Application of new semisynthetic aequorins with long half-decay time of luminescence to G-protein-coupled receptor assay.

Aequorin is a Ca(2+)-binding photoprotein and consists of an apoprotein (apoaequorin) and a 2-peroxide of coelenterazine. Eight new coelenterazine analogues modified at the C2-position were synthesized and incorporated into recombinant apoaequorin with O(2) to yield different semisynthetic aequorins. The luminescence properties and the sensitivity to Ca(2+) of these semisynthetic aequorins were characterized. Two semisynthetic aequorins, namely me- and cf3-aequorin, showed a slow decay of the luminescence pattern with less sensitivity to Ca(2+) and were useful for the cell-based G-protein-coupled receptor (GPCR) reporter assays.