JPS62296899A - Quantitative analysis of proteinase - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention]

3. Detailed Description of the Invention The present invention relates to proteolytic oxygen, especially enzyme-reduced FX,C. 3.4.21. Regarding the method for quantitatively analyzing enzymes such as organ or gland kallikrein, thrombin and plasmin, so-called organ kallikrein or gland kallikrein can be obtained from various organs and glands, such as secretory glands, salivary glands, kidneys, gastrointestinal mucosa, etc. , secreted in the preacetic or active form
Ru. These organ or glandular kallikrein may be chemically and physiologically different from plasma kallikrein, and under certain pathological conditions the organ kallikrein secretion concentration is
Either the value decreases below the normal value or the normal value ELI also increases. That is, for example, urinary kallikrein secretion is substantially reduced, even if the normal value in patients with kidney disease is low. Kallikrein secretion is almost completely suppressed in patients with nephrosclerosis. In patients with idiopathic hypertension, 24-hour urinary kallikrein secretion is effectively reduced by >1 as an average of 50 times the normal value (e.g., Il, a Margol
iusmo W, -Chemistry and
Riology of the Kall
ikrein-Kinin System in
)Earth and Disease”,
1974, pp. 399-409]. Therefore, it is important to rapidly quantitatively analyze organ kallikrein using a simple processing method. For example, certain arginine esters, such as tosyl-arginine methyl ester (TAME
)'! It is known to analyze urine kallikrein by r-esterification. An improved ester decomposition method uses tritium-labeled tosyl-arginine methyl ester and measures the radioactivity of the methanol released by the ester decomposition.
Shira's ester degradation method has the disadvantage that the mononuclear method kakallikrein is non-physiological to the extent that proteolytic oxygen degrades the natural peptide chain to amidolysis rather than to esterolysis. Furthermore, ester substrates have the disadvantage that they are degraded by a large number of other enzymes, ie only kallikrein is degraded in an undefined manner. The method using tritium-labeled TAME is based on the radioactivity of methanol labeled with tritium.
++Before addition, methanol must be extracted from the ester decomposition mixture with a water-immiscible liquid, since residual tritium-labeled TAME still present in the ester decomposition mixture will interfere with the measurements. It is moderately complex. Published without examination 7L rLfc West Germany Patent Application Publication No. 2527932 Format: % Formula % I, -NH-R2 represents a chromophore, X is a phenylalanyl group, I-cyclohexylalanyl group, Phenylglycyl group or tyrosyl group 'fti? The substrates indicated by ) are described. The substrate is very easily degraded by plasma kallikrein, producing a colored degradation product F.
t-NH
It can be measured by the 641 element method, the spectroscopic itl+I method, or the fluorescence side method. Attempts have also been made to use the substrate to analyze organ or glandular kallikrein. However, surprisingly, the substrate is not sensitive to organ or glandular kallikrein, i.e. γ is not degraded by organ or glandular kallikrein.
It is likely that no or only a small amount is degraded.
I made it clear. No. 2,629,067, published without examination, describes tripeptide derivatives useful as substrates for the analysis of specific proteolytic oxygen species, e.g. I'm sad. That is, )(-D-parylleucyl-
Arginyl-p-nitroanilide dihydrochloride and 11-D-)chloroleucyl-lysyl-p-nitroanilide dihydrochloride are mentioned as two examples. The first tripeptide conductor is a substrate for glandular kallikrein and the second tripeptide derivative is a substrate for plasmin. These two compounds are
The enzyme decomposes ILzp-nitroaniline, in which case the amount of p-nitroaniline can be determined photometrically or spectrophotometrically. However, the sensitivity of 1-D-parylleucyl-arginyl-p-nitroanilide dihydrochloride reaches the limit necessary for accurate analysis of urinary kallikrein in unconcentrated urine. However, when the amide substrate is excreted in concentrated urine, the photometric measurement of Jp-nitroaniline is strongly inhibited by the unique color of urine. The first two amino acids in the tripeptide chain of the two tripeptide derivatives described in the above have a water-blocking isozorobyl group in the α- or β-position. In order to increase this water-blocking property and subsequently increase the overall sensitivity to kallikrein, attempts have been made to replace the isopropyl group with an aromatic group, such as a phenyl group, while maintaining the basic structure of the dipezothi single chain. However, this attempt failed. Tripeptide substrates obtained by loading aromatic groups are not degraded by kallikrein at all, or in many cases only to a very small extent. However, IL et al.'s tripeptide substrates with aromatic groups have surprisingly high sensitivity to plasmin. Moreover, it has surprisingly been found that hydrogenation of aromatic groups in the tripeptide plasmin substrates results in a new class of tripeptide substrates containing surprisingly high susceptibility to organ or glandular kallikreins. In order to increase the tripeptide chain, it was found that only naturally occurring amino acids