CS270355B1

CS270355B1 - Method of proteolytic enzymes' activity determination and means for determination of this activity

Info

Publication number: CS270355B1
Application number: CS877522A
Authority: CS
Inventors: Miroslav Ing Csc Rybak; Evzen Ing Csc Kasafirek; Eva Rndr Hrboticka
Original assignee: Rybak Miroslav; Kasafirek Evzen; Hrboticka Eva
Priority date: 1987-10-19
Filing date: 1987-10-19
Publication date: 1990-06-13
Also published as: CS752287A1

Abstract

gešenl sa týká způsobu stanoveni aktivity protoolytických enzymů v čistých a technických preparátech, v biologickém aatoriálu, například v tělních tekutinách, zejména v krvi nabo moči. Podstata řeěení spočívá v tom, že aa zkoumaný vzorek a obsahem proteolytického enzyau uvádí va styk s peptidickým deriváte· p-fsnylsndiaminu obacného vzorca I ve vodném prostředí o hodnotou pH 5' až 10. štěpná reakce so zastaví úpravou pH na hodnotu 0,1 až 4 za přídavku sloučeniny obecného vzorce W-CHO. Po proběhlé p raakci so vzniklé zbarveni analyticky vyhodnotí.gešenl refers to the method of determining activity protoolytic enzymes in pure and technical preparations, in biological aatorial, for example in body fluids, especially in blood or urine. Essence The solution lies in the fact that aa examined sample and proteolytic content the enzyme presents you with the peptide derivative · p-phenylenediamine of Formula I in an aqueous medium having a pH of 5 'to 10. The cleavage reaction is stopped by pH adjustment to a value of 0.1 to 4 with the addition of the compound of formula W-CHO. After passing The resulting color was analyzed analytically evaluates.

Description

CS 270 355 B1 1

The invention relates to a method for determining the activity of proteolytic enzymes in pure and technical preparations, in biological material, eg, in body fluids, especially in blood or urine; The invention also relates to a composition for determining the activity of proteolytic enzymes.

Oak is known to have a rapid and simple demonstration of the activity of proteolytic enzymes, particularly in biological material, of importance in both human and veterinary medicine, especially as an indicator of the biological equilibrium, for longer in biotechnological processes (eg, in the production of bacterial proteinases), in process control using proteinases, in agriculture, in the food industry and in other sectors. More recently, synthetic chromogenic substrates based on peptide derivatives of p-nitroaniline (or other chromophore in the substrate molecule) have been significantly employed in the field of proteolytic enzyme detection. The applicability of these substrates is based on the fact that the proteolytic enzymes in a suitably arranged reaction system are liberated by p-nitroaniline, which characterizes the color of this color, with respect to its intensity, can be used analytically in at least the demonstration of the proteolytic enzyme in the investigated material, under defined conditions and with appropriate instrumentation, then quantitatively determine the activity of the enzyme.

The coloration, which upon cleavage with proteolytic enzymes, provides a chromogenic substrate based on the peptide derivatives of p-nitroaniline, is yellow in nature. In cases where the reaction system itself is yellow in color or the activity of the enzyme is low, the substrate of p-nitroanilides cannot be used at all to detect proteinase.

Synthetic chromogenic substrates have therefore been sought which would not have the disadvantages mentioned, and which would in turn be advantageous for the analysis of less pure samples and, moreover, allow for fast and simple (indicative, ecological) demonstration under reduced conditions, even to less skilled personnel. . Newly synthesized peptidic derivatives of β-phenylenediamine, in which peptide residues are specifically incorporated by reacting crude proteolytic enzymes or a group of these enzymes, provide, under the conditions described in detail below, intense coloration, predominantly red, which facilitates the detection and determination of proteolytic enzymes.

BACKGROUND OF THE INVENTION The present invention relates to a method for determining the activity of proteolytic enzymes in pure and technical preparations, in biological material, for example in body fluids, in particular blood or urine, according to the invention. the p-phenylenediamine derivative of the formula 1 A - B - C - O - CONH - - N CH - W (X), wherein A is hydrogen, pyroglutamic acid, carboxyalkancarbonyl having 4 to 6 carbon atoms, B glycine residue , alanine, proline or zero, C residue glycine, alanine, proline or zero, or histidine, o residue of alanine, valine, leucine, phenylalanine, tyrosine, lysine or arginine, w six-membered aromatic Core or five membered heterocyclic ring containing oxygen or nitrogen As a heteroatom, which nuclei are substituted by at least one nitro, dimethylamino, hydroxy or methoxy or combinations thereof, in an aqueous medium having a pH of 5 to 10, the cleavage reaction in progress is stopped by adjusting the pH to less than 4, with the prior or subsequent addition of

Formula XXI w

CHO (III) 2 CS 270 355 B1 where W knows the same as in formula I, then the resulting color is analyzed analytically. This evaluation can be performed colorimetrically or spectrophotometrically according to the circumstances and requirements, optionally visually by comparison with the standard or by aesthetic analysis.

