Photoprotein

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. 

Chemistry of photoproteins (modified proteins with luminescent covalent-linked heterocyclic fragments) as interface between bioactive molecules and protein function 98PAC2085. 

In our report on the bioluminescence of Meganyctiphanes (Shimomura and Johnson, 1967), the extremely unstable nature of the substance P caused us to interpret the functions of P and F incorrectly, the former as a photoprotein and the latter as a catalyst, as pointed out by Hastings (1968). The error was corrected 28 years later (Shimomura, 1995a), F being unambiguously shown to be a luciferin and P, a luciferase, on the basis that the quantum yield of F is about 0.6 at 0°C, while P can be recycled many times in the luminescence reaction. 

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... 

A cDNA encoding apoobelin was obtained from O. longissima and sequenced (Illarionov et al., 1995). The deduced amino acid sequence of the apoobelin consists of 195 amino acid residues, with a calculated molecular mass of about 22.2 kDa, closely matching the apoproteins of other Ca2+-sensitive photoproteins such as aequorin from the jellyfish Aequorea (Inouye et al., 1985 Prasher et al., 1985) and clytin from the jellyfish Phialidium gregarium (Inouye and Tsuji, 1993). To obtain recombinant apoobelin, the cDNA encoding apoobelin was expressed in E. coli (Illarionov et al., 2000). The recombinant apoobelin produced was purified and converted into obelin by incubation with coelenterazine in the presence of molecular oxygen and 2-mercaptoethanol or dithioerythritol, as in the case of aequorin. 

The small jellyfish Phialidium gregarium (diameter 15-20 mm) used to be abundant at Friday Harbor, Washington, in summer and autumn until about 1990. Levine and Ward (1982) isolated and purified a Ca2+-sensitive photoprotein from this jellyfish and named it phialidin. They extracted the photoprotein from whole specimens with an EDTA-containing buffer. The photoprotein extract was precipitated with ammonium sulfate, and purified by the following methods gel-filtration (BioGel P-150, minus 400 mesh), anion-exchange chromatography (DEAE Bio-Gel A), and gel-filtration (Sephadex G-75, superfine). 

Fig. 4.3.1 Effect of pH on the total light emission of phialidin (A), and the temperature stability profiles of phialidin (minute open circles) and aequorin (solid line) (B). In A, each buffer contained 0.1 M CaCl2 plus 0.1 M Tris, glycine or sodium acetate, the pH being adjusted with NaOH or HC1. In B, the photoprotein samples in 10 mM Tris-EDTA buffer solution, pH 8.0, were maintained at a test temperature for 10 min, and immediately cooled in an ice water bath. Then total luminescence activity was measured by injecting 1ml of 0.1 M CaCl2/Tris-HCl, pH 7.0, to 10 pd of the test solution. From Levine and Ward (1982), with permission from Elsevier.

Ca2+ is slightly lower than that of aequorin, indicating that the photoprotein is suited for measuring higher concentrations of Ca2+ than measurable with aequorin (Shimomura and Shimomura, 1985). 

The cDNA for this photoprotein has been cloned and expressed in E. coli, and the recombinant protein obtained was named mitrocomin (Fagan et al., 1993). Mitrocomin consists of 190 amino acid residues with a tyrosine residue at the C-terminus, and has three Ca2+-binding sites. 

The scyphozoan Periphylla emits light with a luciferin-luciferase reaction using coelenterazine as the luciferin, differing from Pelagia in the same class and all luminous hydrozoans that luminesce with photoproteins. 

Fig. 4.8.2 Comparison of the in vivo and in vitro luminescence of the photoproteins of Mnemiopsis sp. and Beroe ovata (A) in vitro luminescence spectra of mineopsin-1, -2, and berovin (B) in vivo luminescence spectrum of Mnemiopsis, (C) in vivo luminescence spectrum of Beroe. From Ward and Seliger, 1974b, with permission from the American Chemical Society.

On the other hand, the Johnson group isolated the Ca2+-sensitive photoprotein aequorin from the jellyfish (Shimomura et al., 1962), and, after considerable efforts, they isolated two compounds now... 

The rate of protonation may vary according to the structure of the light-emitter and the environment around the light emitter. In the case of chemiluminescence reactions in solutions, the hydrophobicity, permittivity (dielectric constant) and protogenic nature of the solvent are important environmental factors. In the case of bioluminescence involving a luciferase or photoprotein, the protein environment surrounding the light-emitter will be a crucial factor. 

On the basis of the luciferin-luciferase reaction discovered by Dubois (1887), Michelson, Henry and their associates studied the biochemistry of the Pholas bioluminescence for several years beginning in 1970 (Michelson, 1978). They isolated, purified, and characterized the luciferin and the luciferase, and published about a dozen papers in which the luciferin isolated was referred to as Pholas luciferin. Since the luciferin is clearly a protein, later authors called it pholasin following the traditional way of naming a photoprotein... 

Fig. 6.3.7 Luminescence spectrum of a homogenate of the luminous organ of Symplectoteuthis oualaniensis in the presence of 0.5 M KC1 (from Tsuji and Leisman, 1981). A homogenate suspension (1 ml) and 1MKC1 (1 ml), both made with 50 mM Tris-HCl, pH 7.6, containing 1 mM dithioerythritol, were mixed and the spectrum was measured 6 min after mixing. Note that the luminescence of the photoprotein symplectin isolated from the luminous organs showed a maximum at 470—480 nm (Takahashi and Isobe, 1993, 1994).

Extraction and partial purification of photoprotein. The solubility and general luminescence characteristics of the S. luminosa photoprotein are similar to those reported for the S. oualaniensis photoprotein the protein is soluble in buffer solutions containing 0.6-1.0 M salt but not in solutions containing 0.1-0.2 M salt, and the luminescence is pH-dependent. In the extraction of S. oualaniensis,... 

Assay of photoprotein. The activity of the photoprotein was measured in 1ml of 20 mM Tris-HCl buffer, pH 8.0, containing 0.6 M NaCl at room temperature. The intensity and total amount of light emitted were recorded. The luminescence intensity is markedly intensified by adding 5 il of catalase solution (crystalline bovine liver catalase 1.5 mg/ml) and 10 pi of 3% H2O2. 

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