JP2001240855A - Bioluminescent method and luminescent article - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a non-pathogenic and safe photoprotein aequorin derived from Aequorea coerulescens belonging to the order Leptmedusae of the class Hydrozoa of the phylum Cnidaria, free from toxicity, having simple luminous reaction and high sensitivity to calcium and capable of utilizing familiar calcium (city water, sea water, concrete or the like) to luminous reaction as a material for producing light, in which water and light at party event participate, micro- quantitative determination of drinking water and calcium in cow milk, scientific teaching material for elementary, junior, and senior high school students, e.g. action of calcium in the cells of organisms. SOLUTION: This method for carrying out luminous reaction comprises dissolving photoprotein aequorin derived from Aequorea coerulescens into a buffer solution having pH 7.4 and pouring the solution into a substance or water rich in calcium in dark to carry out strong luminous reaction.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The photoprotein aequorin present in O jellyfish exists as a complex of an apoprotein (apoaequorin), a luminescent substrate (coelenterazine), and molecular oxygen. When Ca binds, the structural change of aequorin occurs and emits blue light with decarboxylation,
Produces blue fluorescent protein (BFP: complex of coelenteramide and apoaequorin). In addition, taking advantage of the high sensitivity to calcium and the lack of toxicity problems, various types of parties, events, etc., by causing aequorin to emit light in the dark to calcium (tap water, seawater, concrete, etc.) existing in our everyday life. It can be used as a material to produce safe light at home, and as a science teaching material for elementary and junior high school students, such as a method for micro-quantification of calcium in drinking water and milk, and the function of calcium in cells of living organisms. Since it can be used, the range of use of the present invention is considered to be wide.

[0002]

2. Description of the Related Art Biologically derived LL reaction (luciferin.
The light production material utilizing the luciferase reaction) has a complicated luminescence reaction and its use range is limited. In addition, a light-producing material using a chemiluminescent substance (dialkyl oxalate + H2O2 + fluorescent aromatic polycyclic hydrocarbon) has a problem of toxicity, and is an extremely serious environmental pollutant even in the production process of the chemiluminescent substance. The product is sealed in a container to prevent direct contact with the luminescent liquid.

[0003]

SUMMARY OF THE INVENTION A photoprotein derived from a safe organism that is non-pathogenic and has no toxicity problems. The luminescence reaction is simple, and other luminescent materials may experience similar phenomena. Development of materials for producing light that involves impossible water and light has become an important issue.

[0004]

Means for Solving the Problems 1 mg of aequorin (Aequorin), a photoprotein derived from O jellyfish, which is non-pathogenic and has no toxicity problems and is safe to touch directly
The solution was dissolved in 50 ml of the 7.4 buffer solution, and when the solution was sprayed onto a floor or wall containing calcium in a 500 ml washing bottle in the dark, light like a planetarium spread instantaneously on the floor and wall. 50 ml of a solution in which 1 mg of the photoprotein aequorin was dissolved was directly dropped into 200 ml of tap water or seawater, and the pale cloud was instantaneously spread. To increase the emission, increase the amount of calcium,
The light emission time is short as the light emission is strong. Conversely, when the amount of calcium is reduced, the light emission is weak but the light emission time is long. As a result, the photoprotein aequorin derived from Oan jellyfish was converted to PH.
The method of dissolving in a buffer solution of 7.4 and pouring the solution into a substance or a substance containing a large amount of calcium in the dark to cause a strong luminescence reaction is extremely effective in solving the conventional problems. all right. As a result, according to the present invention, the luminescent substance that enables the production of light involving water and light, which is not possible to experience a similar phenomenon with other luminescent substances, is a photoprotein aequorin (Aequorin) derived from Oan jellyfish )
Was found to be optimal.

[0005]

Next, the method of the present invention and its experimental results will be described by way of examples, but the present invention is not limited thereto.

First Example In the present invention, a natural aequorin (Aequ) is used as a sigma research reagent.
orin) 5 mg was used. As for the adjustment of the reagent, the buffer solution was adjusted to pH 7.4 while adding sodium hydrogen carbonate (Wako Pure Chemicals, special grade) little by little to 50 ml of distilled water. 1 mg of natural aequorin was dissolved in the adjusted buffer solution to obtain a light effect material. The solution, which was a light-producing material, was poured into a 500 ml washing bottle that had been washed with distilled water, and the solution was sprayed little by little on the palm, clothes, walls, and the floor in the dark. As a result, the palms, clothes, and the wall and floor all glow pale, and especially the walls and floors with high calcium content are strong for about 5 seconds,
It glowed pale. The palm and clothes emitted light weaker than the wall or floor, but the light emission time was less than 10 seconds.

