PHASE-DIAGRAMS OF A CRYSTALLINE MEMBRANE-PROTEIN, BOVINE HEART CYTOCHROME-C-OXIDASE, IN THE SALTING-IN REGION
PHASE-DIAGRAMS OF A CRYSTALLINE MEMBRANE-PROTEIN, BOVINE HEART CYTOCHROME-C-OXIDASE, IN THE SALTING-IN REGION
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DOI：
10.1016/0022-0248(92)90226-9
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发表时间：
1992-08-01
影响因子：
1.8
通讯作者：
YOSHIKAWA, S
中科院分区：
文献类型：
--
作者：
ATAKA, M;SHINZAWAITOH, K;YOSHIKAWA, S
根据由于晶体生长导致的上清液中蛋白质浓度随时间的变化，确定了细胞色素 C 氧化酶晶体的相图。当磷酸钠缓冲液（pH 7.4）的浓度在 0.5mM-10mM 范围内降低时，以及当稳定该酶的去垢剂 Brij-35 的浓度在检查范围（0%-14%）内增加时，蛋白质的溶解度会降低。沉淀的蛋白质全部是结晶体，没有任何无定形物质。这些结果表明，在任何固定的 Brij-35 浓度下，由于磷酸根离子，酶会发生“盐溶”。因此，通过添加 Brij-35 或通过浓缩蛋白质溶液而不改变缓冲液浓度（使用超滤装置），该蛋白质最容易结晶。还测定了成核和晶体生长速率。即使初始蛋白质浓度仅比晶体溶解度高 24%，瞬时成核也始终会发生。目前的结果表明，大膜蛋白的晶体生长机制与小水溶性蛋白的晶体生长机制显着不同。
Phase diagrams were determined for cytochrome c oxidase crystals, based on the protein concentration changes with time in the supernatant due to crystal growth. The solubility of the protein decreases when the concentration of sodium phosphate buffer, pH 7.4, decreases in the range of 0.5mM-10mM, as well as when the concentration of Brij-35, a detergent stabilizing this enzyme, increases in the examined range (0%-14%). The precipitated protein was all crystalline without any amorphous materials. These results indicate a "salting-in" of the enzyme due to the phosphate ion at any fixed Brij-35 concentration. Thus, this protein is most readily crystallized either by addition of Brij-35 or by concentration of the protein solution without changing the buffer concentration (with an ultrafiltration apparatus). Nucleation and crystal growth rates were also determined. Instantaneous nucleation always occurred even when the initial protein concentration was only 24% higher than the solubility of the crystal. The present results suggest that the mechanism of crystal growth of large membrane proteins is significantly different from that of small water-soluble proteins.