Properties of Aequorin

Although aequorin is non-fluorescent, the spent solution after luminescence is brightly fluorescent in blue due to the presence of coelenteramide. Although pure coelenteramide is poorly fluorescent in aqueous solutions, it becomes strongly fluorescent in a hydrophobic environment. In the presence of Ca2+, coelenteramide 

According to Charbonneau et al. (1985), aequorin is a single chain peptide consisting of 189 amino acid residues, with an unblocked amino terminal. The molecule contains three cysteine residues and three EF-hand Ca2+-binding domains. The absorption spectra of aequorin and BFP are shown in Fig. 4.1.3, together with the luminescence spectrum of aequorin and the fluorescence spectrum of BFP. 

A concentrated solution of aequorin is yellowish, due to its weak absorption at 460 nm. 

The luminescence of aequorin in terms of total light is efficient in a wide range of pH, from 4.5 to beyond 10 (Fig. 4.1.4). The light intensity is also optimum in a broad pH range of 7-8.5. The time course of the aequorin luminescence reaction is roughly the first order in the presence of various concentrations of Ca2+ (Fig. 4.1.5 Shimomura et al., 1963b). 

Heterogeneity. Natural aequorin is not a homogeneous protein it is a mixture of many isoforms having isoelectric points ranging from 4.2 to 4.9 (Blinks and Harrer, 1975). The isoform composition may vary to some extent by the purification method employed, due to uneven loss of isoforms during purification. Consequently, the properties of each preparation of aequorin may also vary. By anion-exchange 

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Prendergast, F. G., and Mann, K. G. (1978). Chemical and physical properties of aequorin and the green fluorescent protein isolated from Aequorea forskalea. Biochemistry 17 3448-3453. 

Shimomura, O., Johnson, F. H., and Saiga, Y. (1962). Extraction, purification and properties of aequorin, a bioluminescent protein from... 

Shimomura O, Johnson F, Saiga Y. Extraction, Purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 1962 59 223-39. 

Shimomura, O., Johnson, F.H., and Saiga, Y, Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea, /. Cell. Comp. Physiol, 59, 223-239, 1962 Morise, H., Shimomura, O., Johnson, F.H., and Winant, J., Intermolecular energy transfer in the bioluminescent system of Aequorea, Biochemistry, 13, 2656-2662,1974. 

HPLC, about one dozen of the isoforms, aequorins A, B, C, -J, were isolated (Shimomura, 1986a Shimomura et al., 1990). An example of HPLC separation of isoforms is shown in Fig. 4.1.6, and a comparison of the properties of the isoforms is given in Table 4.1.2. 

Table 4.1.1 Main Properties of Natural Aequorin and its Ca2+-triggered Luminescence...

Table 4.1.2 Properties of the Isoforms of Aequorin (Shimomura, 1986a)... 

Table 4.1.3 Properties of Semisynthetic Aequorins prepared from a single batch of Natural Aequorin measured at 23-25°C (Shimomura, 1991a)... 

Hirano, T., et al. (1998). Bioluminescent properties of fluorinated semisynthetic aequorins. Tetrahedron Lett. 39 5541-5544. 

Imai, Y., et al. (2001). Fluorescence properties of phenolate anions of coe-lenteramide analogues the light-emitter structure in aequorin bioluminescence. J. Photocbem. Photobiol., A Chemistry 146 95-107. 

Shimomura, O. (1986a). Isolation and properties of various molecular forms of aequorin. Biochem. J. 234 271-277. 

Shimomura, O., Musicki, B., Kishi, Y., and Inouye, S. (1993a). Light-emitting properties of recombinant semi-synthetic aequorins and recombinant fluorescein-conjugated aequorin for measuring cellular calcium. Cell Calcium 14 373-378. 

In 1975, we demonstrated that treating spent aequorin with coelenterazine in the presence of oxygen results in the regeneration of original aequorin, consequently proving that aequorin contains a coelenterazine moiety. It was an important discovery that provided the basis for producing recombinant aequorin from apoaequorin. In 1978, we proposed that aequorin contains coelenterazine-2-hydroperoxide, based on the chemical properties and reactions of aequorin. The peroxide structure was confirmed by C-NMR spectrometry in 1986."... 

Shimomura O, Musicki B, BCishi Y. Semi-synthetic aequorin an improved tool for the measurement of calcium ion concentration. Biochem. J. 1988 251, 405-410 Shimomura O, Musicki B, Kishi Y. Semi-synthetic aequorins with improved sensitivity to Ca " ions. Biochem. J. 1989 261 913-20 Shimomura O, Musicki B, Kishi Y, Inouye S. Light-emitting properties of recombinant semi-synthetic... 

Li et and Chen et studied the bioluminescence of coe-lenterazine. Whereas the former work focused only on the absorption and emission properties of the product of the reaction (coelenteramide), the latter study also analysed relevant aspects of the mechanism, particularly, the origin and differences among chemiluminescence, bioluminescence and fluorescence in aequorin. On the basis of the TDDFT/ MM findings and the previous CASPT2 results on a small model and TDDFT results of the chemiluminophore in vacuo,the proposed mechanism (Fig. 12) has the characteristics described in the following ... 

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