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 ?Ca !-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.