could be used to prepare IL substrates for degradation by proteolysis. It has been surprisingly found that by using amino acids that do not occur in organ or glandular systems, it is possible to obtain chromophore substrates that easily decompose organ or glandular kallikrein and other proteolytic oxygen, such as plasma kallikrein Kj. Was that. The method of the present invention has a high susceptibility to certain proteolytic oxygens, in particular enzyme reactivates, such as organ or glandular kallikrein, plasmin and thrombin, and therefore It relates to a novel chromophore substrate useful as a substrate for quantitative analysis of enzymes. The substrate has the general formula 'II-D-X-Y-Arg-R, wherein , YU cyclohexylalanyl group or cyclohexyltyrosyl group, hp-nitrophenylamino group, 2-naphthylamino group, 4-methoxy-2-naphthylamino group, 4-methyl-coumaryl-7-amino or Ir! 1.3-(methoxycarbonyl)-phenyl-5-amino group] is a tripeptide derivative represented by 1. The tripeptide derivatives of formula I are resistant to aqueous media and therefore in the form of their salts with acids, in particular mineral acids, such as +IC4, HRr, H2SO4, H3PO4, etc., or organic acids, such as formic acid. , halogenated acetic acids such as acetic acid, zolopionic acid, trimethyl chloride, methoxyacetic acid, trichloro- or nitrifluorophosphoric acid, amine acetic acid, lactic acid, g-acid, malonic acid, citric acid, benzoic acid, substituted in the nucleus. They are preferably used in the form of salts with aromatic acids, such as toluyl, chloro- or brobenzoic acid, methoxybenzoic acid and aminobenzoic acid, phthalic acid and the like. The nature of the nucleic acid is not important as the nucleic acid does not participate in the reaction between the substrate and the enzyme. Substrates of formula l and their salts with acids may contain specific proteolytic oxygen,
In particular, enzyme reactivators g, c, 3.4.21. Enzyme I
-Enzyme Nomenclature”,
Bl 5evier Scientific P
Publishing Company (Amster)
dam) Publishing, 1973, p. 238 (see t1)
, e.g. organ kallikrein and glandular kallikrein, are hydrolyzed by the action of plasmin and thrombin, so that a colored or fluorescent decomposition product of the formula: +(-Nl2 is formed), in which case , the amount of decomposition products can be determined by photometry, spectrophotometry, and fluorescence spectroscopy.
641 can be added by the chemical method. Therefore, this new substrate has a proteolytic oxygen, especially jl'I C,3,4,21-
It is useful for the total quantitative analysis of inhibitors and proenzymes of organ or hz< kallikrein, plasmin, plasma kallikrein, thrombin or the like, as well as other factors involved in the formation or inhibition of the enzymes. be. A predominant group of tripeptide derivatives is where Y represents a cyclohexylalanyl group or a cyclohexyltyrosyl group, and X represents an alanyl group, an α-aminobutyryl group, a valyl group, a norvalyl group, a leucyl group, a norleucyl group or an isoleucyl group. Consisting of a compound of formula l. Following example 3.5.7.9.12.13.36.37.
The tripeptide derivatives described in 38.40 and 41 are particularly useful as substrates for analyzing urine kallikrein. To analyze kallikrein in human expectoration, use the following example 3.5.7.9.12.13.36.37.38.4
The tripeptide conductors described in 0 and 41 can be used. The tripeptide derivatives described in Examples 36, 37 and 41 below constitute a group of substrates that can be used to analyze plasmin. The present invention relates to a method for the quantitative analysis of organ or glandular kallikrein, plasmin and thrombin. The method according to the present invention involves reacting a substance containing the enzyme or in which the enzyme is to be completely formed or consumed with a tripeptide derivative of formula I, and in which the tripeptide derivative is formed by the hydrolytic action of the enzyme. Colored or fluorescent decomposition product R-N142!jk
7t, is characterized by being measured by photometry, spectrophotometry, fluorescence spectrophotometry or electrochemical methods. The method of the present invention can be applied to the enzyme content of the enzyme content of human body fluids and body fluids of mammals, such as floor, intestinal mucus, mammary gland secretions,
This is done by analyzing enzyme concentrations in sweat gland secretions, sputum, and blood. The method of the invention is particularly useful for the quantitative analysis of organ or glandular kallikrein and plasma kallikrein in the body fluids. Using this method, rL7t is formed from free organ kallikrein and mucosal kallikrein or prekallikrein. Kallikrein, as well as physiological or non-physiological inhibitors of kallikrein and physiological or non-physiological activators of pre-kallikrein, can be analyzed for tripeptide derivatives according to the method described below. can be made: (1) Chromophore R is the chromophore R of the C-terminal arginine. xyl group, the α-amino group being protected by a protecting group, such as a carbobenzoxy group or a tert-butoxycarbonyl group, and the δ-guanidyl group being protonated, nitrated or tosyl-based, for example with HC7. protect by The truncated, C-terminal R-Nll group serves as a holding group during the stepwise synthesis of the peptide chain. Other protecting groups can be used, if necessary, to ensure that the desired peptide chain is fully synthesized.
