JPS6048937A - Preparation of compound labeled with radioactive iodine - Google Patents

Abstract

PURPOSE:To provide a compound labeled with radioactive iodine useful for the quantitative determination of proteins, etc., economically, without deteriorating the biochemical properties of the original compound, by adjusting the ratio of the amount of the oxidizing agent added in the preparation of the labeled compound to the amount of the stabilizing agent for the radioactive iodine to a specific level. CONSTITUTION:A compound labeled with radioactive iodine useful for the quantitative determination of proteins, etc. by radio-isotope method, is prepared by using an oxidizing agent such as chloramine-T, hydrogen peroxide, etc. to be added to the reaction system in an amount of >=0.5 equivalent based on the amount of the reducing agent such as sodium thiosulfate, etc. to be added to the radioactive iodine as a stabilizer. The proteins such as mouse EGF (epidermal growth factor), etc. and the constituent amino acids can be labeled with the radioactive iodine having high specific radioactivity in high labeling yield without deteriorating the biochemical properties of the proteins, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Application of the Invention] The present invention relates to a method for quantifying proteins using radioactive iodine, and particularly to a method for preparing labeled proteins for the above-mentioned assay.

[Background of the invention]

In the fields of biochemistry and pharmaceutical production, biological measurement methods (
bioassay) and immunoassay (immunoassay)
The protein is quantified using the assay. The former method uses the reaction between proteins and cells to quantify proteins, and the latter method uses the antigen-antibody reaction of proteins to immobilize proteins.

Methods for quantifying reacted or unreacted proteins by these measurement methods include spectrophotometry, liquid chromatography, and radioisotope methods using radioactive isotopes. Among these, the radioisotope method is widely used because it can quantify even trace amounts of protein.

In the radioisotope method, it is important to prepare *identified proteins (proteins labeled with radioisotopes) without changing their biochemical properties.

Generally, radioactive iodine (Hl) is used to prepare labeled proteins.
1, 126 I), etc. are used as radioactive iodine for protein labeling.
It is manufactured by dj'Juclear (USA), Amersham (UK), and others. (Both are commercially available in Japan by the Japan Isotope Association). This radioactive iodine for protein labeling has a specific radioactivity (iodine unit carbonate).
Since the per unit radiation (ffl'i) is low, when a trace amount of protein is labeled, the biochemical properties of the protein may change, such as the molecular weight of the protein changing.

From the above points, a protein labeling method using radioactive iodine with high specific radioactivity is required.

[Purpose of the invention]

An object of the present invention is to eliminate the drawbacks of the above conventional methods by providing a method for obtaining labeled proteins using radioactive iodine with high specific radioactivity.

[Summary of the invention]

The present invention was developed based on the following experimental facts.

As a result of trying to label proteins using radioactive iodine with high specific radioactivity, it was found that the t of labeled iodine (hereinafter referred to as labeling yield) was as low as 10% or less of the initial value. The inventors have found that the cause of this is sodium thiosulfate (which has Na2 St Os reducing properties) contained as a stabilizer in commercially available radioactive iodine. That is, sodium thiosulfate is
This is because it decomposes oxidizing agents such as clohumin T used when labeling proteins with radioactive iodine.

+ Characteristics of the investigation: 1. As a condition for obtaining a commercial labeling yield, the electrophoretic charge of the source agent is 1.
1, to be above sJu.

[6th Example] Examples of the present invention will be described in detail below. In this example,
As a protein to be labeled, mouse EGF (epidermal
groWth factor) is used as radioactive iodine, carrier-free, and has a specific radioactivity of 120 mC1/μg.
(2mC1/μg for protein labeling)131
We will also take up an example in which chloramine T is used as an oxidizing agent for cooling when labeling a protein with radioactive iodine.

Figure 1 shows the structure of mouse EGF. 1 mouthful: (]
I- is the molecule -6,045, which is a combination of 53 amino acids and, when isolated, forms colorless crystals.

