Purification of prosthetically intact sulfite oxidase from chicken liver using a modified procedure.
A modified procedure was used to purify sulfite oxidase (sulfite:O2 oxidoreductase; EC 1.8.3.1) from chicken liver in high yield. The modifications included dialysis of the enzyme against a buffered solution containing sodium molybdate (prior to ion-exchange chromatography), which apparently reconstituted any demolybdo enzyme present in the extract, and phenyl-Sepharose column chromatography. Analysis showed that the purified enzyme contained Mo and heme in a 1.03:1.00 ratio, indicating that the enzyme was prosthetically intact; exogenous heme and other colored proteins were absent from the final pool. Treatment of the sulfite-reduced enzyme with 50 mM cyanide at pH 8.5 resulted in a gradual loss of catalytic activity with a half-life of 19.7 min. Analysis of the cyanide-inactivated enzyme gave a Mo:heme ratio of 1.02:1.00, providing the first direct evidence that the enzyme does not lose molybdenum when inactivated with cyanide. This modified purification procedure provides enzyme in high yield which is well-suited for experiments requiring prosthetically intact enzyme and which is not contaminated with extraneous heme or with other redox active proteins.
Kipke
C A
CA
Department of Chemistry, University of Arizona, Tucson 85721.
Enemark
J H
JH
Sunde
R A
RA
D000818:Animals; D002021:Buffers; D011232:Chemical Precipitation; D002645:Chickens; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003486:Cyanides / Q000494:pharmacology; D003956:Dialysis; D006358:Hot Temperature; D008099:Liver / Q000201:enzymology*; D010088:Oxidoreductases / Q000302:isolation & purification*; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D011786:Quality Control
D002021:Buffers; D003486:Cyanides; D010088:Oxidoreductases; D050862:Oxidoreductases Acting on Sulfur Group Donors