Luminescence Reaction

In the case of aequorin reacting with Ca +, a conformational change of protein takes place when one molecule of aequorin is bound with two Ca2+ ions (Shimomura 1995b). The conformational change results in the cyclization of the peroxide of coelenterazine into the corresponding dioxetanone, which instantly decomposes and produces the excited state of coelenteramide and CO2 (Shimomura et al. 1974; Shimomura and Johnson 1978). When the energy level of the excited state of coelenteramide falls to ground state, light is emitted. A simplified mechanism of the luminescence reaction is illustrated in Fig. 1.1.

The spent solution of the luminescence reaction of aequorin is a mixture of coelenteramide, apoaequorin, and Ca + that forms a complex called "blue fluorescent protein" (fluorescence emission maximum about 465-470 nm). The dissociation constant of the complex into coelenteramide plus apoaequorin in the presence of 0.5 mM Ca2+ is 7 X 10-6 M at pH 7.4 and 25 °C (Morise et al. 1974; based on the molecular weight of aequorin 21 000). Thus, the luminescence

Aequorin Emission

Fig. 1.1 Schematic illustration of a simplified mechanism of the luminescence and regeneration of aequorin. Aequorin (upper left) is a globular protein that contains peroxidized coelenterazine sealed in its central cavity and has three EF-hand Ca2+-binding sites on the outside. When the protein is bound with two Ca + ions, an intramolecular reaction starts, resulting in the formation of coelenteramide and CO2, accompanied by the emission of blue light (Xmax 460 nm) and opening of the protein shell (upper right). The protein part, apoaequorin (bottom), can be regenerated into the original aequorin by incubation with coelenterazine and molecular oxygen in the absence of Ca2+. In the regeneration reaction, addition of a low concentration of 2-mercaptoethanol increases the yield of regenerated aequorin by protecting the functional cysteine residues of apopro-tein.

Fig. 1.1 Schematic illustration of a simplified mechanism of the luminescence and regeneration of aequorin. Aequorin (upper left) is a globular protein that contains peroxidized coelenterazine sealed in its central cavity and has three EF-hand Ca2+-binding sites on the outside. When the protein is bound with two Ca + ions, an intramolecular reaction starts, resulting in the formation of coelenteramide and CO2, accompanied by the emission of blue light (Xmax 460 nm) and opening of the protein shell (upper right). The protein part, apoaequorin (bottom), can be regenerated into the original aequorin by incubation with coelenterazine and molecular oxygen in the absence of Ca2+. In the regeneration reaction, addition of a low concentration of 2-mercaptoethanol increases the yield of regenerated aequorin by protecting the functional cysteine residues of apopro-tein.

reaction product of aequorin is usually blue fluorescent, unless the concentration ofaequorin used is too low (much less than 1 to form the fluorescent complex. The blue fluorescence of the complex (Amax 465-470 nm) closely matches the bioluminescence emission of aequorin, giving a basis to the postulation that the fluorescent complex is the light emitter ofaequorin bioluminescence (Shimomura and Johnson 1970), although it now seems an oversimplification considering that the conformation of apoaequorin continues to change for several minutes after the light emission.

When the light emission of aequorin is measured in low-ionic-strength buffers containing no inhibitor, the log-log plot of the luminescence intensity versus Ca + concentration gives a sigmoid curve having a maximum slope of about 2.0 for its middle part (Shimomura and Johnson 1976; Shimomura and Shimomura 1982), indicating that the binding of two Ca + ions to one molecule of aequorin is required to trigger the luminescence of aequorin.

Was this article helpful?

0 0

Post a comment