Among these, the amino acid labeled with iodine is tyrosine (T
y r ) and histidine. These amino acids are as shown in Figure 2, and there are two locations at each site, w10, where iodine can be absorbed. If all the stiffness is labeled with iodine, the mouse I =: OF molecule [7315, and more than 20% of the initial molecular rule is 1-! Add. Such an increase in molecular weight may lead to changes in biochemical properties. A specific example of calculation will be shown from the viewpoint of radioactivity (ratio of radioactive iodine to be labeled with respect to changes in molecular weight).

As a condition for the trial calculation, it is assumed that 5 μg of mouse EGF is labeled with 1 mC1 of radioactive iodine and the labeling yield is 50%. These conditions are generally the same as those for labeling proteins and the like. At this time, when using radioactive iodine for protein labeling with a specific radioactivity of 2mC4/μg,
The weight of radioactive iodine is 0.5 μg, and 50% of it, 0.25 μg, is chemically bound to mouse EGF. In this case, the molecular weight of mouse EGF will increase by 5%. Similarly, if the specific radioactivity is 120 mC1, 0.
004 μg chemically binds to mouse EGF. At this time, the molecular weight of mouse EGF fluctuates as O and OS% increase, and biochemical changes in L'j cannot be considered. In addition, the reaction when iodine is 1+'81ip is as follows: nichloramine 1'(r) -) sulfonyl chloride amide sodium molecule $281.691
, iodine ion ■- is oxidized and mouse EGF
Tyrosine t #i: Replaced with mini-histidine.

R, -H+T ”--”-1→It,-I+H”Next,
In the case of labeling mouse E (-JF) with radioactive iodine with a specific radioactivity of 125 mC7/4 g, the labeling yield was determined using the ratio of Naz 8203 t mixed in the radioactive iodine to the addition l of chloramine T as a parameter. The results are shown in Figure 3. Here, Na28
The amount of 203 and chloramine T is 1 based on the redox reaction.
~, each valence is 6, 1, and 1-2, and the equivalent number '5
r: It's a shame. From the results shown in FIG. 3, it can be seen that the cooling of chloramine P is required to be at least 0.5 of the N828203 wrinkle. The amount of chloramine T added is more than 10 times that of 1141, which is commonly used.

In this way, it is important that the biochemical properties of the labeled EOF remain unchanged. To confirm this, 150 samples with known concentrations were tested using the RR, A method (radio
As a result of measurements using chloramine T (receptor assay), there was initial concern that chloramine T-induced
There was no decomposition of GF, which was consistent with the results obtained by other measurement methods.

According to this example, a protein such as mouse E(]F or an amino acid constituting the protein can be labeled with radioactive iodine without damaging its biochemical properties.

In this example, only the example of mouse EGJ- is taken up, but similar effects can be achieved with various amino acids, hormones, hormone related substances, proteins, and the like.

Further, in this example, chloramine T is used as the oxidizing agent, but the same effect can be obtained when an oxidizing agent such as hydrogen peroxide is used.

〔Effect of the invention〕

According to the present invention, the determination of proteins [11
When preparing labeled compounds in the radioisotope method, proteins can be labeled with radioactive iodine without damaging their biochemical properties.

FIG.
Fig. 2 is a state diagram showing the structural formula of the amino acid that is oxidized with radioactive iodine in mouse EGP, and Fig. 3 shows the ratio of the amount of original removal and the amount of oxidizing agent during labeling and the labeling yield. FIG.

10...Iodination position.

Agent Patent Attorney Akio Takahashi Number 1 Diagram 20 C (L) (lθ TyrC kerosene) His (His+Sunno Deaf 3
Figure ~ Kuzsz03L (furnace 1

Claims (1)

[Claims] 1. In the method for preparing a labeled compound using radioactive iodine, the amount of the oxidizing agent that is cooled during preparation of the labeled compound is 0.5 or more of the amount (actual amount) of the reducing agent for stabilizing the radioactive iodine. A method for preparing a compound labeled with radioactive iodine with a percentage of .