Aequorin

Aequorin is a photoprotein isolated from the bioliuninescent jellyfish y4e-quorea victoria. Upon addition of calcium ions (Ca2+) and coelenterazine, a reaction occurs whose end result is the generation of blue light in the 460-470 nm range. 

---

ImmunO lSS iy. Chemiluminescence compounds (eg, acridinium esters and sulfonamides, isoluminol), luciferases (eg, firefly, marine bacterial, Benilla and Varela luciferase), photoproteins (eg, aequorin, Benilld), and components of bioluminescence reactions have been tested as replacements for radioactive labels in both competitive and sandwich-type immunoassays. Acridinium ester labels are used extensively in routine clinical immunoassay analysis designed to detect a wide range of hormones, cancer markers, specific antibodies, specific proteins, and therapeutic dmgs. An acridinium ester label produces a flash of light when it reacts with an alkaline solution of hydrogen peroxide. The detection limit for the label is 0.5 amol. 

Heat stability The Oplophorus luminescence system is more thermostable than several other known bioluminescence systems the most stable system presently known is that of Periphylla (Section 4.5). The luminescence of the Oplophorus system is optimum at about 40°C in reference to light intensity (Fig. 3.3.3 Shimomura et al., 1978). The quantum yield of coelenterazine is nearly constant from 0°C to 20°C, decreasing slightly while the temperature is increased up to 50°C (Fig. 3.3.3) at temperatures above 50°C, the inactivation of luciferase becomes too rapid to obtain reliable data of quantum yield. In contrast, in the bioluminescence systems of Cypridina, Latia, Chaetopterus, luminous bacteria and aequorin, the relative quantum yields decrease steeply when the temperature is raised, and become almost zero at a temperature near 40-50°C (Shimomura et al., 1978). 

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... 

Freeze-drying of aequorin. The process of freeze-drying always results in some loss in the luminescence activity of aequorin. Therefore, aequorin should not be dried if a fully active aequorin is required. The loss is usually 10% or more. The loss can be somewhat lessened by adjusting the buffer composition the use of 100 mM KCl and some sugar (50-100 mM) seems to be beneficial. The buffer composition used at the author s laboratory is as follows 100 mM KCl, 50 mM glucose, 3 mM HEPES, 3 mM Bis-Tris, and at least 0.05 mM EDTA, pH 7.0. 

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. 

Fig. 4.1.3 Absorption spectra of aequorin (A), spent solution of aequorin after Ca2+-triggered luminescence (B), and the chromophore of aequorin (C). Fluorescence emission spectrum of the spent solution of aequorin after Ca2+-triggered bioluminescence, excited at 340 nm (D). Luminescence spectrum of aequorin triggered with Ca2+ (E). Curve C is a differential spectrum between aequorin and the protein residue (Shimomura et al., 1974b) protein concentration 0.5 mg/ml for A and B, 1.0 mg/ml for C. From Shimomura and Johnson, 1976.

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.

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... 

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.

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...

The luminescence is strongly inhibited by bisulfite and p-di-methylaminobenzaldehyde even at micromolar concentrations of the reagents these reagents destroy the ability of aequorin to... 

Fig. 4.1.6 HPLC analysis of a sample of purified natural aequorin on a TSK DEAE-5PW column (0.75 x 7.5 cm) eluted with 10 mM MOPS, pH 7.1, containing 2mM EDTA and sodium acetate. The concentration of sodium acetate was increased linearly from 0.25 M to 0.34 M in 14 min after the injection of the sample. Full-scale 0.02 A. Flow rate 1 ml/min. Reproduced with permission, from Shimomura, 1986a. the Biochemical Society.

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

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. 

Relationship between Ca2+ concentration and luminescence intensity. In the measurement of Ca2+ concentration with aequorin, the calibration of the relationship between Ca2+ concentration and luminescence intensity is essential. However, the application of this relationship is complicated by the chelator used to set the Ca2+ concentration, for the reason noted above. To minimize the complication, we used only a minimum amount of EDTA to protect aequorin in the measurements to obtain the relationship between Ca2+ -concentration and light intensity, and plotted the data as shown in Fig. 4.1.7 (Shimomura and Johnson, 1976). The concentration of EDTA was... 

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.

Effects of various Ca2+ chelators. The effects of various Ca2+ chelators on the luminescence of aequorin are compared in the... 

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.

Aqueous solutions of aequorin also emit light upon the addition of various thiol-modification reagents, such as p-quinone, Br2, I2, N-bromosuccinimide, N-ethylmaleimide, iodoacetic acid, and p-hydroxymercuribenzoate (Shimomura et al., 1974b). The luminescence is weak and long-lasting ( 1 hour). The quantum yield varies with the conditions, but seldom exceeds 0.02 at 23-25°C. The luminescence is presumably due to destabilization of the functional moiety caused by the modification of thiol and other groups on the aequorin molecule. 

---