Aequorin calcium

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. 

Fig. 4.1.4 Influence of pH on the total light emission and initial light intensity of aequorin. Buffer solutions containing 0.1 mM calcium acetate, 0.1 M NaCl, and 10 mM sodium acetate (for pH < 7) or 10 mM Tris-HCl (for pH > 7) were adjusted to various pH with acetic acid or NaOH, and then 2 ml of the solution was added to 3 pi of aequorin solution containing 1 mM EDTA to elicit luminescence, at 22°C. The data shown are a revision of Fig. 9 in Shimomura et al., 1962. The half-total time is the time required to emit 50% of total light.

Fig. 4.1.5 The time course of aequorin luminescence measured with various concentrations of Ca2+. Calcium acetate solution (5 ml) was added to 10 pi of aequorin solution to give the final Ca2+ concentrations of 10 2 M (A), 10-4 M (B), 10-5 M (C), 10 6 M (D), and 10 7 M (E) at 25°C. The dashed line (F) represents the light emitted following the addition of deionized distilled water that had been redistilled in quartz. The concentration of EDTA derived from the aequorin sample was 10 7 M (final cone.). From Shimomura et al., 1963b, with permission from John Wiley Sons Ltd.

The interaction between aequorin and a chelator must be carefully considered when estimating Ca2+ concentrations with aequorin in a calcium buffer containing EDTA or EGTA. This is particularly crucial when using a common calcium buffer system that contains a constant total concentration of a chelator in the buffer solutions of various Ca2+ concentrations in such a buffer system, a buffer of lower Ca2+ concentration contains a higher concentration of the free form of the chelator, resulting in an increased inhibition. 

The slope of 2.0 was also reported by Azzi and Chance (1969), Ashley (1970), and Baker et al. (1971), although several values between 2.0 and 4.0 were also reported by other investigators. The slope of 2 indicates that one aequorin molecule needs to be bound with two Ca2+ for the emission of light to take place the requirement of two Ca2+ was confirmed later by the titration of aequorin with calcium ions (Shimomura, 1995c Shimomura and Inouye, 1996). In Fig. 4.1.7,... 

Fig. 4.1.7 Relationship between the concentration of Ca2+ and the initial maximum intensity of luminescence when 2.5 ml of 2mM sodium acetate (ultrapure grade) containing the indicated amount of calcium acetate was added to 5pJ of aequorin stock solution, at 25°C. The aequorin stock solution contained 0.7mg of aequorin in 1 ml of 2 mM sodium acetate containing 10-5 M EDTA. When no Ca2+ was added the maximum intensity was 1.1 x 109 quanta/s. From Shimomura and Johnson, 1976.

Fig. 4.1.8 Influence of various calcium chelators on the relationship between Ca2 " concentration and the luminescence intensity of aequorin, at 23-25°C (panel A) in low-ionic strength buffers (I < 0.005) and (panel B) with 150 mM KC1 added. Buffer solutions (3 ml) of various Ca2+ concentrations, pH 7.05, made with or without a calcium buffer was added to 2 pi of 10 pM aequorin solution containing 10 pM EDTA. The calcium buffer was composed of the free form of a chelator (1 or 2mM) and various concentrations of the Ca2+-chelator (1 1) complex to set the Ca2+ concentrations (the concentration of free chelator was constant at all Ca2+ concentrations). The curves shown are obtained with 1 mM MOPS (A), 1 mM gly-cylglycine ( + ), 1 mM citrate (o), 1 mM EDTA plus 2mM MOPS ( ), 1 mM EGTA plus 2 mM MOPS ( ), 2 mM NTA plus 2 mM MOPS (V), and 2 mM ADA plus 2 mM MOPS (A). In the chelator-free buffers, MOPS and glycylglycine, Ca2+ concentrations were set by the concentration of calcium acetate. Reproduced with permission, from Shimomura and Shimomura, 1984. the Biochemical Society.

Stability of aequorin. Information on the stability of aequorin is important when using this photoprotein as a calcium probe. As already noted, aequorin is always emitting a low level of luminescence, thus... 

Fig. 4.1.14 Relationship between Ca2+ concentration and the initial light intensity of various recombinant semisynthetic aequorins and w-aequorin J (a semisynthetic natural aequorin made from isoform J). The curve number corresponds to the number of semisynthetic aequorin used in Table 4.1.4. A sample aequorin (3 (Ag) was in 3 ml of calcium-buffer solution containing 1 mM total EGTA, 100 mM KC1,1 mM Mg2+ and 1 mM MOPS (pH 7.0), at 23-24°C. From Shimomura etal., 1993a, with permission from Elsevier.

To prepare apoaequorin from aequorin, lOmM calcium acetate is added dropwise to a solution of aequorin (l-2mg/ml) containing less than 2-3 mM EDTA until its ability to luminesce is completely... 

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