Extraction and Purification of Aequorin

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 

Assay of aequorin. The assay of aequorin is simple. To a vial containing a small amount of aequorin sample (1-100 xl), 1 ml of 10 mM calcium acetate solution is injected, measuring the total amount of light emitted. The amount of the total light is proportional to the amount of aequorin in the sample. 

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. 

A sample of aequorin (purity 80%) is first luminesced by adding a sufficient amount of Ca2+. To the spent luminescence solution, ammonium sulfate is dissolved to a concentration of 1M, and then the solution is added onto a column of Butyl Sepharose 4. The apoaequorin adsorbed on the column is eluted stepwise with buffer solutions containing decreasing concentrations of (NH4)2S04 starting from 1M. Apoaequorin is eluted at a (NH4)2SC 4 concentration lower than 0.1 M. The apoaequorin eluted is regenerated with coelenter-azine in the presence of 5 mM EDTA and 2 mM 2-mercaptoethanol 

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