Ca. Reilly et al., EVIDENCE FOR A PROTEIN-PROTEIN COMPLEX DURING IRON LOADING INTO FERRITIN BY CERULOPLASMIN, Archives of biochemistry and biophysics, 354(1), 1998, pp. 165-171
The formation of a protein-protein complex for the loading of iron int
o ferritin by ceruloplasmin was investigated. Ferritin stimulated the
ferroxidase activity of ceruloplasmin unless the ferritin was fully lo
aded, in which case it inhibited the ferroxidase activity of cerulopla
smin. The apparent association constant for the interaction of ferriti
n and ceruloplasmin was 24 nM. Isothermal titration calorimetry indica
ted that the interaction of ceruloplasmin and ferritin was endothermic
, driven by positive changes in entropy. The association constants for
complex formation between ferritin and ceruloplasmin were 4.5 +/- 0.7
x 10(5) and 9.5 +/- 0.3 x 10(4) M-1 for the reduced and oxidized form
s of ceruloplasmin, respectively. The oxidized form of ceruloplasmin w
as retained on an affinity column with ferritin immobilized as the lig
and and remained bound to the column with mobile phases of increased h
ydrophobicity, but was eluted with increased ionic strength. The abili
ty of ceruloplasmin to remain bound to the affinity resin was affected
by the species from which ceruloplasmin was isolated, Gradient ultrac
entrifugation also provided evidence that the two proteins were associ
ated, since ferritin promoted migration of ceruloplasmin through the g
radient. Including ferrous iron in the gradient resulted in reduction
of ceruloplasmin and increased the mobility of ceruloplasmin with ferr
itin, These data provide evidence that ferritin and ceruloplasmin form
a protein-protein complex during iron loading into ferritin, which ma
y limit redox cycling of iron in vivo. (C) 1998 Academic Press.