Kemple M D, Ray B D, Jarori G K, Rao B D, Prendergast F G
Biochemistry. 1984 Sep 11;23(19):4383-90. doi: 10.1021/bi00314a022.
Aequorin is a Ca-activated bioluminescent protein from jellyfish. This protein contains two sulfhydryl groups, one of which is essential for its bioluminescence. Little information concerning the structure of and relationship between the metal binding sites of aequorin and the sulfhydryl group(s) is known. Aequorin was modified by attachment of either a maleimide spin-label [studied by electron paramagnetic resonance (EPR)] or the fluorescent label Acrylodan at the essential sulfhydryl in order to gain such information. These modifications caused destabilization of the chromophore of aequorin. Both of the attached labels showed considerable freedom of motion. The spin-label was quite accessible to the solvent, and the fluorescent label was less so. In addition the metal binding properties of the spin-labeled aequorin were studied by Mn(II) EPR. One tight Mn(II) binding site per spin-labeled aequorin was found. The distance between the Mn(II) binding site and the spin-label is at least 20 A. Furthermore, the relative affinity of spin-labeled aequorin for various metal ions was found to be in the order Pr(III) greater than Mn(II) greater than Ca(II) greater than Mg(II).
水母发光蛋白是一种来自水母的钙激活生物发光蛋白。这种蛋白质含有两个巯基，其中一个对其生物发光至关重要。关于水母发光蛋白的金属结合位点与巯基之间的结构及关系，目前所知甚少。为了获取此类信息，通过在必需的巯基上连接马来酰亚胺自旋标记（通过电子顺磁共振（EPR）研究）或荧光标记丙烯罗丹对水母发光蛋白进行了修饰。这些修饰导致水母发光蛋白发色团的不稳定。所连接的两种标记都表现出相当大的运动自由度。自旋标记对溶剂相当易接近，而荧光标记则不然。此外，通过锰（II）EPR研究了自旋标记的水母发光蛋白的金属结合特性。发现每个自旋标记的水母发光蛋白有一个紧密的锰（II）结合位点。锰（II）结合位点与自旋标记之间的距离至少为20埃。此外，发现自旋标记的水母发光蛋白对各种金属离子的相对亲和力顺序为镨（III）大于锰（II）大于钙（II）大于镁（II）。
