JPH03167287A - Method for sensitized luminescence of aequorin by water-soluble high-molecular compound - Google Patents

Abstract

PURPOSE:To make it possible to sensitize the luminescence of aequorin and keep aequorin in a stable state by using a water-soluble high-molecular compound in combination with a bioluminescent system of aequorin. CONSTITUTION:A method for emitting light by using aequorin and its derivative, wherein a water-soluble high-molecular compound is used in combination therewith. As the water-soluble high-molecular compound, a synthetic high-molecular compound, a natural high-molecular compound, and modified high-molecular compound are used. Examples of the synthetic high-molecular compound include polyethylene glycol and polyvinyl alcohol. Example of the natural high-molecular compound include polysaccharide and protein. An example of the modified high-molecular compound is a polysaccharide derivative. Example of the aequorin derivative include a variant aequorin, a semisynthetic aequorin, and a semisynthetic variant aequorin.

Description

[Detailed description of the invention] [Industrial application field]

The present invention relates to a method for sensitized luminescence of aequorin, which is characterized by the coexistence of a water-soluble polymer compound. [Prior art and its problems] The photoprotein aequorin is a calcium-binding protein isolated from the luminescent Aequorina jellyfish.In nature, the protein part apoaequorin and the substrate part coelenterazine form a complex via molecular oxygen. is formed. Luminescence occurs when calcium binds to this complex. Calcium concentration can be measured using this luminescence. The present inventor used recombinant DNA techniques to clone the cDNA of 7-poaequorin from luminescent Aequorina jellyfish,
The primary structure was clarified (JP-A-61-135.5
811), and then succeeded in producing apoaequorin inside and outside the bacterial cell using Escherichia coli as a host using the cDN^ of
-102,895), and established a purification method for it (JP-A-1-132,397). Furthermore, he created an aequorin gene combined with a functional gene and made efforts to produce its fusion protein (Japanese Patent Application Laid-Open No. 64-39,990, Patent Application No. 63-3).
08.424) and established its purification method (Patent Application No. 1999).
-69,862). Then, we developed a metal detection method and an immunoassay method using these aequorins and their fusion proteins (Japanese Patent Application Laid-Open No. 82-281.942, Japanese Patent Application No. 1-74
, 742). The present invention is a report on a sensitized luminescence method for aequorin using a water-soluble polymer compound. By the way, the usefulness of Aequorin is well known to those skilled in the art.
Aequorin's luminescence can be used to detect various substances, that is, it can be applied to all kinds of measurement and detection systems such as immunoassays, DNA probes, and biosensors. It is expected to be useful as a test drug. In view of the above-mentioned technical circumstances, as a result of research, the present inventor has
We were able to develop a sensitized luminescence method for aequorin using a water-soluble polymer compound. As is clear from the above explanation,
The purpose of the present invention is to provide a luminescence sensitization technique for applying aequorin to a more ultrasensitive measurement method.

[Means to solve the problem]

