A Simple and Sensitive Method for Detection of L-Asparaginase by Polyacrylamide Gel Electrophoresis n-Asparaginase (n-asparagine amidohydrolase, EC 3.5.1.1) is an enzyme which has been applied as a potent antiproliferative drug in neoplastic diseases, particularly in leukemias. The enzyme was demonstrated to be most effective in the therapy of acute lymphoblastic leukemia (1).
Most clinical studies employ the enzyme isolated from Escherichia coli. Two forms (designated EC-l and EC-2) of n-asparaginase, only one (EC-2) of which is effective in the treatment of leukemia, were found in strains of E. coli (2). The two forms differ in solubility in ammonium sulfate solution, chromatographic properties, and effect of pH on the enzyme activity.
Isoenzymic patterns of E. coli L-asparaginase were also investigated by acrylamide gel electrophoresis. However, the method involved gel slicing and determination of enzyme activity in individual gel sections (5). A method for detecting this enzyme in the gel was, therefore, desirable.
The present communication describes such a simple and sensitive procedure. The procedure is based upon the forming of insoluble complex of sodium tetraphenylboron-NaB (C&H,) ,-with NH,+ ion (4). Materials and Methods. Serratia marcescens PCM 501 was obtained from the Polish Collection of Microorganisms, Wroclaw, and E. coli 055 B5 was our laboratory strain. Microorganisms were grown as previously described (7). Cells were washed in distilled water, suspended in 0.1% Cetavlon (Cetyltrimethylammonium bromide, Chemapol, Prague, Czechoslovakia), and disrupted by freezing and thawing (repeated five times). After centrifugation L-asparaginase activity (8) and protein content (6) were measured in crude extracts. Purified E. coli n-asparaginase (Crasnitin) was obtained from Bayer. Sodium tetraphenylboron-NaB (C,H,) 4-was obtained from the Institute of Organic Chemistry, PAN, Warszawa, and was recrystallized twice from ether, before use. Recrystallization procedure: the reagent was dissolved in 317