Musci G, Bonaccorsi di Patti M C, Petruzzelli R, Giartosio A, Calabrese L
Department of Biochemical Sciences, University of Rome La Sapienza, Italy.
Biometals. 1996 Jan;9(1):66-72. doi: 10.1007/BF00188092.
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.
通过用锰进行金属置换以及利用电子顺磁共振光谱监测钙对结合锰的竞争性置换，研究了钙与人及羊血浆铜蓝蛋白的结合情况。发现钙的解离常数（Kd）为1.4 mM。镁也能与血浆铜蓝蛋白结合，人血浆铜蓝蛋白的Kd为0.3 mM，羊血浆铜蓝蛋白的Kd为0.7 mM。通过差示扫描量热法研究发现，血浆铜蓝蛋白的热稳定性受钙影响，但不受镁影响。在有钙存在的情况下，观察到羊血浆铜蓝蛋白的熔点（Tm）值从73.8℃显著升高至83.1℃。人血浆铜蓝蛋白的Tm值仅因钙而略有改变（从85.1℃变为87℃）。钙会削弱血浆铜蓝蛋白与其分离所用的色谱材料（用氯乙胺衍生化的琼脂糖4B）之间的相互作用。这使我们能够建立一种新的纯化方案，通过相同的单步色谱法从血浆中高效分离出血浆铜蓝蛋白和凝血酶原。
