JPH099997A - Stable plasmin solution - Google Patents

Abstract

PURPOSE: To enable the rapid and simple measurement of a blood coagulant fibrinolytic factor without deteriorating plasmin activities and bonding activities of the plasmin to an a 2PI even when preserving the plasmin in 8 solution state by adding an ollgopeptide of a specific amino acid thereto. CONSTITUTION: A oligopeptide (a dipeptide, a tripeptide, etc.) comprising one or more amlio acids selected from lysine, arginine, glycine, alanine, aspartlic acid and methionine is contained in a plasmin solution. The content of the oligopeptide is usually 1-20wt.%, preferably 5-20wt.%.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a stable plasmin solution useful as a reagent for measuring blood coagulation / fibrinolysis factor.

[0002]

2. Description of the Related Art Enzymatic reactions in a living body are controlled and regulated by an activating substance thereof and an inhibiting substance which inhibits the reaction to regulate the function. For example, in the blood coagulation mechanism, there are enzyme inhibitors that inhibit blood coagulation reactions and excessive hypercoagulation / fibrinolysis in areas other than vascular injury sites, which control / regulate blood coagulation / fibrinolysis. It is being appreciated. In hematological tests for examining the progress of thrombus formation, the measurement of enzyme inhibitors is important and is a good indicator of the state of coagulation / fibrinolysis. Therefore, antithrombin III (ATIII), α2-plasmin Inhibitor (α
2PI) and other enzyme inhibitors have been measured.

Of these, α2PI has been clarified to be the most important factor among fibrinolysis-inhibiting factors that regulate the fibrinolytic phenomenon of blood, and is used as an index for knowing the in-vivo enhanced state of fibrinolysis. Attention has been paid. Further, the blood level thereof varies depending on various diseases and symptoms such as generalized intravascular coagulation (DIC) and markedly decreased in liver diseases. Therefore, screening, pathological analysis, prognosis and fibrinolysis of these diseases are performed. It is used as an index for determining drug efficacy during therapy.

Conventionally, as a method for measuring such an enzyme inhibitor, the enzyme inhibitor is measured by reacting the enzyme inhibitor to be measured with an excess amount of enzyme and measuring the remaining enzyme. Has been done. For example, when measuring the amount of α2PI in a biological sample (specimen), α2P
Utilizing the fact that I inhibits the enzyme plasmin, a certain amount of plasmin is reacted with α2PI in the sample, and then the activity of the remaining plasmin is measured to obtain α2PI.
The amount is being determined. And in this case,
The activity of plasmin is determined by a method such as measuring the hydrolysis rate of a chromogenic synthetic substrate by measuring the change in absorbance.

However, most of such enzyme inhibitors are serine protease inhibitors, and the enzyme for measuring such enzyme inhibitors is Senri protease. Many proteases, such as serine proteases, have a site that serves as their own substrate within their own molecules, so that they rapidly decompose in solution, and a decrease in protease activity or binding activity with protease inhibitors may be observed. is there. For example, in the case of human-derived plasmin used for the measurement of α2PI, 72% of protease activity is inactivated when left at 37 ° C. for 1 hour,
Degradation occurs in both H and L chains (KNN Redd
y, Biochem. Biophys. Res. Com
mun. , 92, 1016-1022 (1980)).

On the other hand, the protease activity of plasmin is
Fibrinogen, ε-aminocaproic acid, high ionic strength, addition of glycerol (J.Jespersen, T.
hrmb. Res. , 37, 395-404 (198
6)), and addition of ε-aminocaproic acid or lysine (KNN Reddy, Progress in F).
ibrinolysis, 374-379 (198).
1)) and the like are known to improve stability. However, these methods can achieve stabilization for an extremely short time, and if left at 37 ° C. for 1 hour, the activity is significantly reduced. Further, even if the stability of the protease activity is relatively maintained like 50% glycerol, there is a problem that the binding activity with the protease inhibitor is reduced (M. Shim).
okawa, Analytical Science,
10, 533-536 (1994)).

[0007] Therefore, such a reagent for measuring an enzyme inhibitor is manufactured as a lyophilized product because plasmin cannot be stored in a solution state, and it requires preparation at the time of measurement, which is economical. There was a problem in terms of operability, operability, and speed. Moreover, in the conventionally used plasmin solution, since the viscosity is high and the plasmin activity and the α2PI binding activity of plasmin are significantly reduced,
It was difficult to apply it to an automatic analyzer as a PI measuring reagent.

