Substrate for measuring tryptase activity

A trypsin and activity determination technology, applied in the direction of microbial determination/inspection, measuring devices, biological testing, etc., can solve difficulties and fail to eliminate technical problems

Pending Publication Date: 2022-04-12
NAT UNIV CORP TOKYO UNIV OF AGRI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unfortunately, however, this excellent study published in 2001 has never been i Practicalization of an easy-to-use format such as Boc-Arg-Asn-Arg-MCA
[0007] As mentioned above, so far, tripeptides that can be used as tryptase substrates have been reported, but as far as the inventors know, there is no report that the substrates are used to directly measure blood samples mixed with proteases. Further, Since this substrate can also be cleaved by other proteases such as thrombin, technical difficulties cannot be eliminated in synthesizing a substrate for measuring tryptase activity that can be directly measured in blood samples mixed with proteases

Method used

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  • Substrate for measuring tryptase activity
  • Substrate for measuring tryptase activity
  • Substrate for measuring tryptase activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] 1. Screening of Tryptase Substrates

[0071] A total of 17 MCA substrates were chemically synthesized by using the reported literature information on the amino acid sequence of the tripeptide-MCA substrate and the simple library method used for tryptase. For the commercially available tryptase isolated from human lung, The best sensitivity and physical properties were found i Boc-Ala-Ala-Arg-MCA. proceed with the following steps i Synthesis of Boc-Ala-Ala-Arg-MCA_Pmcs.

[0072] (1) Under ice-cooling, N,N'-dicyclohexylcarbodiimide ( DCC) (450 mg, 2.2 mmol), stirred for 1 hour. AMC (manufactured by Tokyo Chemical Industry Co., Ltd., 400 mg, 2.2 mmol) was added to the formed symmetrical acid anhydride, and the reaction was carried out at room temperature overnight to obtain Fmoc-Arg(Pmc)-MCA.

[0073] (2) by conventional methods i Boc-Ala-OH (self-made, 1.89g, 10mmol) and H-Ala-OBzl_HCl (self-made, 2.12g, 10mmol) were condensed to prepare i Boc-Ala-Ala-OBzl. Furthe...

Embodiment 2

[0092] 2. Synthesis of macromolecular substrate of Suc-Ala-Ala-Arg-MCA bonded to tryptase indigestible water-soluble polymer

[0093] A substrate for measuring tryptase activity to which poly(L-lysine), which is a tryptase indigestible water-soluble polymer, was bonded was synthesized by the following method.

[0094] (1) Fmoc-Arg(Pmc)-MCA (self-made, 1.1 mmol) was dissolved in 20% piperidine / DMF (10 mL), and reacted for 90 minutes. DMF was distilled off to obtain the free amine as a white solid. It was dissolved in DMF (5 mL), added Boc-Ala-Ala-OH (self-made, 1.2 mmol), and condensed by HBTU / HOBt method. After 4 hours of reaction, Boc-Ala-Ala-Arg(Pmc)-MCA was isolated and purified by silica gel chromatography [yield 505 mg (62%)].

[0095] (2) Boc-Ala-Ala-Arg(Pmc)-MCA (440 mg) was dissolved in TFA (5 mL), and reacted for 10 minutes. TFA was rapidly distilled off and crystallized with diethyl ether.

[0096] (3) Dissolve TFA_H-Ala-Ala-Arg(Pmc)-MCA (390mg) in DMF (5mL), add...

Embodiment 3

[0113] 3. Use the substrate of the present invention to measure the activity of tryptase in serum

[0114] Using the Suc-Ala-Ala-Arg-MCA-bonded poly(L-lysine) prepared in Example 2, it was tested whether the tryptase activity in serum could be specifically measured.

[0115] (1) 20.0 mg of Suc-Ala-Ala-Arg-MCA-bonded poly(L-lysine) was dissolved in 50 mL of 50 mM Tris-HCl (pH 8.0).

[0116] (2) 2 μL of commercially available human serum (manufactured by BioWest) and 2 μL of PBS(-) (pH 7.4) (control) or 2 μL of 10 μM Nafamostat (tryptase inhibitor, manufactured by Tokyo Chemical Industry Co., Ltd. ) or 2 μL of 10 U / mL hirudin (thrombin inhibitor, manufactured by Sigma Aldrich), and after standing at room temperature for 10 minutes, each mixed solution was added to 100 μL of the solution prepared in (1) above, and the enzyme activity was measured immediately.

[0117] The results are shown in Table 3 below.

[0118] [table 3]

[0119] Comparison of human serum enzyme activitie...

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Abstract

The present invention addresses the problem of providing a method for accurately and rapidly measuring tryptase activity in a blood sample by a simple operation in order to accurately assess the disease condition of a disease in which mast cells are involved. According to the present invention, the tryptase activity in a blood sample can be directly measured by a tryptase activity measurement substrate comprising a tripeptide in which a dye marker is bonded to a C-terminal peptide selected from the group consisting of the following formulae (1) to (3): (1) Lys-Ala-Arg-X; (2) Ala-Ala-Arg-X, and (3) (3) Abu-Ala-Arg-X (In the formula, X is a dye marker whose fluorescence characteristics or color development characteristics change by cleaving a peptide bond to Arg, and Abu is 2-aminobutyric acid).

Description

technical field [0001] The present invention relates to a substrate for the determination of tryptase activity, characterized in that it comprises a specific tripeptide in which the C-terminal peptide is bonded to a dye marker, and by cleaving the C-terminal peptide bond of the aforementioned tripeptide, the fluorescent properties of the aforementioned dye marker or A change in chromogenic properties; a method for measuring tryptase activity in a blood sample using the substrate; and a kit for measuring tryptase activity in a blood sample comprising the substrate. Background technique [0002] Mast cell activation syndrome is diagnosed in allergic diseases and tumors, retinopathy of prematurity and other diseases in which mast cells are involved in the disease state. Among various mediators released by mast cells, tryptase, which is a proteolytic enzyme, is used in disease state evaluation due to its specificity. Currently, its quantification is performed worldwide using an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K5/083C07K5/09C07K5/02C09K11/06C12Q1/37G01N21/64
CPCC12Q1/37G01N33/58G01N33/582G01N33/583G01N2333/96433C07K5/0806C07K5/0808C07K5/0815
Inventor 松田浩珍西野宪和
Owner NAT UNIV CORP TOKYO UNIV OF AGRI & TECH
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