The principle of the method of determination according to the invention can be schematically represented as follows:

In the first step, the hydrolysis reaction is carried out by treatment with a proteinase in an aqueous medium having a pH of 5 to 10 (depending on the nature of the enzyme) at 20 to 37 ° C. The aqueous medium may form a suitable buffer or buffer system. Addition of substances that suitably affect the assay (surfactants, polymers) is also possible. In the second stage, which proceeds in the same environment and at the same temperature as the first stage and begins by lowering the pH of the reaction system to below 4, the liberated monosubstituted p-phenylenediamine derivative is reacted with an aldehyde of formula III which can be added beforehand or with or after the acidifying agent. This reactive disubstituted p-phenylenediamine derivative forms! intense color, predominantly red. This color is long enough to be analytically evaluated by any of the known methods, as discussed above. Intensitaining is proportional to enzyme activity in the analyzed material. Grade 1 specificity and sensitivity (hydrolysis) is determined by the suitably selected amino acid sequence of the substrate (ABCD-) with respect to the enzyme being analyzed. Also, the chromophore residue affects the cleavability of the substrate by the enzyme and, in some cases, is higher in some cases, lower in some cases relative to the corresponding p-nitroanilide. The second stage of the reaction is highly specific and sensitive, so that very small amounts of enzyme can be detected in the sample to be tested. In the second stage, the aldehyde of formula III which serves to form a colored product does not need to be aldehyde whose moiety is part of the parent molecule. the peptide derivative of p-phenylenediamine of general formula (I), that is, the meaning of the substituents may be different. Which alternative is chosen, depends! the user. <Al <has already been said. It is possible to carry out the method of the invention under considerably variable conditions, from simply forming a colored product on a paper foil, a microtiter plate, a tube, and the like, to exact results obtained by instrumentation. However, particularly preferred is the method for which practical reasons for simplicity and speed give priority in certain cases, J. the use of indicator means comprising, according to the nature of the proteolytic enzyme of interest, any of the compounds of formula I. Indication means known and used, their shape, the material of which they are made, and their spatial arrangement are chosen as desired. Especially preferred are such indicator means which are inexpensive, readily available and well adaptable to the widest possible range of the present invention, made of a suitable capillary material, which must be inert to both the general type I and the intermediate. - drugs, test samples containing proteinases, reagents, and the environment in which they are assayed.

Thus, a further feature of the present invention is a means for determining the activity of proteolytic enzymes which consists of an inert organic CS 270 355 B1 3 or an inorganic material having capillary properties comprising a peptide derivative of p-phenylenediamine of formula I as a chromogenic substrate, an agent maintaining the pH of 5-10, an agent lowering the pH of the system below 4 and a compound of formula III in an amount of at least one equivalent of the compound of formula I. Usually, in the form of absorbent material, such as paper, with a suitable substrate and sensitive product detection, highly sensitive detection of proteolytic enzymes, as well as multi-components simultaneously (and already enzymatic or non-enzymatic), allows the use of multiple enzymes located in the biological material leukocytes) to one site. The major advantage of this is that, in order to evaluate the results of the analysis, a device device is required, in particular, it is valuable where rapid screening is involved. The product resulting from the reaction illustrated in Schemes 1) and 2) is so intensely colored, that it is enough to compare with the chromatic standard to evaluate.