[0008]

Therefore, an object of the present invention is to provide a plasmin solution in which the plasmin activity and the binding activity of plasmin with α2PI can be stably maintained for a long time even when plasmin is stored in a solution state. It is in.

[0009]

Under such circumstances, as a result of intensive studies by the present inventors, plasmin is stable for a long period of time when an oligopeptide consisting of a specific amino acid produced by plasmin cleaving a substrate is used. And α
The present invention has been completed by finding that a plasmin solution useful as a reagent for 2PI measurement can be obtained.

That is, the present invention provides a plasmin solution containing plasmin and an oligopeptide consisting of an amino acid selected from lysine, arginine, glycine, alanine, aspartic acid and methionine.

As the plasmin used in the present invention,
Any of methionyl type (the N-terminal is methionine), glutamyl type (the N-terminal is glutamic acid), lysyl type (the N-terminal is lysine) and the like are commercially available from Chromogenics Inc. You can use what you have. These plasmins can be used alone or as a mixture and have an activity of 0.1-10 nk.
It is preferably used in the range of at / ml, particularly 0.3 to 5 nkat / ml. In addition, 1 nkat refers to the amount of plasmin that decomposes 1 nmol of plasmin synthetic substrate (S-2251) per second.

Examples of amino acid oligopeptides used in the present invention include dipeptides, tripeptides and the like in which one or more amino acids selected from lysine, arginine, glycine, alanine, aspartic acid and methionine are combined. . Of these, a dipeptide or tripeptide consisting of one kind of amino acid is preferable, and glycylglycine, glycylglycylglycine, and alanylalanine are particularly preferable.

These oligopeptides may be used either individually or in combination of two or more. The oligopeptide is preferably contained in an amount of 1 to 20% by weight (hereinafter, simply expressed as%), particularly 5 to 20% in the total composition. Further, it is preferable to add it in the range of 1 to 2000 mg / nkat, particularly 10 to 700 mg / nkat, relative to the plasmin in the solution.

Further, these oligopeptides can be used in combination with one or more amino acids selected from lysine, arginine, glycine, alanine, aspartic acid and methionine, and a more stable plasmin solution can be obtained. be able to. In this case, these amino acids are preferably blended in an amount of 1 to 20%, particularly 5 to 20% in the total composition.

The plasmin solution of the present invention can be further blended with a polyhydric alcohol to obtain a more stable plasmin solution. Here, as the polyhydric alcohol, glycerol, ethylene glycol, polyethylene glycol and the like are preferable. When a polyhydric alcohol is added, it is preferably added in an amount of 1 to 50%, particularly 5 to 30%, based on the total composition.

The plasmin solution of the present invention is useful as a measurement reagent for α2PI, a standard solution for plasmin, and the like. When used as a measurement reagent, α2PI in a sample can be accurately quantified by using a commonly used chromogenic synthetic substrate. As a chromogenic synthetic substrate,
There is no particular limitation, and for example, S-2 manufactured by Chromogenic Co.
251 (HD-Val-Leu-Lys-paranitroaniline), S-2403 (Glu-Phe-Lys-).
Paranitroaniline) and the like can be preferably used.

[0017]

INDUSTRIAL APPLICABILITY The plasmin solution of the present invention is stably maintained without being reduced in plasmin activity and α2PI binding activity even after long-term storage, and is useful as a reagent for measuring α2PI. Further, since it can be stably stored in the state of a solution, it can be used as it is at the time of measurement, it is excellent in economical efficiency and operability, and the measurement can be carried out easily and quickly. Furthermore, since it has a low viscosity, it can be suitably used for an automatic analyzer.

[0018]

EXAMPLES Next, the present invention will be further described with reference to examples, but the present invention is not limited to these examples.

Example 1 To 250 μl of the first reagent having the following composition, 3 μl each of a normal plasma sample (containing a predetermined amount of α2PI) or a physiological saline solution was added.
In addition, the mixture was reacted at 37 ° C for 5 minutes, and then the second composition of the following composition
After adding 100 μl of the reagent, the change in absorbance per minute at a wavelength of 405 nm was measured. The amount of change in absorbance when using a physiological saline solution shows plasmin activity, and shows the titer of hydrolyzing the synthetic substrate (S-2251) of plasmin. Further, the value obtained by subtracting the residual activity of plasmin when a normal plasma sample was reacted from the plasmin activity of physiological saline indicates the α2PI binding activity of plasmin.
In addition, each plasmin solution of the second reagent is shown in Table 2.
A 20% glycerol solution containing the additives shown in 1 was used. The pH of these solutions is 7.4 with sodium hydroxide or hydrochloric acid.
After dissolving the plasmin, the half was measured on the day of preparation using the half, and the other half was sealed and left at 37 ° C. for 4 days, and then measured. For comparison, 50% glycerol and 20% glycerol were used. Table 2 shows the results.

