Shimomura O
Marine Biological Laboratory, Woods Hole, MA 02543.
Biochem J. 1995 Mar 1;306 ( Pt 2)(Pt 2):537-43. doi: 10.1042/bj3060537.
Aequorin emits light in the presence of Ca2+, decomposing into apoaequorin, coelenteramide and CO2. Semisynthetic aequorins, produced by replacing the coelenterazine moiety in aequorin with analogues of coelenterazine, showed widely different sensitivities to Ca2+ as well as certain spectral variations. A group of semisynthetic aequorins, e-type aequorins, showed bimodal luminescence, with peaks at 400-405 nm and 440-475 nm in various intensity ratios, whereas all other aequorins luminesced with only one peak, in the range 440-475 nm. The cause of the spectral variation was studied by various experiments including: (1) comparison with the fluorescence of the spent solution and the luminescence of the spent solution produced by added coelenterazine; (2) luminescence in 2H2O; (3) the rate of conformational change of apoaequorin; (4) the rates of regeneration in the presence and absence of O2. The results suggested that the spectrum of Ca(2+)-triggered luminescence is strongly affected by the ionic charge on the amide N atom of the coelenteramide that is bound to apoaequorin. When the amide N atom is negatively charged, light is emitted with a 440-475 nm peak. In the case of e-type aequorins, the negative charge on the amide N atom is less because of the structure of e-coelenterazine involved, resulting in the emission of a 400-405 nm peak from the uncharged form of coelenteramide; the intensity ratio of 400-405 nm peak to 440-475 nm peak is determined by the amount of negative charge resting on the amide N atom of e-coelenteramide at the time of light emission. Most of the spectral variations in luminescence and fluorescence can be explained on the basis of ionic and hydrophobic interaction between a coelenteramide and apoaequorin.
水母发光蛋白在钙离子存在的情况下会发光，并分解为脱辅基水母发光蛋白、腔肠素酰胺和二氧化碳。通过用水母发光蛋白中腔肠素部分的类似物替代腔肠素而产生的半合成水母发光蛋白，对钙离子表现出广泛不同的敏感性以及某些光谱变化。一组半合成水母发光蛋白，即e型水母发光蛋白，表现出双峰发光，在400 - 405纳米和440 - 475纳米处有不同强度比的峰值，而所有其他水母发光蛋白仅在440 - 475纳米范围内有一个发光峰值。通过各种实验研究了光谱变化的原因，包括：（1）与用过的溶液的荧光以及添加腔肠素后产生的用过的溶液的发光进行比较；（2）在重水中的发光；（3）脱辅基水母发光蛋白的构象变化速率；（4）在有氧气和无氧气存在时的再生速率。结果表明，钙离子触发发光的光谱受到与脱辅基水母发光蛋白结合的腔肠素酰胺酰胺氮原子上的离子电荷的强烈影响。当酰胺氮原子带负电荷时，发出的光在440 - 475纳米处有峰值。在e型水母发光蛋白的情况下，由于所涉及的e - 腔肠素的结构，酰胺氮原子上的负电荷较少，导致不带电荷的腔肠素酰胺形式发出400 - 405纳米的峰值；400 - 405纳米峰值与440 - 475纳米峰值的强度比由发光时e - 腔肠素酰胺酰胺氮原子上的负电荷数量决定。发光和荧光中的大多数光谱变化可以基于腔肠素酰胺和脱辅基水母发光蛋白之间的离子和疏水相互作用来解释。
