Improved Aequorin for Ca Assay in Functioning Cells

光蛋白aequorin（最早于1962年由P.I.分离）已被广泛用作活细胞中的钙指示物。然而，过去使用的水麒麟制剂是含有杂质的异水麒麟混合物，无法确定。最近，在P.I.中制备了两种改进型的aequorin第一种是由单分子物种组成的纯异水麒麟；第二种是修饰了官能团的半合成光蛋白e-水麒麟。e-aequorin的发光光谱显示两个峰（405 nm和465 nm），峰强度之比为ıCa ！-依赖于pCa在5 - 7的范围内，因此可以直接从两个峰的强度比来测定Ca浓度。本项目的主要目的是建立与其他钙指标相比，使用异钙素和e-钙素测量细胞内钙的优势；这将通过与细胞生物学家合作实际使用这些材料来完成。为此目的，并应其他调查人员的要求，将生产足够数量的异丙四酚和e-丙四酚。为了提高更有用类型的异等分激素的产量，将研究异等分激素之间的相互转化。第二个目标是制备其他有用形式的半合成萘醌，优于异萘醌和e萘醌；这将通过进一步修改官能团来实现。

The photoprotein aequorin (initially isolated by the P.I. in 1962) has been widely used as a Ca -indicator in living cells. However, the preparations of aequorin used in the past were undefinable mixtures of isoaequorins containing impurities. Recently, two improved types of aequorin were prepared in the P.I.'s laboratory: the first is pure isoaequorins consisting of single molecular species, and the second is a semi-synthetic photoprotein designated e-aequorin in which the functional group is modified. The luminescence spectrum of e-aequorin shows two peaks (405 nm and 465 nm) and the ratio of the peak intensities is ıCa !-dependent in the pCa range of 5 - 7, thus allowing the determination of Ca concentration directly from the intensity ratio of the two peaks. The primary objective of this project is to establish the advantage of using isoaequorins and e-aequorin in measuring intracellular Ca , compared with other Ca -indicators; this will be accomplished by the actual use of those materials by collaborating cell biologists. Sufficient amounts of isoaequorins and e-aeqourin will be produced for this purpose and also to comply with requests from other investigators. To increase the yields of more useful types of isoaequorins, interconversion between isoaequorins will be investigated. The second objective is to prepare other useful forms of semi- synthetic aequorin which excel isoaequorins and e-aequorin; this will be done by further modification of the functional group.