Regeneration of Aequorin from Apoaequorin

During the process of regeneration, apoaequorin in a somewhat unfolded conformation is converted into a rigid conformation around a core of coelenterazine. It appears that coelenterazine has a strong capability to refold the unfolded apoaequorin molecules. In fact, apoaequorin denatured under various drastic conditions, such as treatment with 1 M HCl, 1M NaOH, and 6 M urea, or heating at 95°C, can be regenerated into aequorin with yields over 50% (Shimomura and Shimomura, 1981). 

The regeneration of native-type aequorin is not hindered by the presence of coelenteramide. Therefore, apoaequorin in a spent solution of luminescence can be regenerated into aequorin by simply adding an excess amount of EDTA to chelate the existing Ca2+, together with coelenterazine and some 2-mercaptoethanol, followed 

Yield of regenerated aequorin. Regeneration of aequorin is a slow reaction, which takes 25-30 minutes for a half completion, and about 3 hours for a 90% completion at 5°C the reaction is essentially complete in 12 hours. The concentration of apoaequorin has important effects on both the rate of regeneration and the yield of regenerated aequorin, as shown in Fig. 4.1.11. The relationship 

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