SU515471A3 - Method for quantitative determination of hydrolytic enzymes - Google Patents

Description

(54) METHOD OF QUANTITATIVE DETERMINATION OF HYDROLYTIC ACTION ENZYMES

concentration of SHsmvia in homogeneous media (such as serum or tissue extracts

To methoxypated compounds pre; respectfully add buffer, provide-. Maintaining an optimal or close to its pH value for a given enzyme. If the compound is solubilized, it is also possible to add propylene glycol. This mixture is mixed with a homogenate containing the enzyme to be determined, after which the enzyme is provided to provide hydro-. roliz connection.

After hydrolysis is carried out for a certain period of time, a denaturing compound, such as hydrochloric or trichloroacetic acid, is added to stop the hydrolysis. After removing any type of precipitate that may form, add a means capable of forming a dye with any 4-methoxy 2 aphthylamine, which is available due to enzymatic hydrolysis of the substrate.

Typical agents for dye formation are durable blue (tetra ™ zoated dlL anisidine), durable garnet (diazotized 4 Ogidazo o. Toluidine), durable blue (diazotized 4.-lmins 2,5-diethoxybenzinilide) and hexotonic pararosaniline.

YlociK dye formation is determined by the dye concentration: photometrically by passing a light beam of a given intensity through the mixture holding the dye, after which the intensity of the light beam passing through the mixture is measured. This enzyme concentration is determined by comparing with a standard ™ curve obtained by making a series of similar determinations with known enzyme concentrations.

Example: The mixture to be incubated is prepared by draining 1.5 ml of the substrate — buffer of one of the types listed.

below, O, 3 ml of the preparation of tissue or sterotka and 1.2 ml of 0.85 n. saline solution in test tubes.

The tubes are capped and incubated for 2 hours at 37 ° C in a water bath,

After incubation, remove the caps and stop the reaction with the enzyme by adding 0.5 ml of trichloroacetic acid solution, and the resulting precipitate is removed by centrifugation,

1.0 ml of an aqueous solution was introduced into each tube, and contained in 1 ml of 1 mg Crastel durable blue.

Then the tubes are subjected to vibration for 15-30 seconds,

then, after 5 minutes, spectrophotometric determination was performed at 520 mmk relative to the empty sample. The amount of 4-naphothyme released and 2-naphthylamine is counted using a previously prepared standard curve, graded with 4-A1toxy ™ 2 naphthylamine in the presence of formulations containing serum or tissues,

In the above process, the following substrate buffers can be used.

To determine the total activity of aminopentidase: leucyl-41 1-tox-2, 2-naphthylamide, 32.3 mg of the above compound are added to 50 mp of water and mixed with the same volume of O, 2 M phosphate buffer, pH 7, O, to form a substrate buffer solution.

Lysyl lanyl1 ... 4-methoxy-2- is used to determine the activity of dipept & dilimichopetidase (activity level P) found in lyso1; ah cells of imitation gland. naphthalamide, 43.4, mg of the above compound is added to 50 ml of water and mixed with an equal volume of O, 2 M sodium cacodylate buffer, pH 5.5, containing Ou. 5 ml of mercaptoethylamine hydrochloride salt in the form of a 1, O M solution, to form a substrate buffer solution,

To determine the activity of dipeptidyl-aminoputidase (activity level 1 cathepsin C), propylargynyl.4-methoxy-2-naphthylamide, 52, O mg of the above compound is added to 50 ml of water and -mixed with an equal volume of O, 2 M sodium coxidine. buffer, pH 5.5, containing 0.5 ml of a 0.1 M solution of mercaptoethylamine hydrochloride, to form a substrate-buffer solution.

To determine the activity of dipeptidyl-aminopentidase (activity level 1U), glycylpropi-I-4 is used. A-etoxy-2-y ™ tilamide. 43.0 mg of the above compound is added to 50 ml of water and mixed with an equal volume of 0.1 M Tris-HCl buffer, pH 7,5, to obtain a substrate-buffer solution (Tris 2-amine-2-hydroxymethyl 1,3-propandiol, supplied by a foreign firm, Eastman Chemicals).

To determine the active dipeptidyl-aminopeptidase (activity level III), arginylaryl-.4-methoxy-2i ac. thalamide, 59.5 g of the said compound

is added to 5 O ml of water and mixed with an equal volume of Tris-HCl

Claims (1)

buffer in the form of 0, 2 M solution for obrado ,. Vani substrate “-buffer solution. For the determination of trypsin and trypsin-like enzymes, such as cathepsin B and the trypsin-like enzyme found in the substance of the spromatose heads that contain 6 sperm, carbobenzox digly is used; -1 "Arginyl 4" methoxy 2- naphthylamide. To determine the concentration of cathepsin B, 50 ml of water is added with such a copolymer of the above-mentioned amide to obtain a 2.0 M solution, and mixed with an equal volume of 2, O M solution of sodium and dilute buffer, pH 4, 8, containing 2-mercaptzethanol , to obtain substrate-buffer solution. Due to the change in the pH value of this solution to 6, 7, it can be used to determine the concentration of an enzyme like trypsin found in the secretion of cell grains. To determine the trypsin concentration, the amount of amide mentioned is added to 50 ml of water in order to obtain a 2, O mM solution which is mixed with an equal volume of O, 2 M tris buffer at pH 7 to charge the substrate buffer solution. To facilitate the dissolution of the amide, propylene glycol can be added to 50 ml of water. - Formula of the invention. A method for the quantitative determination of enzymes of hydrolytic action. by analyzing the sample of the substrate-buffer solution, incubating the mixture with the subsequent introduction into the resulting solution of the dye-forming agent, photometric and determining the enzyme according to a calibration schedule, characterized in that, to improve the accuracy of determination, enzymes are used as substrates for the hydrolytic action of enzymes -amino acid derivatives of 4-l-etoxy-.2-naphthyamide with the general formula O I O where R contains, at least one amino acid group.