Rapid purification of ferritin from lysates of red blood cells using proteinase-K.
A simple, rapid, and novel procedure for purifying ferritin from the postnuclear supernatant of red blood cell lysates is described. This report establishes the resistance of commercially available holo- and apo-ferritins to proteinase-K digestion, and documents how the use of this enzyme, in conjunction with the well-documented resistance of ferritins to heat denaturation (75-80 degrees C for 10 min), makes it possible to obtain high yields (greater than 90%) of pure, undegraded ferritin from the postnuclear supernatant of hypotonically or Triton X-100 lysed red blood cells. The resultant purified ferritin contains the same amount of iron as ferritin not treated with proteinase-K and, as judged by one- and two-dimensional gel electrophoresis and electron microscopy, consists of intact ferritin with a subunit isoform composition identical in molecular mass and isoelectric points to that obtained from ferritin prepared in the absence of this enzyme.
Atkinson
B G
BG
Department of Zoology, University of Western Ontario, London, Canada.
Dean
R L
RL
Tomlinson
J
J
Blaker
T W
TW
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K; D004912:Erythrocytes / Q000032:analysis*; D005293:Ferritins / Q000097:blood / Q000302:isolation & purification*; D008854:Microscopy, Electron; D011784:Quail / Q000097:blood*; D012697:Serine Endopeptidases / Q000378:metabolism*
D005293:Ferritins; D012697:Serine Endopeptidases; D019286:Endopeptidase K