Purification of bovine liver rhodanese by low-pH column chromatography.
We report a purification of bovine liver rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) using column chromatography under conditions that take advantage of recent information regarding the structure and stability of this enzyme. At low pH (e.g., pH 4-6), rhodanese is stabilized against inactivation processes. By maintaining rhodanese at low pH, column chromatography, and especially ion-exchange chromatography, becomes practical, without loss of enzymatic activity. A purification method involving the sequential use of cation-exchange, size-exclusion, and hydrophobic-interaction chromatography was developed, and rhodanese was purified with good yield to electrophoretic purity and high specific activity. Previous methods for purifying bovine liver rhodanese employ repeated ammonium sulfate fractionations and crystallization of the rhodanese. In these methods, it is difficult to separate rhodanese from yellow-brown contaminants in the final stages of the procedures. Here, yellow-brown contaminants, which copurify with rhodanese on the first two columns, are completely resolved by hydrophobic interaction chromatography. This method can be readily scaled up, requires no special equipment, eliminates the variability inherent in previous methods, and is less dependent upon experience.
Kurzban
G P
GP
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
Horowitz
P M
PM
D000818:Animals; D002417:Cattle; D005591:Chemical Fractionation; D002845:Chromatography / Q000379:methods*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003956:Dialysis; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology*; D013884:Thiosulfate Sulfurtransferase / Q000302:isolation & purification*
D013884:Thiosulfate Sulfurtransferase