can be selectively removed to conjugate other amino acid derivatives. Finally, the remaining protecting group -i
, completely decomposes without It-NH-funding (e.g., A4klosfiodansky et al., "
Peptide 5ynthesis-, Inters
Published by science Publishers, 1966,
pp. 163-165). (2) First, the peptide chain f (method by Hodansky et al., in the above cited text) is synthesized. At this time, the C-terminal hydroxyl group of arginine is replaced with a commonly used ester group, such as a methoxy group, an ethoxy group. or a benzyloxy group (in the case of arginine), or a t-butoxy group (in the case of lysine). In the case of zolotonization, the ester group is removed enzymatically using trypsin, in which case no racemization occurs. The chromophore R-NH-'i is bonded to the chromophore R-NH-'i.The δ-guanidino group of arginine is protected by a nitro group or a tosyl group, and the N-terminal α-amino group of the tripeptide g4 is protected by a carbobenzoxy group or tdp-methyl group. -1p-methoxy- or Up-chlorobenzyloxycarbonyl group or t-
When protecting with butoxy groups, all protection funds are removed at the same time. This removal can be carried out by treating the protected tripeptide derivative with anhydrous HF' at room temperature, thus in this case all said protecting groups of the amine and δ-guanidino groups are removed. Sanru. This removal can also be performed using 2NH in glacial acetic acid if the protected tripeptide derivative does not contain a protected nitro or tosyl group.
It can also be carried out by treatment with Hr at room temperature. The preparation of the tripeptide derivatives used in the method of the invention is detailed in the following examples. Analysis of the bath effluent and product obtained in Example 1 is as follows:
Glass plate pressed with silica gel (Merck, F2
γ was carried out by thin single layer chromatography using 54). The a-layer chromatogram was developed using the following solvent system: A chloroform/methanol (9:4R
n-pronozonol/ethyl acetate/water (7:1:21
Cn-butanol/axic acid, 'water (3:1:1) The following abbreviations are used: A c () fl = acetic acid Ala = alanine A, rg = arginine soc = t-butoxycarbonyl Rut = α-amino butyric acid Cbo -calgebenzoxy CIA = cyclohexylalanine CHO = 7 chlorohexylglycine CHT - cyclohexylglycine:: p-hydroxycyclohexylalanine DMF = dimethylformamide r) PA = 1.3-di(methoxycardynyl) )-Phenylly5)-amide TLC = thin layer chromatography P] 13N = ) ethylamine + 1MP'l''A = phosphoric acid N, N, N', N
', N'', N''-hexamethyltriamide 4e = inroinone Leu = leucine SS = solvent-based Lys = = lysine MCA = 4-methyl-coumaryl-(7)-amide MeO
H = methanol 4-MeO-2-NA = 4-methoxy-2-naphthylamide 9 Nleu = = norleucine Nval = norvaline (JpNP = p-nitrophenoxyPhe - phenylalanine Ph' (JIy = phenylglycine Pip = pipecolic acid pNA = p-Nitroanilide Pro = Proline THF - Tetrahydrofuran Tyr = Tyrosine Vat = Valine Unless otherwise stated, the amine g in the peptide chain has the L-form. dried) Cbo-Arg-OH, HC416, Oy
(47.0 mmol) at 20°C anhydrous) LMPTA
The atmosphere in the flask was kept moisture-free. A (T and τ) at room temperature to m class: t3N Il, 74 in IIMPTA 10-
, (47,0 mmol) of vinegar solution was added, then (1
00% excess) p-nitrophenyl isocyanate h 1
6.4. Added all drops (100 mm IJ moles). 20℃
After a reaction time of 24 hours, most of the IIMPTA was removed by distillation in vacuo. 30 to this residue
Extracted several times with %A c OH. This residue was discarded. The combined acetic acid extracts were equilibrated with a total of 30% Ac0) and
0% A c OH to release m “Sephadex (5
Further K& was prepared by passing it through a column of ephadex)()-15' (registered trademark). Fractions of the A c OH eluate, which had been degraded by treatment with tridicine to liberate p-nitroaniline, were lyophilized. Thus, a homogeneous amorphous powder in SSC of 12.6 as shown by TLC, fL
friend. After elemental analysis and calculations from the empirical formula C2oI125N605Ct, the following values were obtained (values from the empirical formula are in parentheses): C = 51.29% (51,67%
l, H=5.48% (5,42%), N=17.92%
(18,08%), CL = 7.50 (7,63%)
). Compound 1a4-65y (10 mmol) was treated with 40 ml of 2 N HBr in glacial acetic acid at 20° C. for 1045 min in the absence of moisture. The amino acid conductor was bath-deposited under the generation of C02. This reaction mixture was added dropwise to 250 d of water ether under strong stirring. 1 ri of this solution, 2HBr, produced a precipitate of H-Arg-pNA. The entire ether phase was filtered with suction and the solid phase was then washed four times with 100 g of anhydrous ether in order to remove the benzyl bromide formed as a by-product (excess of 18 r) and Ac 04 %. This residue was dissolved in 50°C of methanol, the pH was adjusted to 4.5 by adding Bt3N, and the solution was dissolved in vacuo at 30°C.
