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Novel chromogenic substances and use thereof for the determination of carboxypeptidase activities

A technology of carboxypeptidase and substrate, which is applied in the field of new chromogenic substrate and its application in the determination of carboxypeptidase activity, and can solve the problems of long time and difficulty in automation of color development method

Inactive Publication Date: 2006-11-08
DIAGNOSTICA STAGO SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0030] - Or the color development method takes too long, is limited, and is not easy to automate

Method used

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  • Novel chromogenic substances and use thereof for the determination of carboxypeptidase activities
  • Novel chromogenic substances and use thereof for the determination of carboxypeptidase activities
  • Novel chromogenic substances and use thereof for the determination of carboxypeptidase activities

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0152] Synthesize a group of azoformyl compounds of the present invention

[0153] Table 1 below lists the chemical formulas of 8 preferred compounds among the compounds of formula (I) of the present invention. Their absorption spectra measured with a UV-visible spectrophotometer (UVIKON-KONTRON) are shown in FIG. 1 .

[0154] The synthesis of one of the compounds, compound 4 (MxPAFFR), is described in detail below. The procedure for the preparation of this compound described below is the same as for each of the other compounds, except for compound 5 (MxPAFFK), for which a complementary step of lysine deprotection is essential (see Reaction Scheme and Explanation).

[0155] Table 1:

[0156] Mode

[0157] Compound 1: 2,3-DMPAFFR

[0158]

[0159] 2,3-Dimethylphenylazoformylphenylalanylarginine

[0160] Compound 2: 2,4-DMPAFFR

[0161]

[0162] 2,4-Dimethylphenylazoformylphenylalanylarginine

[0163] Compound 3: 2,5-DMPAFFR

[0164]

[0165] 2,5-Dimethylphenylaz...

Embodiment 2

[0414] 1- Assay principle and main reagents used

[0415] TAFIa Enzyme Activity Assay Principle

[0416]

[0417]

[0418] 2-Example of the protocol for measuring TAFI activity according to the method of the present invention

[0419] a) The first mode of operation: the thrombin-thrombomodulin method

[0420] The test plasma sample was divided into two equal parts, one part was added with PIC (Calbiochem-Réf. 217359), and the other part was added with Hépès buffer.

[0421] The two aliquots were then treated identically according to the following principles:

[0422] TAFI activation

[0423] 150 μl of plasma diluted 1 / 20 in Hépès buffer (or 13 μg / ml of purified TAFI in Hépès buffer);

[0424] 10 µl PIC or H 2 O;

[0425] 5 minutes at room temperature;

[0426] 150 μl coagulation activator buffer;

[0427] 10 minutes at room temperature;

[0428] 100 μl PPACK + 100 μl MxPAFFR (substrate) (5 mM).

[0429] TAFI activity test

[0430] 30 minutes at room temperatur...

Embodiment 3

[0469] Establishing a Calibration Curve in the Thrombin-Thrombomodulin Method

[0470] under image 3 is a calibration curve established by spiking pure TAFI to TAFI-free plasma to obtain a TAFI concentration of 0-26 μg / ml.

[0471] The compound of formula I used was MxPAAFR at a concentration of 5 mM.

[0472] TAFIa concentration (μg / ml)

0

1.63

3.25

6.5

13

26

Measured ΔOD

0.168

0.217

0.273

0.399

0.624

0.995

[0473] Such as image 3 As shown, the calibration curve is linear over the concentration range studied. This fully covers the prescribed concentration range, ie, the concentration of TAFI in vivo of 5-15 μg / ml.

[0474] Thus, the methods of the invention can detect deficient and abnormally high levels of TAFI.

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Abstract

The present invention relates to chromogenic compounds and their use in the determination of carboxypeptidase N and carboxypeptidase U family enzymes. The present invention more particularly relates to the compound of formula (I), wherein A=(1) or (2) or (3) or (4) or (5),-R1, R2=H,-CH3,-CH(CH3 )2, -OCH3, -Cl, -CF3, -OCF3, -SCH3, R3 = amino acid group hydrolyzable by carboxypeptidase A, R4 = basic amino acid group. ∴

Description

technical field [0001] The invention relates to a chromogenic compound and its application in the determination of carboxypeptidase N and carboxypeptidase U family enzymes. More specifically, the present invention relates to the use of said compound in the determination of TAFI (thrombin-activated fibrinolysis inhibitor) activity in blood samples and the corresponding determination method. Background technique [0002] Carboxypeptidases (CPs) constitute a group of enzymes in the exopeptidase family. They are enzymes that cleave the amide bond of a polypeptide chain at the last COOH-terminal amino acid. They include serine carboxypeptidases, cysteine ​​carboxypeptidases and metallocarboxypeptidases. [0003] Many carboxypeptidases have been isolated and sequenced from bacteria, yeast and plants. These enzymes are also found in various mammalian tissues. [0004] Carboxypeptidases have been isolated from pancreas and mast cells. Other carboxypeptidases circulating in plas...

Claims

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Application Information

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IPC IPC(8): C07K5/06G01N33/68C12Q1/37C07K5/062C07K5/065
CPCC07K5/06026C07K5/06034C07K5/06043C07K5/06052C07K5/06078C07K5/06
Inventor G·昆廷
Owner DIAGNOSTICA STAGO SA