The present invention has the following configurations (1) to (4). (1)
A sensitized luminescence method using aequorin and its derivatives, which is characterized by coexisting with a water-soluble polymer compound. (2) The sensitized luminescence method according to item 1 above, which uses a synthetic polymer compound, a natural polymer substance, or a modified polymer compound as the water-soluble polymer compound. <3) The sensitized luminescence method according to item 1 above, in which polyethylene glycol or polyvinyl alcohol is used as the synthetic polymer compound, a polysaccharide or protein is used as the natural polymer substance, and a polysaccharide derivative is used as the modified polymer compound. (4) The sensitized luminescence method according to item 1 above, wherein the aequorin derivative is a mutant aequorin, a semisynthetic aequorin, or a semisynthetic mutant aequorin. The structure and effects of the present invention will be explained in detail below. The present invention is a sensitized luminescent method for aequorin using the catalytic effect and sensitizing effect of a water-soluble polymer compound, and can be carried out, for example, by the method shown in the Examples below. The dioxetanes that are relevant to the method of the present invention are 's1
Coelenterazine is a compound having a four-membered peroxide structure as shown in Figure 2, and aequorin is a complex having a structure as shown in Figure 3. Aequorin derivatives include mutant aequorin in which the apoaequorin part is replaced with a mutant apoaequorin, semisynthetic aequorin in which the coelenterazine part is replaced by a coelenterazine derivative, and semisynthetic mutant aequorin in which both are replaced. Examples of water-soluble polymer compounds include compounds shown in Table 1 below. The present invention will be explained with reference to the accompanying drawings and tables. Figure 1 shows the structure of dioxetanes, where 1 shows the structure of the basic skeleton of dioxetane, and 2 to 5 are examples of dioxetane luminescent materials. ．． 2C shows the structure of coelenterazine. Figure 3: Schematically shows a complex of quorin. Figure 4.5 shows a complex of polyethylene glycol (
This is the result of tracking the time course of aequorin regeneration in the coexistence of PEG. Figure 'M6 shows the results of tracing the time course of the stability of regenerated aequorin. The 70th one strengthens the mechanism of aequorin's luminescence and regeneration. Table 1 below shows water-soluble polymer compounds, and Table 2 below shows classifications and representative examples of fluorescent substances. Table 3 shows the results of tracking the time course of sensitized luminescence of aequorin in the coexistence of a water-soluble polymer compound. ☆ 'M1 Table (Various water-soluble polymer compounds) 》 帛a η 5 TOS Table 2 (Various fluorescent substances'x) Note. N8D: 'J-nitrobenzofurazan derivative, SBD
: 7-Sulfonylbenzofurazan derivative Aequorin or an aequorin derivative coexists with a water-soluble polymer compound as follows. As aequorin, regenerated aequorin or natural aequorin, which is obtained by mixing and reacting apoaequorin with coelenterazine under predetermined conditions, is used. Preferred reaction conditions for reacting coelenterazine are 4°C and Is~20 hours. The obtained aequorin and aequorin derivatives are mixed with a water-soluble polymer compound as exemplified in Table 1 under predetermined conditions to obtain a luminescent agent for use in the present invention. The amount of light emitted by mixing the luminescent agent and Ca'' is measured in accordance with a known method. By using the sensitized luminescence method of the present invention obtained as described above, a trace amount of a substance can be extracted with ultrahigh sensitivity. As shown in Figure 7, the luminescence of aequorin is caused by the cleavage of a dioxetane intermediate, so the chemiluminescence of coelenterazine and dioxetanes is also highly water-soluble. It is thought that the sensitizing effect of molecular compounds can be expected.
Furthermore, the sensitizing effect of these luminescence is expected to be further enhanced by the coexistence of fluorescent substances. [Effects of the Invention] The usefulness of the method for sensitized luminescence of aequorin of the present invention will be obvious to those skilled in the art. Furthermore, by coexisting with an appropriate fluorescent substance, it becomes possible to sensitize the luminescence of aequorin. Such materials are well known to those skilled in the art. The above disclosure enables one skilled in the art to practice the claimed invention. However, in order to increase the understanding of this technology, the procedure used for the sensitized luminescence of aequorin, which is important to the present invention, will be clarified by an example. [Example] Example 1 [Time course of aequorin regeneration in the presence of polyethylene glycol (PEG)] JP-A-61-24
Produced by the production method described in No. 9,098, patent application No. 1983-
It was purified by the purification method described in 291 and 440, and separated using a silica-based Cosmosil 10C4 (10 x 150 mm) column by applying a 20 to 80% acetonitrile gradient in a 0.1% trifluoroacetic acid, water-acetonitrile system. Aboaequorin silk preparations were used in the following experiments. Aboequorin (10nl/μA) aqueous solution SOμjl
, 200aM Trls HCI (pH 7.6) 1
00 M EDTA buffer - 50 μA1 coelenterazine (200 g/mA) methanol solution 5μ℃, 2-mercabutoethanol 5μ41, PE04000 (20
%) aqueous solution in an appropriate amount, and finally the temperature was adjusted to 500μ℃ with water. After mixing, the mixture was incubated in ice water at 25°C and 37°C. Collect 10 μA at appropriate intervals and divide into cubes}-
(IOX 75), and transfer it to a photometer (T
D4000, Labo Science Co., Ltd.)
mM CaClt. 30mM Trls HCI (p
