Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1997-12-22
1999-03-09
Tsang, Cecilia J.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
435188, 435212, 530300, 530331, 530332, 514 2, C12N 968, A61K 3804
Patent
active
058799230
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a stable plasmin solution useful as a reagent for the quantitative measurement of a blood coagulation and fibrinolysis factor.
BACKGROUND ART
The reaction of an enzyme in the living body is controlled and regulated by its activating substance or a substance inhibiting its reaction, whereby its function is adjusted. For example, in the blood coagulation mechanism, there exists an enzyme inhibitor which inhibits the blood coagulation reaction at the sites other than a vascular injury site or inhibits hypercoagulability and hyperfibrinolysis and by the inhibitor, blood coagulation and fibrinolysis is being controlled and regulated. In the hematological test to investigate the advance of thrombus formation, the quantitative measurement of an enzyme inhibitor has an important meaning. Since the amount of the enzyme inhibitor serves as a suitable index showing the condition of coagulation and fibrinolysis, quantitative measurement of an enzyme inhibitor such as antithrombin III (ATIII) or .alpha.2-plasmin inhibitor (.alpha.2PI) is carried out.
It has been made cleared that among such enzyme inhibitors, .alpha.2PI is one of the most important fibrinolysis inhibiting factors which can control fibrinolysis in the blood and it has attracted attentions as an index for finding hyperfibrinolysis in the living body. The blood level of .alpha.2PI is different according to the kinds or symptoms of the diseases. For example, it shows a marked reduction in the case of disseminated intravascular coagulation (DIC) or liver troubles. The blood level of .alpha.2PI has therefore been used as an index for screening of such a disease, analysis of the morbid state or judgment of prognosis and also an index for judging drug efficacy at the fibrinolytic treatment.
The quantitative measurement of such an enzyme inhibitor has conventionally been carried out by reacting the enzyme inhibitor with an excess amount of an enzyme and then measuring the residual amount of the enzyme. For example, based on the fact that .alpha.2PI inhibits the enzyme plasmin, the amount of .alpha.2PI in a biosample (specimen) is measured by reacting .alpha.2PI in the specimen with a certain amount of plasmin and then measuring the remaining plasmin activity. In this case, the activity of the plasmin is determined, for example, by measuring the hydrolysis rate of a chromogenic synthetic substrate from a change in the absorbance.
Many of such enzyme inhibitors are serine protease inhibitors and an enzyme for measuring such enzyme inhibitors is serine protease. Proteases such as serine protease each has a site which will become its own substrate within its molecule so that prompt decomposition in the solution and lowering in the protease activity or binding activity with a protease inhibitor are sometimes observed. For example, a human-derived plasmin used for the quantitative measurement of .alpha.2PI loses 72% of its protease activity when allowed to stand at 37.degree. C. for one hour and decomposition occurs both in the H chain and L chain (K. N. N. Reddy, Biochem. Biophys, Res. Commun., 92, 1016-1022(1980)).
It is known, on the other hand, that the protease activity of plasmin exhibits improved stability under the presence of fibrinogen, .epsilon.-aminocaproic acid and glycerol or at high ion strength (J. Jespersen, Thromb. Res., 37, 3955-404(1986) or under the presence of .epsilon.-aminocaproic acid and lysine (K. N. N. Reddy, Progress in Fibrinolysis, 374-379(1981)).
The above methods are however accompanied with the drawbacks that they bring about stabilization only for extremely short time and if plasmin is allowed to stand at 37.degree. C. for one hour, its activity lowers considerably; and that even if 50% glycerol permitting the relative retention of the stability of protease activity is added, the binding activity with a protease inhibitor lowers (M. Shimokawa, Analytical Science, 10, 533-536 (1994)).
The reagent for the quantitative measurement of such an enzyme inhibitor is prepared as a lyophili
REFERENCES:
patent: 4361652 (1982-11-01), Uemura et al.
patent: 4361653 (1982-11-01), Watanabe et al.
patent: 4442213 (1984-04-01), Heber et al.
Emmi Mari
Yago Hirokazu
Daiichi Pure Chemicals Co. Ltd.
Mohamed Abdel A.
Tsang Cecilia J.
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