Concentrate and dry. Obtained product f MeOH75m
The mixture was passed through a column of C3ephadex (C3ephadex) LH-20' (cross-linked dextran gel) equilibrated with 1% eOH. From the fraction of this eluate, a homogeneous amorphous compound]b4.18 (91.6% of the theoretical value) was obtained in SSC as shown by TLC. Element phase separation and empirical formula C
The calculation from l2H2oN603Br2 gave the following 1 shift: C=31.15% (31,60
%), H= 4.3 5% 4 4.4 2%
), N=18.84% (18,43%) and B
r = 34.81% (35,03%). lc, Cbo-CHA-Arg-pNA, ) 18°C
Compound] b 4.56, (10 mmol) was dissolved in 30 mmol of freshly distilled DMF, and this solution was dissolved in 10 mmol of freshly distilled DMF.
Add f.: r 3 N to the solution cooled to ℃ under stirring.
1.40 ml (10 mmol) was added. The formed E t 3N,I IRr was removed by filtration and washed with a small amount of cold 1)NIF. Add Cbo-CILA-OpNP 4-69 y to this P solution at -10℃.
(4 mmol) and the reaction was carried out in the absence of moisture for 2 to 4 mmol.
The reaction was continued for 3 hours, and the temperature of the in situ reaction solution gradually decreased to about 2
The temperature reached 0°C. This bath liquid was cooled again to -10°C.)
; t3N O-70 ml' 5 mmol), -
The reaction was carried out at 10°C for about 2 hours, and then at room temperature for about 3 hours. This method i) was repeated using -(t3N O, 70 mZ) and the reaction bath was concentrated to dryness in vacuo at 50 °C for 16 h. The residue was dissolved in 75 ml of 50% AcOH and equilibrated with 50% AcUII. 1 cephaladex (5
It was purified by gel filtration using an ephadex) G-15' column. The main fraction of the AcOH eluate was concentrated to dryness in vacuo at 40 °C. This residue was mixed with MeOH1
Dissolve in 50 ml, concentrate again and dry. The resulting residue was dried over P2O5 at 60 °C in a full vacuum desiccator and produced by TLCK to give a homogeneous amorphous compound in SSC (5.85%, 88.3% of theory). I got it. After elemental analysis and calculation from the empirical formula C and H4oN706Rr, the following value was obtained: A*:C=52.28%f
52.57%l, H=6.16% (6,09%)
, N = 15.09%I 14.80%) and Rr
= 4.85% (12, f16%). Compound 1 c 5-30 y (8 mmol) was prepared in 32 rnl of 2 N IIRr in 1 m of ice at 20°C under stirring.
for 40 minutes. The dipeptide conductor gradually dissolved under the evolution of CO2. This reaction solution was added dropwise to 250° of anhydrous ether under vigorous stirring. This solution is 21(B
The ether phase resulting in a precipitate of r, tl-CIIA-Arg-pNA is filtered off with suction and the solid phase k Mli formed as fresh benzyl bromide as well as excess (D H
Anhydrous ether 10 to remove Br and AcOH
It was washed 4 times with 0- children. This residue was dissolved in MeOH50-. The pH was adjusted to 4.5 using Et3N, and the solution was concentrated to dryness in vacuo at 30°C. The obtained residue was dissolved in 50 ml of MeOH and equilibrated with MeOH.
