Life Sciences, National Central University, Chung-Li, Taiwan, ROC. lsrong@cc.ncu.edu.tw
Yeh
H Y
HY
Cheng
S C
SC
Chow
L P
LP
Lee
T C
TC
D001147:Arsanilic Acid / Q000737:chemistry*; D015153:Blotting, Western; D002847:Chromatography, Agarose / Q000379:methods*; D006801:Humans; D007624:KB Cells / Q000201:enzymology; D011770:Pyruvate Kinase / Q000302:isolation & purification*; D017421:Sequence Analysis
D011770:Pyruvate Kinase; D001147:Arsanilic Acid