DD153242A1 - METHOD FOR DETERMINING THE PROTEASE ACTIVITY IN BIOLOGICAL MATERIALS - Google Patents

Abstract

The method according to the invention is used in medical and biological laboratories. The aim of the invention is to provide an improved method for determining the protease activity in biological materials. The object of the invention is to provide novel chromogenic enzyme substrates in which the chromophore residue is easily extractable with organic solvents and in which the visual observation of the protease effect is better enabled. According to the invention chromogenic enzyme substrates are used, which are constructed according to the general formula. In the formula, R & ind2! an alpha-amino acid or peptidyl residue, which is cleaved from the rest of the substrate portion when exposed to a transferase or protease specific for that residue, optionally with the addition of a suitable acceptor such as glycylglycine. R & ind1! stands for H, CH & ind3! Ethyl, n-propyl, n-butyl or such a cycloalkyl group that the amino nitrogen becomes part of a piperidine or pyrrolidine ring.

Description

Method for determining protease activity in biological materials.

Field of application of the invention

The invention relates to a method for the determination of protease activity in biological materials, as obtained for example in laboratory diagnostics. The determination of protease and transferase activity in human serum, urine and tissues may have diagnostic significance, such as, for example, the activity of serum glutamyltranspeptidase in the presence of congestive itch and liver carcinoma is particularly high.

Characteristic of the known technical solutions.

Easy-to-use and sufficiently sensitive methods are very useful for determining enzyme concentrations in biological materials for diagnostic purposes. Protease and transferase activities are frequently studied by laboratory diagnostics with amino acid and peptidyl-4-nitroanilides as chromogenic substrates. ; ,

The 4-nitroaniline released in the course of the hydrolytic reaction can be determined spectrophotometrically in its concentration at 4o5 nm. Due to the location of the UV-VIS absorption bands of 4-nitroaniline, a visual preselection for below a threshold enzyme concentrations can not be found in the often cloudy, colored sample material. Sensitivity enhancement or more suitable absorption bands can be achieved with the 4-nitroanilides only by complicated chemical operations, for example by azo couplings. "In most other colorimetric methods, one of the residues primarily eliminated by the enzyme is removed by means of a subsequent chemical operation converts a product having a suitable UV-VIS absorption band (3).

Fluorogenic substrates containing a fluorescent moiety resulting from the enzymatic reaction can only be used for laboratory diagnostic purposes if there are devices for the spectral filtering of excitation and emission radiation * (4)

Object of the invention

The aim of the invention is an improved method for the determination of the protease activity which becomes highly sensitive in colored, strongly turbid samples, which are often found among biological materials.

Explanation of the invention of the invention. ·

It is an object of the invention to provide novel chromogenic anilide-type enzyme substrates with an aminoacyl substituent specific for the transferase or protease activity to be determined on the anilide nitrogen.

According to the invention, specific enzyme substrates of the general formula

NHR.

used.

In the formula, Rp denotes an o-amino acid or peptidyl radical which is split off from the remaining substrate part when it is exposed to a transferase or protease specific for this radical, optionally adding a suitable acceptor, such as glycylglycine. R ₁ is H, CH-. Ethyl, n-propyl, n-butyl or such a cycloalkyl group that the amino nitrogen becomes part of a piperidine or pyrrolidine ring.

The action of the proteases and transferases contained in the biological materials under such external conditions as are commonly used for the determination of these enzymes releases a substituted aniline whose

spectral characteristic is long-wavy compared to the chromogenic substrate shifted. The course of the substrate cleavage can be followed by the usual spectrophotometric methods at 45o - 55o nm, preferably at 49o nm, but also due to the particularly long-wave UV-VIS absorption bands of the substituted aniline, with suitable substituents R ₁ and R ~, as a threshold visually capture.

An important advantage of the chromogenic subclass

 "ir

strate consists of the fact that, because of its molecular structure and unlike the substrate, the '' cleaved chorophoric radical '' can be extracted very well at pH values of greater than 5'with organic solvents which can form multiphase systems with aqueous media in specific concentration ranges (US Pat. for example, halogenated hydrocarbons, esters, ethers, higher alcohols). Depending on the ratio of organic phase, aqueous phase, this process can be associated with an enrichment of the enzymatic cleavage product. At the same time, the separation of turbidity and self-absorption of the biological material disturbing the spectrophotometric determinations is also possible.