The fractions of the MeOH eluate, which was purified on a -20 column and digested by treatment with trypsin to liberate p-nitroaniline, were concentrated to dryness in vacuo at 30°C. The resulting residue was purified with P2O in a vacuum desiccator at 40°C.
Dry at 5 and dry at 88 C.
' A homogeneous amorphous compound 1 d 4.48 y (91.9% of the theoretical value 1 was obtained. Elemental analysis and empirical formula C
21) The following values were obtained by calculating 135N704Br: C=41.80%(4]-,39ch), H=
5.86% (5,79%), N=16.31% (16,
09%) and Br = 25.85% (26,23%)
. 3.05 mmol of compound 1d (5 mmol) was freshly distilled and dissolved in 9 TAMF20-, and the solution was heated at -10°C.
It was cooled to To this solution was added 70 ml of Et3N O (5
mmol) was added under stirring. Formed E t 5
N. Remove the HBr by filtration and cold D
Washed with a small amount of MF. Add Cbo- to this P solution at -10°C.
1) -Val, 0pNP 2.0 5 y (
5.5 mmol) was added under stirring. The reaction mixture was allowed to react for 2-3 hours in the absence of moisture, with the temperature of the reaction solution gradually reaching approximately 20°C. The solution was cooled again to -10°C and diluted with Et3N O,3
5 rat (2,5 mmol) and at -10 °C.
The reaction was continued for an additional 3 hours at room temperature. This method was repeated photometrically to determine the amounts of t, N O, and 3S ra, and after 16 hours the reaction bath fL was evaporated in vacuo for 50 minutes.
It was concentrated and dried at 0°C. This residue was mixed with 50% AcO850m
It was purified by gel filtration using a column of 0-15' Sephadex, equilibrated with 50% AcOH, and treated with trypsin to release p-nitroaniline. The main fraction of the tXc04 eluate was digested by 40 min in vacuo.
It was concentrated and dried at °C. The residue was dissolved in MeOHlood and the solution was concentrated again to dryness. The resulting n9 residue was dried in a vacuum desiccator at 60°C over P2O5 and T
A homogeneous amorphous compound 1e3.15y (theoretical value 82.7y) was obtained at 88C as shown by LC. Elemental analysis and % [Formula Cs 444 q N807
1 (by calculation from r, the following value is obtained: Ai:C=52.
88% < 53.61%), H = 6.54 excellent (6,4
8%), N = 15.08% (14,71%) and Br = 10.25% (10,49%) If,
24Br, +! -D-Vat-C1(A-Arg-pN
Compound 1 e 2.29 y (3-”') mol)
was treated with stirring in a 12rnl 2N jar of iced water at 20°C for 40 minutes in the absence of moisture. The tripeptide fatty conductor gradually dissolved under decarboxylation and evolution of CO2. This reaction solution was added to 120 g of anhydrous ether under vigorous stirring. This Fj liquid has 2HHr,
A precipitate of 11-1)-Val-CHA-Arg-pNA was produced. The ether phase was thoroughly filtered with suction, and the solid phase was washed four times with 50 parts of anhydrous ether. The resulting residue was completely dissolved in 40-40% MeOH. The pn was adjusted to 4.5 using Et3N and the solution was concentrated to dryness at 30° C. in full vacuum. Meal (3
0 ml of MeOH, purified on a column of "Sephadex Li1l-20" equilibrated with Meal (7t), and decomposed by treatment with trypsin to liberate p-nitroaniline. Fractions of the eluate were concentrated to dryness in full vacuum at 30°C. For post-purification, the prepurified residue was dissolved in 33% AcOH and purified with "Sephadex" equilibrated with 33% AcOH. Purified by gel filtration on a column of 5ephadex) G-15-.The main fraction of the AcOFl bath solution was digested by treatment with trypsin to liberate p-nitroaniline in total vacuum. The resulting residue was concentrated and dried at 40°C in a desiccator at 40°C.
The amorphous compound was dried over P2O5 and shown by TLC to be homogeneous in SSC] f1.84, (
A theoretical value of 86.4φ) was obtained. Elemental analysis and empirical formula C2
The following value is obtained by calculation from 6444N805Br2: n4: c = 43.60% (44,08%), 4 = 6
．． 41chi (6.26%), N = 16.15% (
15,82%) and Br = 22,21% (22,
56%). Amino acid analysis confirmed the presence of the desired amino acid in the following exact ratio rLfc: Arg: 1.0O-C)1A: 0.96-
1) -CHG: 0.98oRr value 0.46°Example 2 Commercially available Cbo-Arg-MCA, HCtl 3.0,
(25.9 mmol) was deblocked according to Example 1b using a solution of 2 N HBr in glacial acetic acid (208 mmol). This dried residue f MeOH400WLtK
Dissolve this solution (IL-1 Cephadenx (5ep)
hadex) LH-20'' column. 4-
MeOH was decomposed by treatment with trypsin to liberate methyl-7-amino-coumarin! The eluate fractions were dried in vacuo at 30°C 4'. The resulting n9 residue was dried over P2O5 at 40 °C in a vacuum desiccator and analyzed by TLC.