The process of the invention can be varied and simplified, for example, by carrying out the last step of the synthesis of the compound of formula (I) in situ by reducing the starting peptide derivative of p-nitroaniline, preferably with chloride in hydrochloric acid and reacting with an aldehyde of formula III, which can be used in excess for the reaction to proceed after the enzyme hydrolysis of the substrate. Reagent films for the determination of proteinase esterases released from leukocytes, used to detect lysucocytes in biological material based on the esterolytic properties of proteinases, are known, manufactured and used in practice. For example, some commercially available films contain peptide-aryl esters when applied, the cleaved phenol or pyrrole is coupled with the diazo salt to a more or less colored compound. Oal is a known method for the detection of trypsin by collagen with bound dye or chymotrypsin (Certificate No. 217 802). The other method (U.S. Pat. No. 4,156,916) is based on the incorporation of aromatic aldehydes (e.g., benzaldehyde and substitution derivatives thereof) with aromatic amines such as 2-naphthylamine to form a fluorometrically detectable compound.

Compared to the known methods, the process according to the invention is more specific and more sensitive, with the use of a simple, rapid, reliable indicator device, with very wide application areas, including many fields (health, agriculture, food industry). The usefulness and convenience of demonstrating proteinases in biological materials follow from the following examples: The presence of leukocytes in urine indicates a pathological progression in urogenital tract. The site of the laborious and inaccurate determination of leukocyte counts by a microscope (does not include disintegrated leukocytes) provides the method of the invention with rapid information on their number (3 to 5 leukocytes in 1 µl). The presence of leukocyte proteinases in the lung lavage fluid indicates a pathological process in the lungs. The presence of leukocyte enzymes in milk, or the presence of alpha -antitrypsin, indicate an inflammatory process (mastitis) in the milk-producing organs of both cattle and humans.

The process of the present invention allows for rapid control of biotechnological processes in which proteinases are produced for industrial purposes (e.g., proteinases produced in Bacilus subtilis). The method also allows for a rapid demonstration of impaired blood coagulation, fibrinolytic and kinin-producing balance in the blood, allowing urinary kinase to be detected and many others. More details of the method according to the invention are given in the following examples, which are merely illustrative but not limiting. Example 1 a) Preparation of trypsin substrate. 100 ml of a solution (0.01 mol / l) of pGlu-His-Arg-p-nitroanilide in water are mixed with 100 ml of 4 CS 270 355 B1 0.1 mol / l SnCl2 dissolved in 1 mol / L HCl and left to mix react about 1 hp at room temperature. Thereafter, 20 ml of 0.1 mol / l p-dimethylaminobenzaldehyde dissolved in methanol was added to the reaction mixture and stirred for 1-2 hours. The Jepak reaction mixture was adjusted to pH 8.2 to precipitate Sn2 + and Sn4 + as its own. pGlu-His-Arg-CONH-CgHg-N «> CH-CgH5-N (CH3) 2 - trypsin substrate and unreacted aldehyde added in excess are present in the solution. Preparation of the Reagent Film and Used to Determine Trypsin The Substrate Solution Obtained is Directly Used to Saturate the Reagent Strip The reagent strip can be paper free of reactive groups, or organic synthetic tissue (poured, cleaved), or inorganic intact material used for chromathographic The strip is saturated with a suitable buffer (Tris pH 8, 2 containing 0.025 mol / l CaCl 2. Optionally, 0.05 mol / 1β-dlmethylaminobenzaldehyde in 50% methanol is added to the reagent strip and the film is dried) a. Trypsin Contact with the substrate on the film and after hydrolysis (1 to 5 min) the color is induced by acidification (HCl, citric acid) to form IV. By addition of acid, the hydrolytic reaction is simultaneously stopped, allowing relatively accurate discoloration, and tlm activity. Color 3e subtracts after 30 to 60 sec. c) For the preparation of the reagent strip, the pure substance (substrate - which is prepared from 0.01 to 0.005 mol / l solution. The reagent film in which the buffer was previously placed is saturated. It is saturated with the solution of the substance I - the substrate. by adding a reagent containing 0.05 mol / l of p-dimethylaminobenzaldehyde (causing red color) or p-dimethylaminocinamaldehyde (causing blue coloring) in 1 mol / l cyan hydrochloric acid.

