Purification of a uridine-specific acid nuclease from chicken liver by fast protein liquid chromatography.
A rapid purification procedure for a novel uridine-specific nuclease from chicken liver based on the Pharmacia fast protein liquid chromatography (FPLC) system is presented. The purification was achieved by applying crude extract to a Mono S cation-exchange column equilibrated with 10 mM potassium phosphate buffer (pH 6.0). The enzyme was eluted in 20 min with a potassium chloride gradient at a flow-rate of 2 ml/min. The enzyme was then chromatographed on a Superose 12 size-exclusion column in less than 1 h at a flow-rate of 0.5 ml/min (Kav = 0.77). The enzyme was re-chromatographed on a second Mono S column to concentrate the protein. The uridine-specific nuclease hydrolyzed poly(U) and Escherichia coli 5S RNA. Poly(A) was hydrolyzed by the nuclease less efficiently (about 10% of the poly(U) activity). No hydrolysis was detected with poly(C), poly(G), poly(dT) or poly(dA) as substrate. The speed with which each purification step could be carried out facilitated the determination of optimal chromatographic conditions. We found that the resolution of the Mono S and Superose 12 columns was superior to that of conventional ion exchangers and size-exclusion columns respectively.
D000818:Animals; D002645:Chickens; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D004722:Endoribonucleases / Q000302:isolation & purification*; D007202:Indicators and Reagents; D008099:Liver / Q000201:enzymology*; D011506:Proteins / Q000032:analysis
D007202:Indicators and Reagents; D011506:Proteins; D004722:Endoribonucleases; C046343:uridine-specific acid nuclease