Aequorin method

Johnson, F. H., and Shimomura, O. (1978). Introduction to the bioluminescence of medusae, with special reference to the photoprotein aequorin. Method. Enzymol. 57 271-291. 

S Lizano, S Ramanathan, A Feltus, A Witkowski, S Daunert. Bioluminescence competitive assays for biotin based on photoprotein aequorin. Methods Enzymol 279 296-303, 1997. 

Blinks used a different method to dislodge the particles of active material from the strips cut off from the jellyfish (Blinks et al., 1978). The strips are shaken in cold seawater, and the particles dislodged are harvested by filtration on a Buchner funnel with the aid of Celite. The filter cake is first washed with 50 mM EDTA, pH 8.0, containing (NH4)2S(>4 at 75% saturation, to remove seawater. Then, the particles are cytolyzed and aequorin is extracted in situ by washing the filter cake with cold 50 mM EDTA, pH 8.0. The filtrate is clear and slightly greenish. The active matter in the filtrate is precipitated by saturation... 

Purification of aequorin. The purification method of aequorin reported by Shimomura et al. (1962) was essentially the repetition of column chromatography on DEAE-cellulose, the only usable, efficient chromatographic adsorbent available at the time. Since then, various different types of chromatographic media have been developed, and the purification method has been steadily improved. 

The methods and techniques presently available for the purification of aequorin are summarized below. In the description, all buffers are pH 6.5-8, and chromatography is performed at 0-5°C. 

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... 

An improved method of producing recombinant aequorin was devised based on the fact that the expression of the peak amount of apoaequorin in bacterial cells occurs several hours before the secretion into culture medium (Shimomura and Inouye, 1999). The cells containing apoaequorin in the periplasmic space, before secretion, are extracted under a very mild condition and, at the same time, converted into aequorin. The purification of the extract by two steps of column chromatography yields a high-purity preparation of recombinant aequorin. 

X-ray structure of aequorin (Head et al., 2000). X-ray crystallography was performed with the recombinant aequorin prepared by the improved method described above. The crystals of recombinant aequorin were grown in a high concentration of ammonium sulfate. The results revealed that aequorin is a globular molecule containing a... 

The second procedure is to measure the luminescence intensities at various Ca2+ concentrations and plot log (light intensity) against —log [Ca2+] for each aequorin. Examples of this method are shown in Fig. 4.1.14. This method provides more detailed information on the sensitivity of each aequorin. Generally, an increase in Ca2+ sensitivity shifts the curve to the left. 

Minute amounts of coelenterazine can also be measured utilizing apoaequorin or apoobelin (Campbell and Herring, 1990 Thompson et ah, 1995). In this method, a sample containing coelenterazine is treated with an excess amount of apophotoprotein (apoaequorin or apoobelin) to convert it to a Ca2+-sensitive photoprotein (aequorin or obelin). The photoprotein formed is assayed by luminescing it with Ca2+ to determine the amount of coelenterazine originally existed. With this method, the luminescence reaction is fast and usually complete in a few seconds, in contrast to the slower luminescence reactions with luciferases that sometimes require a few minutes to complete. However, the formation of photoprotein from apoaequorin is slow and not necessarily quantitative, and the overall accuracy of the photoprotein method does not compare favorably with that of the luciferase method that directly measures coelenterazine. The author recommends using a luciferase if the enzyme is available. 

Blinks, J. R., et al. (1978). Practical aspects of the use of aequorin as a calcium indicator Assay, preparation, microinjection, and interpretation of signals. Method. Enzymol. 57 292-328. 

Kihara, Y., and Morgan, J. P. (1989). A comparative study of three methods for intracellular loading of the calcium indicator aequorin in ferret papillary muscles. Biochem. Biophys. Res. Commun. 162 402—407. 

Masuda, H., et al. (2003). Chromatography of isoforms of recombinant apoaequorin and method for the preparation of aequorin. Protein Expression and Purification 31 181-187. 

Prasher, D. C., McCann, R. O., and Cormier, M. J. (1986). Isolation and expression of a cDNA coding for aequorin, the calcium-activated photoprotein from Aequorea victoria. Method. Enzymol. 133 288-298. 

Yamaguchi, A., Suzuki, H., Tanoue, K., and Yamazaki, H. (1986). Simple method of aequorin loading into platelets using dimethyl sulfoxide. Thromb. Res. 44 165-174. 

Today, bioluminescence reactions are used as indispensable analytical tools in various fields of science and technology. For example, the firefly bioluminescence system is universally used as a method of measuring ATP (adenosine triphosphate), a vital substance in living cells Ca2+-sensitive photoproteins, such as aequorin from a jellyfish, are widely utilized in monitoring the intracellular Ca2+ that regulates various important biological processes and certain analogues... 

Finally it should be noted that while classical methods for analysis tend to be little used, there are certain colorimetric methods for calcium which are of value. Thus, determinations with arsenazo III involve a detection limit of 10-7 mol dm 3. Calcium may also be determined using the photoproteins aequorin and obelin, which emit light on interaction with Ca2+ and have detection limits around 5 x 10-8 mol dm-3. These methods have had some interesting in vivo applications. 

Immunometric assays for TSH are available commercially as manual kit procedures or for use on automated systems. For practical reasons, nonisotopic methods dominate the market and have replaced radioactive tracer methods in most routine laboratories. The majority of immunometric TSH assays label the detection antibody with chemiluminescent labelled molecules other labels include peroxidase or alkaline phosphatase and sensitive photo-metric and fluorescenri molecules. Other assays are based on the use of fluorescent labels using europium chelates chemiluminescent compounds such as acri-dinium esters or ruthenium or bioluminescent molecules such as recombinant aequorin. ... 

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