[0007]

EFFECT OF THE INVENTION The present invention is a safe photoprotein derived from living organisms which is non-pathogenic and has no toxicity problems, and is present near us by utilizing its simple luminescence reaction and high sensitivity to calcium. Aequorin reacts with calcium (tap water, seawater, concrete, etc.) by emitting light, and can be used as a light production material that involves safe water and light at various parties and events. Since it is suitable as a science teaching material for elementary, middle and high school students, such as micro-quantification method and the function of calcium in cells of living organisms, it is considered that the range of application is widened.

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[Submission date] May 2, 2000 (2005.2)

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[Title of the Invention] Bioluminescent method and luminescent article

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[Correction target item name] Claims

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[Claims]

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[0001]

BACKGROUND OF THE INVENTION The calcium-emitting photoprotein is a naturally-occurring or recombinant photoprotein selected from the group consisting of the calcium-binding photoprotein aequorin (aequorin), clintine, oberin, mitrocomin, mineospin and belvoin. Certain manufactured goods are sensitive to calcium, and calcium that is present in our daily lives (tap water, seawater, concrete, sweat, etc.)
And a material that generates fluorescence is a naturally-occurring or recombinant fluorescent protein selected from the group of GFPs and BFPs derived from soft aquatic organisms or flower insects. Since the activity of the product is stable for a long time and there are no toxicity problems, etc.
BB bullet for air gun, BB bullet for air gun, fish bait,
Ornamental fish baits, fishing baits, health supplements, ice cubes, science teaching materials,
There are a wide range of uses, such as simple calcium analyzers, party goods, water guns, and toys.

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[0004]

The present invention is non-pathogenic, has no poison productivity, is safe to touch directly, has a simple and fast luminescence reaction, and has stable activity. A group consisting of the calcium-binding photoprotein aequorin (aequorin), clintine, oberin, mitrocomin, mineopsin and belboin, which are directing materials that involve water and light, which are incapable of experiencing a similar phenomenon with other luminescent substances. Articles made of naturally-occurring or recombinant photoproteins selected from the group consisting of natural or recombinant fluorescent proteins selected from the group of GFPs and BFPs derived from soft aquatic organisms or flower worms can solve conventional problems. Extremely effective.

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First Example The photoprotein and fluorescent protein used in the present invention were obtained from commercial suppliers. That is, the natural photoprotein aequorin was purchased from Funakoshi Co., Ltd., and the recombinant aequorin was purchased from Molecular Probes. The recombinant fluorescent protein GFP and its mutant GFP were purchased from Clonetech, Inc. For the preparation of the reagents, 0.1 ml of recombinant aequorin and 0.9 m of EDTA (chelating agent) were added to 1 ml of phosphate buffer.
was dissolved, and 0.01 mg of the recombinant fluorescent protein GFP was added to 2 ml of the solution to prepare a reagent. 1 ml of the reagent was adsorbed on a polyvinyl alcohol crosslinked polymer superabsorbent polymer manufactured by Nippon Synthetic Chemical Co., Ltd. for 2 hours, and the polymer with the adsorbed reagent was accommodated in a household refrigerator. Another reagent 1m
1 was ice cubes, and after 4 weeks, the polymer and ice cubes were put into water containing calcium carbonate in the dark to emit light, and when they emitted light, they emitted green light first, then changed to blue light emission and emitted light for about 15 minutes. No difference was observed in the luminescence of both manufactured articles.

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[0007]

Industrial Applicability The present invention is non-pathogenic, has no productivity of poison, is safe even when touched directly, has a simple and fast luminescence reaction, and has a stable activity. A group consisting of the calcium-binding photoprotein aequorin (aequorin), clintine, oberin, mitrocomin, mineopsin and belboin, which are directing materials that involve water and light, which are incapable of experiencing a similar phenomenon with other luminescent substances. GFs derived from naturally occurring or recombinant photoproteins selected from the group
Articles made of natural or recombinant fluorescent proteins selected from the P and BFP groups are expected to be widely used in luminescence playground equipment, science teaching materials, health supplements, and fisheries resource surveys.

Claims (2)

[Claims] 1. A method for producing a light effect producing material for scientific teaching materials for the purpose of allowing a photoprotein aequorin derived from Oan jellyfish, which is a soft aquatic organism of the phylum Cnidaria, to undergo a luminescent reaction with calcium. 2. A method for producing a light for a scientific teaching material for producing a luminescence reaction of calcium with a photoprotein aequorin derived from Oan jellyfish, which is a soft aquatic organism of the order Cnidaria hydroid.