A uniform amorphous compound 3 b 4.2 (87.7% of theory) was obtained in SSC as shown by 1. Element nominal and empirical formula 〇161425N503F3r2
From the calculation, the following value is obtained: nfC: C=39.4
0% (38,96%), H=4.61% (4,70%
), N = 14.48% + 14.20%) and Or =
31.90% (32,40%). Compound 2b 4.93, (10 mmol) and C
Bo-CIIA-OpNP 4-69y (4 mmol) was added to freshly distilled DMF 75-. -1
After cooling to 0 °C, Bt, N total concentration 1.40 under stirring
m (10 mmol) and then 0.70 mt (5 mmol) were added. This mixture is mixed in the absence of moisture.
] The reaction was carried out at 0°C for 3 hours, and then at room temperature for 4 hours. This reaction@liquid was cooled again to -10°C and diluted with Et3N O
, 70- and stirred overnight at 20°C. The reaction mixture was condensed to dryness in vacuo at 50°C, and the residue
It was dissolved in 0% Ac 0 H 200 g, and the solution was purified with a Sephadex-15' column. Both parts of the AcOH eluate, which had been digested by treatment with trypsin to liberate 1-methyl-7-amino-coumarin, were concentrated to dryness in vacuo at 40°C. The residue thus obtained was purified with P2O at 60°C in a vacuum desiccator.
5.95 y (theoretical value of 85.0) of a crystalline compound 2 homogeneous in SSC as indicated by TLCK. Elemental analysis and empirical formula C331 (calculated from 43N606Br yielded the following value: C = 56.33% (
56,65%), +1 = 6.28% (6,19%)
, N=12.25% (12,01%) and Rr=4.
30chi (4,42%). Compound 2c5.60. (8 mmol) in 2N glacial acetic acid
Example] dK Eri deblocking was performed using IIHr 32-. The obtained crude product was dissolved in 100 ml of MeOH,
Add this solution to “Sephadex+5eph-dex)”
Both fractions of the 7'CMeOH eluate were purified by treatment with trypsin to liberate 4-methyl-7-amino-coumarin. The resulting residue was evaporated with P2O in a vacuum desiccator at 40°C.
5 and showed by TLC.
Amorphous compound homogeneous in 2 d 4.76. (92.1% of the theoretical value). Elemental analysis and empirical formula C
25H58N604 Rr 2, the following values were obtained: C = 47.02% (46,45%), H
= 6.02% < 5.93%), N = 13.21% (1
3,00%) and Rr = 24.48% (24,72
blood ). Compound 2d 3.23 , (5 mmol) in Example 1e
Eri Cbo-D-Vat -0pNP 2.05 y
(5.5 mmol). The crude product obtained was dissolved in 5°C of AcOH, and the solution was purified on a column of 8-15' Sephadex. With the release of 4-methyl-7-amino-coumarin, Decomposed by treatment with trypsin A
Both fractions of the ft-AcOH eluate were concentrated to dryness in vacuo at 40°C and the residue f4 was dried over P2O5 at 60°C in a vacuum desiccator and filtrated by TLC.
3.21 g of homogeneous amorphous compound 2e in SC (
80.4% of the theoretical value) was obtained. Elemental analysis and empirical formula C,
Calculations from 8852N, 07Br gave the following values: C = 57.05 chi (57,14%), H = 6
．． 61chi (6,56chi), N=12.49% (12,2
8%) and Br = 9.82 inches (10,00 inches). 2f, 2HBr, HD-Vat-CHA-Arg
-MCC compound 2d2.40, (3 mmol) was deblocked in Example 1f using 2NHRr12- in glacial acetic acid. The obtained fL7'n crude product was dissolved in MeOH50
- and add this solution to 1 cephadex (5 epha).