Microplate plates can also be used in place of the reagent strip, and the resulting reaction product can be dissolved by the addition of an organic solvent, preferably methanol, and evaluated by measuring the absorbance at 405 nm with a suitable instrument. Example 2

The procedure is as in Example 1a. The prepared pGlu-His-Arg-p-phenylenediamine p-dimethylaminobenzaldehyde substrate is used for the spectrophotometric determination of trypsin activity. The hydrolyzed monosubstituted p-phenylenedlamine derivative absorbs at a maximum of 405-420 nm. Example 3

Proceed as in Example 1 when Glt-Ala-Ala-Pro-Val-p-nitroanilide is used as the starting material for preparing the leukocyte elastase substrate. The first condensation is carried out with p-dimethylaminobenzaldehyde and, after hydrolysis, the product is condensed with p-dimethylaminocinamaldehyde on a blue colored fabric. The pH of the hydrolysis is 7.2 to 7.4 maintained by phosphate buffer. Example 4

It repetits as in Example 1, leaving as the Outcrop! Thrombin Substrate Preparation Z-Gly-Pro-Arg-p-nitroanilide (0.1 mol / l pH 8.2 phosphate buffer) and the first p-dimethylaminobenzaldehyde condensation agent are used. After hydrolysis, the product is condensed with p-dimethylaminocinamaldehyde to a blue product. The substrate serves to determine some coagulation parameters (thrombin generation test) and together with other reagents (thromboplastin, cephalin, Ca 2+) to demonstrate coagulation factors VII and II.

Claims

Example 270 Proceeds as in Example 1 when a p-nitroanilide amino acid sequence specific for coagulation factor X, such as Bz-Ile-Glu-Arg-, is used as the starting material. Factor X Demonstration Performed After Its Activation by Jede Viper.Russel, Kefalin and Ca2 +. Substrate Prepared with p-dimethylaminobenzaldehyde and detection after hydrolysis of Jep by one of p-dimethylaminocinamaldehyde. EXAMPLE 6 Proceed as in Example 1 when H-D-Pro-Phe-Arg-p-nitroanilide is used as the starting material, hydrolysis is carried out by plasmatic quallikrein at pH 8.2 (0.1 mol / l phosphate). Condensation after hydrolysis is carried out with "p-diethylaminobenzaldehyde", while the preparation of the substrate with p-dimethylaminobenzaldehyde. The substrate allows the determination of precallikrein in human blood plasma upon activation by a suitable activator (e.g., Hageman Factor fragment, dextran sulfate and the like). Example 7 The procedure of Example 1 was followed, using Suc-GiyGlyPhe-p-nitroanilide as the starting material to obtain a chymotrypsin substrate. The first condensation is carried out using p-dimethylaminobenzaldehyde, the second condensation is then followed by p-dimethylaminocinamaldehyde. Example 8 10 µl of 0.005 mol / l Glt-Ala-AlaLeu-p-nitroanilide is contacted with 10 µl of a solution of neutral proteinase from Bacillua subatilis at pH 7.4 (phosphate 0.1 mol / l) on the reagent strip. ) for 2 to 5 min. Hydrolysis of the liberated p-nitroaniline (yellow) is converted to the blue product by the addition of a reagent composed of 0.05 mol / L chloride chloride and 0.01 mol / l of p-dimethylaminocinamide in 1N HCl. The resulting color is evaluated as in the previous examples. OF THE INVENTION

What is claimed is: 1. A method for determining anthoolytic enzymes in preparations, in biological material, e.g., blood and urine, characterized in that the test solution containing the proteolytic enzyme is contacted with a p-phenylenediamine derivative of the formula (I) ABCD-CONH. -N-CHW (I) wherein A is hydrogen, pyroglutamic acid, carboxyalkancarbonyl of 4-6 carbon atoms, glycine residue, alanine, proline or zero, C glycine residue, alanine, proline or zero, or histidine, □ alanine residue, valine, leucine, phenylalanine, tyrosine, lysine or arginine W six-membered aromatic ring or a five-membered oxygen or nitrogen-containing heterocyclic ring heteroatom substituted by at least one nitro, dimethylamino, hydroxy or methoxy group in an aqueous medium with a pH of 5 to 10, the ongoing hydrolysis reactions are stopped by adjusting the pH to 0.1 to 4 with the addition of a compound of formula III 6 CS 270 355 B1 W-CHO (III). where W knows the same as in formula I, then the resulting color is analyzed analytically.

2. A composition for the activity of proteolytic enzymes according to claim 1, characterized in that it consists of an inert material with capillary properties, e.g. the peptidyl derivative of the p-phenylene diamine of the formula I as a chromogenic substrate, a buffer maintaining the pH of the system at 5-10, for example tri9, a phosphate buffer, a pH lowering agent of 0.1 to 4, for example hydrochloric acid, citric acid and a compound of formula III in an amount of at least one equivalent based on the compound of formula I.