The ^ -amino acid or peptidyl radicals Rp of er. Substrates according to the invention contain the sequences listed in Table 1, wherein the transferases or hydrolases or their isoenzymes specific for these residues are also listed.

  - 5 _

The rest R _? is linked to the aryl amine with the C-terminus

Table 1 . ·

R. enzyme

Pro-Arg, His-Ser. - DAP I

Lys-Ala. ·. DAP II

Arg-Arg-DAP III

As-Pro DAP IY

-Cys-Cys (dianilide) oxytocinase

i- Glutarayl % - Glut amyl trän s-

peptidase

GIy-Ala-AI elastase

Leu Leueinaminopeptidase

Glutaryl-leu-Phe-chymotrypsin

In Table 1, the following common abbreviations were used: Ala (alanine), -Arg (arginine), Cys (cysteine), His (histidine), lys (lysine), Leu (leucine), Pro (proline), Phe (phenylalanine ), DAP (dipeptidyl peptidase).

Some of the substrates listed in Table are salts

(for example Hydrochloric!, tosylate).

Each of these substrates when cleaved under conditions which are generally typical of the enzyme is cleaved leaving the aniline with Rp = H and exhibiting its typical visible light absorption properties. For R ₂ = H and R '. = CH ₃ in H ₂ O (pH 7), an absorption maximum for this aniline of 468 nm, 1,2-dichloroethane of 492 nm, whereby a red-violet color is due.

The substrates themselves (R ₁ = CH-, R ₂ = aminoacyl) absorb, relatively independent of the type of Aminoacylrestes (A. _max approximately 41 nm).

The invention will be explained in more detail below with reference to six exemplary embodiments.

Example 1 ' .

Dipeptidyl peptidase IY can be determined using GIy-Pro-2-nitro-4-dimethylamino-anilide.2 HCl as a substrate in tissue homogenates. '

The "Reagenzlösuagen containing 2 mM substrate 1oo mM Tricine buffer pH 7.5 at 37 ⁰ C 1oo mg to 1 g of the homogenate is added to a total volume of 1o ml. To this solution, depending on the tissue type and a certain time," reacted together Then, by means Acetate buffer stopped at pH 5 and 2 ml of 1,2-dichloroethane was added After shaking, the substituted aniline is 98 % in the organic phase and can be determined spectrophotometrically at 492 nm ( ί = .3130).

BeisjDiel_2

The substrate used in Example Ί Gly-Pro-2-nitro-4-dimethylamino-anilide-HCl can be prepared by dissolving BOC-Gly-Pro-OH (o, o1 mol) in 15 ml of THP and with o, o1 moles of N-methyl-morpholine added. Add to this - 15 ⁰ C

Isobutyl chloroformate (o, p1.Mol) and allowed to stand at this temperature for eight minutes. "Subsequently ¹ is reacted with 2-l-titrated-4-dimethylaminoaniline (0.9 mol) in 30 ml of THP and worked after two hours of stirring The solvent is removed in vacuo, taken up in ethyl acetate, and the ester phase is washed with 5% citric acid, 5% strength ITaHCO 3, solution and with water. The BOC group is split off by means of HCl / glacial acetic acid (2 m).

The peptide dihydrochloride is chromatographically uniform (n-butanol / glacial acetic acid / HgO and n-butanol / glacial acetic acid / -sigester / H ₂₀ ). The H-UMR spectrum showed the expected signals. (DMSO-dg) '.

Example 3

Dipeptidyl aminopeptidase IV can be determined using GIy-Pro-2-nitro-4-dimethylaminoanilide ~ 2HCl as a substrate in body fluids (serum, plasma, urine, seminal fluid) by a kinetic method. ·

Reagents Amount i_.Ansatz Final Concentration i.Tes

Tricine buffer pH = 7.4 500 pl 175.4 mmol

Substrate. 50 μl 1.0 mmol

Sample. 20} il

(Dilute sperm with physiological ITaCl 1 + 9) The reaction can be started with the sample.

