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

Aequorin is a Ca2+-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 O2 to yield different semisynthetic aequorins. The luminescence properties and the sensitivity to Ca2+ 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 Ca2+ and were useful for the cell-based G-protein-coupled receptor (GPCR) reporter assays.