Fluorescent Proteins - Aequorin
Aequorin was originally obtained by purification from the jellyfish Aequorea victoria, but now it is prepared more efficiently using recombinant techniques. The active aequorin is formed by a complex between apoaequorin (APO), oxygen and coelenterazine. On binding calcium, the complex is broken emitting light and rendering the free apoaequorin and coeleteramide.
Active aequorin can be directly microinjected or obtained by reconstitution of apoaequorin with coelenterazine in transfected cells. In both cases, coelenterazine is needed to obtain the active complex (coelenterazine is burn out after light emission and is needed for reconstitution of aequorin). When using cells transfected with apoaequorin cDNA, the rate-limiting step in the reconstitution is the cell permeation of coelenterazine.
Reconstitution of aequorin can be done using the native coelenterazine or analogs (some analogs with quicker response and more intese signal have been described and are comercially available).
Although this protein was very popular for quantifying calcium in the past 30 years, it has been substituted by GFP derivatives.
New derivatives combining aequorin and GFP or RFP (red fluorescence protein) have been developed based on the chemiluminescence resonance energy transfer process (CRET or BRET).