-dex) Purified with LH-201 column. 4-
Fractions of the MeOH eluate were concentrated to dryness in vacuo at 30°C. Furthermore, for afJJ purposes, pre-purification t'1. A was prepared by dissolving 50 μm of the fc product in 40 μm of AcOH, and gel-filtering this solution through a 1-5 Ephadex 0-15 column. The main fraction of the 'l'Schiff'-AcOH eluate, which was resolved by treatment with trypsin to liberate 4-methyl-7-amino-coumarin, was concentrated to dryness in vacuo at 40°C and the residue was evaporated in vacuo. Dry over P2O5 at 40°C in a desiccator and TL
An amorphous compound 2 f 1.73 homogeneous in ssc, denoted by CK. (77.3% of the theoretical value). Elemental analysis and empirical formula C
3oH47N705B

[After calculating from 2, the following values were obtained: C = 48.12% < 48.33%), H
= 6.4 3% (6.35%), N=13
．． 38 Chi (13,15% + and Rr=21-18
% (21,44chi). The presence of the desired amino acid was confirmed in an accurate ratio for amino acid analysis: Val: 1.00-Arg: 1.02-D-C
HA: 0.97 FLf value: 0.42° Example 3 24Br, H-1)-Val-CIA-Arg-DPA
Anhydrous Cbo-Arg-OH, IC/, 34-48 y
(0,1 mol) klooo-〇 anhydrous DMF freshly distilled in a three-necked flask] 50- and anhydrous THF 30-
0- at 20°C. Et, N 10.21 (0.1 mol) was added to the solution cooled to -10° C. under stirring in the absence of any total moisture. Furthermore, T) IF50m
/ isobutyl chloroformate 13.65, (0,1
mol) solution was added dropwise over a period of 20 minutes, after which the reaction temperature never exceeded -5°C. -】O℃~-5℃
After an additional reaction time of 10 min at a temperature of 1) MF 7
Dimethyl 5-amino-isophthalate in 5d20.
92, (0.1 mol) was added dropwise over 30 minutes,
Thereafter, the reaction temperature was always kept below -5°C. The reaction mixture was allowed to react for an additional 1 hour at -5°C. The reaction mixture was stirred overnight at 20° C. and then cooled to -15° C. in order to photometrically crystallize the amount of Et3N, HC. All the I et 3N and I lcZ that were formed were separated and washed with a small amount of cold friends D to IF. All of this p solution and cleaning solution
Concentrate and dry at 50℃ in vacuum. 50% of this residue
Ac0HI is dissolved in 000 g, and this solution is dissolved in 50 g of Ac0HI.
5 ephadex equilibrated with OH
A was purified by gel filtration on a 1()-15" column.A was degraded by treatment with trypsin to liberate dimethyl 5-amino-isophthalate.
The main fraction of the cOH eluate was concentrated to dryness in vacuo at 40°C. This residue was heated to P2O5 at 50°C in a full vacuum desiccator.
Homogeneous amorphous compound 3a 24-61 at 88 C as indicated by 'I'LC'
(Theoretical value of 45.9 inches) All written. Elemental analysis and empirical formula C
By calculation from 24H3°N507Ct, the following values were obtained: C = 53.21% (53,78%), H
= 5.71% (5.64%), N = 13.20% (1
3,07%) and Ct = 6,52% (6,62%) 0 Compound 3a21-44r (40 mmol) was deblocked in Example 1b. After the usual work-up, the crude product obtained was dissolved in 250 ml of MeOH and this solution was purified by gel filtration on a column of V-Sephadex LH-20". Dimethyl 5-amino-isophthalate The fractions of the free fcMeOH eluate were concentrated to dryness in vacuo by treatment with trypsin. The residue was dried over P2O5 at 40°C in a vacuum desiccator and analyzed by TLC. A homogeneous amorphous compound 3b of 19.63% (93.1% of theoretical yield) was obtained in SSC using the following procedure. Elemental analysis and empirical formula C16
By calculation from 5N505Br2, the following value is obtained 1
Did: C=3 6.8 2%
(36,45%), H=4.67
Chi f 4.7 8%), N=13.45
%r13.28%) and Br = 29.85% (3
0.31%). Compound 3 b 5.27 , (10 mmol)'! i
l-Example IC to EliCbo-CHA-○pNP 4.69
, (4 mmol). The crude product obtained after normal treatment is 50% AcOH200-, and this M'/Qi'fc-Sephadex<5eph
adex) 0-15' column. Decomposed by treatment with trypsin to liberate dimethyl 5-amino-isophthalate:n-7t The fractions of the AcOH eluate were concentrated to dryness in vacuo at 40°C. The residue was dried in a vacuum desiccator at 60°C over P2O5 and T
Amorphous compound homogeneous in SSC as shown by LC (82.6% of theory)
I got it. Elemental analysis and empirical formula C33H45N6088r
After calculating from , the following value was obtained: C=53.7
4 chi (54,02 section', H = 6-28% + 6.18%
), N=4.90% (4,46%) and Br=10.6
8% (10,89%). 3d, 2HRr, ) (-CHA-Arg-DPA compound 3c 5-87 y (8 mmol)) was deblocked in glacial acetic acid using 2N I ([3r 32-) in Example 1dK. The crude product f' obtained after conventional treatment is M
The core solution was purified with a column of "Sephadex LH-20" and treated with trypsin to liberate dimethyl 5-amino isophthalate. Decomposed γ Me
Both portions of the OH eluate were concentrated to dryness in vacuo at 30°C. The residue was dried in a vacuum desiccator at 40° C. over P2O5 and 1'LC showed a uniform amorphous compound in SSC of 534.79 (theoretical value 88.0).