Measuring temperature, for example 37 ° C. The released amine was determined at 492 nm. By multiplying the extinction per

Minute with the factor 1-.O6.'1O ~ ² (for sperm applies factor 0.106) one obtains the enzymatic ^ activity of the sample in U / L.

Example 4-. ,

Dipeptidyl aminopeptidase IV can be determined discontinuously using Gly-Pro-2-nitro-4-dimethylaminoanilide-2HCl as a substrate in body fluids. . '

Reagents ^ ^ _-

Tricine buffer pH = T _s 3

substratum

IT acetate buffer pH 5 * 5

dichloroethane

0.2 mmol of saturated solution

Pipette into reaction vessels

Tricine-buffer

substratum

sample

physiological HaCl

Close the vessels at 37 ° C

sample

400 μl 100 μl • 20 μl

blank

400 μΐ 100 μl

20 pl

Incubate for one hour

Place sat z

Sample blank

Na-acetate buffer '100 ul 100 pi dichloroethane. * 1000 μl 1000 nl

Seal the vessels and shake for 5 minutes, then

'Sf-''.' *

centrifuge briefly for phase separation.

Measure the absorbance of the organic phase of the sample against the organic phase of the blank.

Calculation of enzymatic activity:

( ^E sample ~ ^E blank) χ 266.2 = U / L

Sample dilution from 80 U / L (= 10% sales)

Example 5

Cystinaminopeptidase (serum oxytocinase EC 3.4.1.2.) Can be determined using the substrate S-benzyl-L-cysteine-2-nitro-4-dimethylaninoanilide-2-HCl in body fluids.

Sample materialί Serum

Reagents: "

Substrate staining solution = 3 »3 ml & ol in DIvU ¹ Tris buffer« 100 mM pH = 7.4

Probenvprbereitung:

Working solution: Mix buffer and substrate stock solution 6: 1

-10-

Be.stimmungsansatz:

Wavelength 492 nm, temperature 37 ° C in the reaction vessel pipette

Sample blank

Working solution 1000 μΐ 1000 μΐ

Serum 50 μΐ

physiological KaCl solution - 50 μΐ

Extinction difference per sample for samples and blank value. '.

( ^S sample " ^B blank") χ 7.77. 1θ " ³ = U / I

example

Determination of dipeptidyl-indenopeptidase IV can be determined using GIy-Pro-2-nitro-4-dimethylaminoanilide-2-HCl as a substrate by a visual threshold method.

Reagents: Stock solution:

Tricine buffer pH = 7.3 0 _$ 2 m

Substrate 10.0 mmol

Ua-acetate buffer pH = 5.5. · Saturated solution Dichloräthän

~ 11

is ¹

'- 11 -

Probenyor preparation ·

Pipette into a reaction vessel.

! Sample comparative value

Tricine buffer 400 μΐ. 400 μΐ substrate 100 'μΐ 100 μΐ

Sample * 20 μΐ. '-

Standard sample - 20 μΐ

Close the tubes, incubate at 37 ° C.

Be ^ mmmmi.g s ansatz '. ,

Comparison sv / ert

Na acetate buffer 100 μΐ 100 μΐ

Dichloroethane 20 μΐ 20 nl

Close the vessel and shake for 5 minutes, then centrifuge briefly for phase separation.

The color of the organic phase is assessed in comparison to with ge guide te 11 standard.

Claims (1)

1JL - ¹ B- Invention Claims -1. Method for determining the protease activity in biological materials, characterized in that specific enzyme substrates of the general formula be used wherein R ₂ means a% i -Aminosäure- relational • as peptidyl ₅ can be cleaved from the rest of the substrate member when it is exposed to a specific for this radical transferase or protease, optionally with a suitable acceptor is added as glycylglycine , and R _? , is H, CEU, ethyl, n-propyl, η-butyl or such a cycloalkyl radical that the amino nitrogen becomes part of a piperidine or pyrrolidine ring which reacts with the proteases and / or transferases contained in the biological materials, wherein the released substituted aniline is spectrophotometrically at 45o - 55o nra be true, is. 1.1. A method for determining the protease activity in biological materials according to item 1, characterized in that the formed substituted aniline at pH values greater than 5 extracted with such organic solvents +4 which form with Y / ass multiphase systems, wherein the extracted aniline is determined visually as a threshold or spectrophotometrically at 450-550 run.