[0020]

[Table 1] First reagent: HD-Valyl-L-leucyl-L-lysyl-p-nitroaniline (S-2251) 1.2 mM 25 mM Tris buffer (pH 7.4) Second reagent: plasmin 3 nKat / ml Each plasmin solution (pH 7.4)

[0021]

[Table 2]

As is clear from the results in Table 2, the plasmin solution of the present invention containing glycylglycine was stably maintained without a decrease in plasmin activity and α2PI binding activity. The viscosity of the plasmin solution was also low.

Example 2 Using various plasmin solutions having the compositions shown in Table 3, Example 1
In the same manner as above, plasmin activity and α2PI binding activity of plasmin at the time of production and after storage at 37 ° C. for 2 days were measured. The results are shown in Table 3.

[0024]

[Table 3]

As is clear from the results of Table 3, the plasmin solution of the present invention containing glycylglycine was stably maintained without a decrease in plasmin activity and α2PI binding activity. The viscosity of the plasmin solution was also low.

Example 3 Example 1 was carried out using various plasmin solutions having the compositions shown in Table 4.
In the same manner as above, the plasmin activity and the α2PI binding activity of plasmin at the time of production and after storage at 37 ° C. for 25 days were measured. The volume of physiological saline and normal plasma sample was 5 μl. The results are shown in Table 4.

[0027]

[Table 4]

As is clear from the results of Table 4, the plasmin solution of the present invention containing glycylglycine was stably maintained without a decrease in plasmin activity and α2PI binding activity. The viscosity of the plasmin solution was also low.

Example 4 Using various plasmin solutions having the compositions shown in Table 5, Example 1
In the same manner as above, the plasmin activity and the plasmin α2PI binding activity at the time of production and after storage at 37 ° C. for 9 days were measured. The volume of physiological saline and normal plasma fluid sample is 5
μl. Table 5 shows the results.

[0030]

[Table 5]

As is clear from the results shown in Table 5, the plasmin solution of the present invention containing glycylglycine was stably maintained without lowering the plasmin activity and α2PI binding activity. In addition, No. The viscosity of the plasmin solution containing 10% glycylglycine and 10% glycerol of 2 was 1.89 cp when measured at 25 ° C. using VISCOMATE (manufactured by Yamaichi Denki Kogyo KK).
Met. On the other hand, the viscosity of the plasmin solution containing 50% glycerol at 25 ° C was 6.98 cp. The plasmin solution of the present invention has a low viscosity and can be used in automatic analyzers.

Example 5 Using a 10% glycylglycine plasmin solution containing 10% glycerol in the same manner as in Example 1, 0 to 20 was used.
0% α2PI sample (n = 2 measurements) and 50 or 1
The activity of the α2PI sample (n = 10 measurements) at a concentration of 00% was measured to examine the calibration curve and reproducibility. In the examination of the calibration curve, since the stock solution of the normal plasma sample was set to 200% concentration as α2PI, the sample amount was set to 10 μl. In addition, in reproducibility studies, 1 stock solution of normal plasma sample was used.
Since it was set as α2PI with a concentration of 00%, the sample amount is 5
μl. The results are shown in FIG. 1 and Table 6.

[0033]

[Table 6]

From the results shown in FIG. 1, the calibration curve is good,
From the results of Table 6, reproducibility was also good.

[Brief description of drawings]

FIG. 1 is a diagram showing a relationship between α2PI concentration and a measured value (ΔAbs / min) when a calibration curve was examined in Example 5.

Claims (5)

[Claims] 1. A plasmin solution containing plasmin and an oligopeptide consisting of an amino acid selected from lysine, arginine, glycine, alanine, aspartic acid and methionine. 2. Further, lysine, arginine, glycine,
The plasmin solution according to claim 1, which contains at least one amino acid selected from alanine, aspartic acid, and methionine. 3. The plasmin solution according to claim 1, further comprising a polyhydric alcohol. 4. A method for stabilizing a plasmin solution, which comprises adding an oligopeptide consisting of an amino acid selected from lysine, arginine, glycine, alanine, aspartic acid and methionine to the plasmin solution. 5. Further, lysine, arginine, glycine,
The method for stabilizing a plasmin solution according to claim 4, wherein at least one amino acid selected from alanine, aspartic acid and methionine is added.