H7.6) An aqueous solution was added at 100μ℃, and the amount of luminescence was measured. The results are shown in Figure 4.5. In the case of ice water, regeneration was completed after 10 hours in the system without PEG, and no decrease in aequorin activity was observed until 26.5 hours later (Figure 4 only shows up to 22 hours). ). Compared to the additive-free system, in the PEG-added system (0.4-10%), an increase in aequorin activity was observed even after 10 hours, suggesting that regeneration was not yet complete. Furthermore, this tendency became more pronounced as the PEG concentration increased, suggesting that the coexistence of PEG was an obstacle to aequorin regeneration. However, very interestingly, 0.4% and 1% PE
The G-added system exceeds the aequorin activity value of the non-additive system at all times (here from 30 minutes to 22 hours), and compared to the non-additive system, the aequorin activity value after 22 hours is 0.4
Aequorin activity was 1.6 times higher in the % addition system and 1.7 times higher in the 1% addition system. From the above, it has become clear that the coexistence of PEG slightly hinders the regeneration of aequorin, but increases the luminescence of aequorin itself. This means that the maximum effect of increasing luminescence can be obtained in a short period of time by regenerating aequorin in an additive-free system and then adding PEG after the regeneration is complete to cause a luminescent reaction. In the case of 25°C, a decrease in aequorin activity was confirmed in all systems with a peak time of 3 to 4 hours. This is thought to be due to thermal decomposition of the substrate coelenterazine.
However, as in the case of ice water, 0.4% and 1% PEG
An increase in aequorin activity was observed in the additive system compared to the non-additive system. From the above, it is clear that aequorin regeneration is faster at 25°C than in ice water, but at the same time, the decomposition of the substrate coelenterazine proceeds rapidly, so not all apoaequorin is regenerated. We show that sufficient aequorin regeneration is possible even at room temperature by coexisting with coelenterazine in excess. This is important when applying aequorin as a test drug. At 37°C, aequorin activity became O in all systems after 2 hours because the decomposition reaction of coelenterazine was extremely rapid. This means that the regeneration temperature of aequorin cannot be raised too much, and room temperature was considered a reasonable line from an application standpoint. Example 2 [Temperature stability of regenerated aequorin] PEG-free regenerated aequorin regenerated in Example 1 (22
．． (regenerated for 5 hours) was incubated in ice water at 25°C, 37°C and k. At appropriate times, aequorin activity was measured in the same manner as in Example 1. The results are shown in Figure 6. 25℃
When I experienced this, aequorin activity decreased to 60% in 3.5 hours. Furthermore, when treated at 37°C, there was no activity at all after 1 hour. This means that the regenerated aequorin was decomposed by the temperature increase. Conversely, regenerated aequorin is stable at low temperatures (about 0 to 4°C). fact,
It remained stable in ice water for 26.5 hours after completion of regeneration. Example 3 [Time course of aequorin-sensitized luminescence in the coexistence of water-soluble polymer] Apoequorin (10 ng/μ) aqueous solution 50μ°C, 2
00sM Dingr1s HCI(1)H7.6) 10
0+sM EDT^ Puffer - 50μl 1 Coelenterazine (200mg/■°C) 5μ of methanol solution, 5μ of 2-mercabutoethanol, and finally water to make a 500μ square. After mixing, incubate at 4℃ for 26 hours.50
Aliquot 1 μl into separate tubes, and add dextrin to each tube. Dextran, bovine serum albumin (8S
A) 50 ti1 of aqueous solutions of PEG8QQQ and PEG4000 of various degrees of crushing were added, mixed, and then incubated in ice water. Sample 10μl at appropriate intervals, transfer it to Kievet (1OX7s quantity garden), set it on a Lumiphotometer (D4000), and incubate for 30s.
M CaCl. , 30mM TrlsHCI (+)
H7.6) 100 μl of the aqueous solution was added, and the amount of luminescence (equorin activity) was measured. The results are shown in Table 3. It was found that in the coexistence of water-soluble polymer compounds other than dextran, a 1.2 to 1.9-fold sensitizing effect was obtained, and aequorin remained stable even after 15 hours.
In addition, in the system without additives, aequorin decomposition progressed gradually, and aequorin activity decreased to 28% after 15 hours. This means that in ice water, aequorin activity decreased by about 30% within 41 hours from the start of regeneration.
It can be seen that in the additive-free system, aequorin activity decreases after a regeneration time of 20 hours. However, in a coexisting system with water-soluble polymer compounds other than dextran, even after 41 hours,
It can be seen that aequorin does not undergo decomposition and remains stable. As described above, by coexisting a water-soluble polymer compound with the bioluminescent system of aequorin, it becomes possible to enhance aequorin luminescence and maintain the stability of aequorin.

[Brief explanation of the drawing]

1 to 7 are explanatory diagrams of the present invention. Figure 1 shows the chemical structure of dioxetane. Figure 2 shows the chemical structure of coelenterazine. Figure 3 schematically shows the structure of aequorin. Figures 4 and 5 show the results of tracking the time course of aequorin regeneration in the coexistence of PEG. Figure 6 shows the results of tracing the thermal stability of regenerated aequorin over time. Figure 7 shows the mechanism of aequorin's luminescence and regeneration. that's all

Claims (4)

[Claims] (1) A sensitized luminescence method characterized by coexisting a water-soluble polymer compound in a luminescence method using aequorin and its derivatives. (2) A synthetic polymer compound as a water-soluble polymer compound,
The sensitized luminescence method according to claim 1, which uses a natural polymer substance or a modified polymer compound. (3) The sensitized luminescence method according to claim 1, in which polyethylene glycol or polyvinyl alcohol is used as the synthetic polymer compound, polysaccharide or protein is used as the natural polymer substance, and polysaccharide derivative is used as the modified polymer compound. (4) The sensitized luminescence method according to claim 1, wherein the aequorin derivative is a mutant aequorin, a semisynthetic aequorin, or a semisynthetic mutant aequorin.