Divalent cation binding to ceruloplasmin.
Binding of calcium to human and sheep ceruloplasmin was investigated by metal substitution with manganese and competitive displacement of bound manganese by calcium monitored by electron paramagnetic resonance spectroscopy. The Kd for calcium was found to be 1.4 mM. Magnesium also bound to ceruloplasmin, with Kd = 0.3 and 0.7 mM for the human and sheep protein, respectively. The thermal stability of ceruloplasmin, as studied by differential scanning calorimetry, was affected by calcium but not by magnesium. A considerable increase of the Tm value, from 73.8 to 83.1 degrees C, was observed for sheep ceruloplasmin in the presence of calcium. The Tm value of the human protein was only slightly altered by calcium (from 85.1 to 87 degrees C). The interaction of ceruloplasmin with the chromatographic material used for its isolation, Sepharose 4B derivatized with chloroethylamine, was weakened by calcium. This allowed us to set up a novel purification scheme that made it possible to efficiently isolate ceruloplasmin and prothrombin from plasma with the same single-step chromatography.
Musci
G
G
Department of Biochemical Sciences, University of Rome La Sapienza, Italy.
Bonaccorsi di Patti
M C
MC
Petruzzelli
R
R
Giartosio
A
A
Calabrese
L
L
D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002151:Calorimetry; D002413:Cations, Divalent / Q000378:metabolism*; D002570:Ceruloplasmin / Q000378:metabolism*; D002845:Chromatography; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008345:Manganese / Q000378:metabolism; D011485:Protein Binding; D011516:Prothrombin / Q000378:metabolism; D012756:Sheep
D002413:Cations, Divalent; D008345:Manganese; D011516:Prothrombin; D002570:Ceruloplasmin; D002118:Calcium