JPS59104556A - Protein stabilizing agent - Google Patents

Abstract

PURPOSE:To prevent denaturation of protein for a long period of time by adding the cyclized oligosaccharide to blood, enzyme, etc. CONSTITUTION:Various studies are made with respect to saccharides in order to prevent the denaturation of protein in blood and eyzme, and as a result, it is found that the cyclized oligosaccharide having the constitutional formula shown in the figure, that is, cyclodextrin consisting of 1.4 bond of dextrose, provides an excellent effect as a protein stabilizer for blood and enzyme. More specifically, if the cyclized oligosaccharide of the constitutional formula shown in the figure is added to blood, enzyme, etc., the denutration of the protein can be prevented for a long period of time, and in the case of, for example, blood, the detection of the hemoglobin saccharified by the denatured protein as a positive error is obviated in a clinical measurement of a saccharified hemoglobin value. The decrease in the activity of enzyme is obviated in the stage of a measurement relating to enzyme.

Description

[Detailed description of the invention] The present invention relates to a protein stabilizer for surface fluids, enzymes, etc.

Aldoses such as glucose have traditionally been added for the purpose of preventing denaturation of proteins such as +m[ and enzymes.

However, aldoses such as glucose usually have an active pyrolyl group, and the active water group and the amine 7 in protein.
The protein reacts non-enzymatically at room temperature (5-30°C), resulting in protein denaturation. For example, glucose reacts with the amino/terminus of the β chain of hemoglobin (Hb) A, and even when blood is stored at 4°C, Hb undergoes glycation [(bA+a+
b.

In this way, reducing sugars easily react with amino/terminal and lysine residues in proteins, so for example, human 11b and #-1
HbA and glucose react both inside and outside the body and undergo saccharification, increasing t(bAlc).The same occurs with enzymes, resulting in low F of enzyme activity.

The purpose of the present invention is to eliminate such drawbacks and prevent protein denaturation in blood, enzymes, etc., and its gist is that the general formula (however, in the above formula, n is an integer from 6 to 8) ) consists of a protein stabilizer consisting of a cyclized oligosaccharide represented by

Next, the protein stabilizer of the present invention will be explained in detail.

Its constituent components are protein molecules such as blood and enzymes, and its three-dimensional shape is maintained by secondary bonds. However, the secondary bonds are susceptible to cleavage and the protein loses its properties.

By the way, Ilb is subject to clinical tests such as measurement of Hbtf in blood, blood IIb concentration, and measurement of glycation HbIfi degree, but Ilb also causes degeneration. For example, in the measurement of glycated Hb, tib is denatured during storage of the measurement sample, and denatured Hb
In high-performance liquid chromatography, the most difficult separation measurement involves dissolving glycated Hb at position 1 (11), so there is an apparent denaturation at position 1 (11).
(As b increases, a positive error occurs with respect to the glycated H concentration number. Therefore, the accurate glycated H that was originally contained in the sample
In order to measure the degree of BA, it is necessary to prevent the generation of Hb during storage and use of the specimen. Among the aldoses that have been used until now, [the amino terminal of Hb]
Since it undergoes IJ cosylation and becomes glycated hemoglobin, a positive error occurs with respect to the concentration of glycated Hb originally contained in the sample. For this reason, aldoses lack applicability as protein stabilizers.

Therefore, in order to prevent the denaturation of proteins in blood and enzymes, the inventors of the present invention conducted various studies on sugars, and found that cyclized OJ having the following structural formula was used to prevent the denaturation of proteins in blood and enzymes. It has been found that the effect as a stabilizer is <4'J when t-<'tt.

i.e. In the L formula, the value of n is 6, 7 and 8.

The above-mentioned cyclized oligosaccharide is a cyclodextrin consisting of 14 bonds of dextrose.

To obtain the above-mentioned cyclized oligosaccharide, starch is decomposed by the action of amylase, and a mixed solvent of ethylene tetrachloride and ethane tetrachloride is added to the decomposed product to precipitate a cyclodextrin mixture.

Next, when the precipitate was dissolved in water and P-cumene was added, only β-cyclodextrin (n = 7) and γ-cyclodextrin (n = 8) were precipitated, and α-cyclodextrin (n = 8) was precipitated in the solution. n=6) and some β-cyclodextrin and γ-cyclodextrin remain. Furthermore, three types of cyclodextrins can be isolated from the starch decomposition product by separately precipitating α-cyclodextrin from fluorobenzene and γ-cyclodextrin from anthracene.

α-cyclodextrin, β-cyclodextrin, γ
- Cyclodextrins may be used alone or in combination.These are used as stabilizers for blood and enzymes, in which case the amount used is 0.001 mol to 0. f)
It is preferable that

According to the present invention, the cyclized oligosaccharide of the above structural formula (blood,
By being added to enzymes, etc., protein denaturation can be prevented for a long period of time, and for example, in the case of blood, glycated Hb due to protein denaturation will not be detected as a positive error in clinical measurements of glycated Hb levels. Then, the enzyme (
1 will not cause low F activity.

Distilled water 100m7! 101 liters of anticoagulated blood from a healthy individual was mixed with 15 ml of a solution containing 1-1 octylphenoxypolyethoxyethanol as a hemolytic agent and 0.52 ml of β-cyclodextrin as a protein stabilizer.
After shaking briefly, the mixture was stored at 24°C, and after 0.5 and 10.15 hours, the OHbA and a-1-b values were separated and measured using a high-performance liquid chromatography device. The results are shown in the solid line graph in FIG.

For comparison, the HbA+1+b value was measured under the same conditions using a solution containing only β-cyclodextrin, and the results are shown in the dotted line graph in FIG.

As is clear from these results, when β-cyclodextrin is used, there is less change in protein over time compared to when it is not used, and the value after 15 hours shows that when β-cyclodextrin is used, It was reduced to about 60% compared to the case without it.

Example 2 In Example 1, α-cyclodextrin was replaced with α-cyclodextrin.
The IbA+a+1) value in the blood was separated and measured by high performance liquid chromatography in the same manner as in Example 1 except that cyclodextrin 21 was used. The result was 3.6%, which was 87% of the value when α-cyclodextrin was not used. Met.

Example 3 The HbA+a4-b value in blood was separated and measured by high performance liquid chromatography in the same manner as in Example 1 except that 0.22% of γ-dextrin was used instead of β-dextrin in Example 1. Results 3. It was 9%, and 86% when γ-dextrin was not used.

Example 4 An ammonium sulfate suspension of 5/- β-galactosidase (hereinafter abbreviated as β-Gal) was centrifuged at 2000 f for 15 minutes, and the precipitate was mixed with 0.01 M containing 1 mM magnesium chloride and α1M sodium chloride. Phosphate buffer (PH value 7.0)
1- to prepare a β-Gal solution. Further, the β-Gal solution diluted 121 times with phosphate buffer and β-cyclodextrin phosphorus buffer solution were mixed at a volume ratio of 1:1, and 1 t each was placed in a test tube.
Incubated for 20 hours at 37°C. Then β−
Gal enzyme'+ activity was measured and the decrease in activity was compared.

To measure the enzyme activity, add 50 μl of the enzyme solution to 500 μl.
7! l 0.1 heavy R5?5 ortho-nitro 7enyl β
-■) - 0.0000. containing galactopyranoside, 1mM magnesium chloride, 0.1M sodium chloride, 01% by weight bovine blood lr1 albumin, 0.1% by weight sodium azide.
Mix with 02M phosphate M buffer (PH ratio 7.2) and add 30
After reacting for 30 minutes at °C, the enzymatic reaction was stopped by adding 2 ml of 0.1 M sodium carbonate, and the absorption of ortho-nitrophenol produced by the decomposition of ortho-nitrophenyl β-D-1f lactopyranoside was measured at a wavelength of 420 nm. It was determined by its absorbance using a visible spectrophotometer. The absorbance ratio of each sample containing this additive to the blank was determined to determine the enzyme stabilizing effect.

In this case, the concentration of β-cyclodextrin tio, ooi
iI stabilization rate (sample absorbance/blank absorbance 'i) in moles, tL44, and at a concentration of 0.01 molar, the stabilization rate was 154.

[Brief explanation of drawings]

FIG. 1 is a graph showing the relationship between HbA, a+b values, and sample storage time when β-cyclodextrin was used and not used in Example 1. Patent applicant Sekisui Chemical Co., Ltd. Representative Motoshi Fujinuma

Claims (1)

[Claims] t A protein stabilizer comprising a cyclized oligo moe represented by the general formula (where n is an integer of 6 to 8 in the above formula)