%) was obtained. Based on the elemental analysis and calculation from the empirical formula C25l (4oN606Rr), the following value was obtained: C = 43.75%
I 44.13%), H=5.88% (5,93%),
N=12.69% (12,35%) and Rr=23
．． 22% (23,49%). 3e, Cbo-D-Val-CIA-Arg-DP
A, HBr compound 3 d 3.40, (5 mmol) Example] Eli Cbo-13-Val-0pNP
2.05 y (5.5 mmol). The crude product obtained after conventional work-up was diluted with 50% Ac0I-4.
The solution was dissolved in 0.00 liters of water and separated on a column of 5-ephadex (+-15'') in this bath/pj. min yyt s
A fraction of gold X obtained from the Ac04 bath was condensed and dried in air at 40°C. This residue was heated to P2 in a vacuum desiccator at 60°C.
Dry over O5 and SS as shown by TLC.
A homogeneous amorphous compound 3 e 3.25 y (78.1% of theory) in C - elemental analysis and empirical formula obtained C3
a454N709) (By calculation from r, the following 1st shift is obtained fLfc: C = 53.95% (54,80% ',
”=6.65% (6,54%), N=12.07 (4
, 77%) and Br = 9.38%! 9.59%
). 3f, 2HBr, HD-Val-CHA-Arg
-DPA compound 3e2.50r (3 mmol) was deblocked using 2N HF3r 12- in glacial acetic acid according to Example 1f. The crude product obtained after conventional processing was M
Dissolve in eOH50- and add this solution to 1 sefdex (
5 ephadex I LH-20" column. 1 Me
The fractions of the O)I eluate were condensed to dryness in vacuo at 30°C. This prepurified product was converted to 50% Ac0)-150
ml and purified by gel filtration on a column of 5-ephadex G-15. The main fraction of the AcOH eluate was concentrated to dryness in vacuo at 40°C. The residue was dried over P2O5 at 40°C in a vacuum desiccator and
The uniform amorphous appendage 3r in the SSC was 1.93 (82.6% of the theoretical value) in the TLCK process. Elemental analysis and empirical formula c30H49N7o7” 2
In the calculation, the following value was obtained: C=46.8
4% (46,22%l, H=6.42%(6,34%l
, N = 12.16% (12,58%) and Rr = 2
0.22% (20,50 chi). Amino acid analysis confirmed the presence of the desired amino acid in the correct ratio: D-Val: 1. QO-Arg: 0.98-(
JIA: LO2°Rr value: 0.4. @. Example 4 Commercially available Cbo-Arg-2-NA, HCl2.40,
(20 mmol) in Example 1bK erythriacetic acid 2N
React with a solution of 80 ml of HBr. The fL7 obtained after the usual work-up was dissolved in the product fMeOH]50- and this solution was dissolved in 5 ephadex l L.
Purification was performed on a l+-20' column. 2-naphthylamine was decomposed by treatment with trypsin to liberate it.
The L 7CMeOH eluate fractions were concentrated to dryness in vacuo at 30°C. Pour this residue at 40°C in a vacuum desiccator.
dried over 2O5 and shown by 1'LC
A homogeneous amorphous adduct 4b8.60,1 (93.2% of theory) was obtained in SC. Elemental town and empirical formula C16I
(Eli calculation from 23N50Br2, the following value is obtained 1
Nine: C= 42.08% (41,67%), 4=
5.12chi (5.03%), N = 14. ．． 68
%+15.19%) and Br=33.96%+3
4.65%). Compound 4 b 4.6 , (10 mmol) ft Example 1
c to EriCbo-CHA-OpNP 4.69,
I 1 1 mmol).9. rLfc crude product obtained after conventional treatment with 50% AcOH]
50- and this solution was added to 1 sepadex (5 ep).
It was purified with a column of hadex)0-15'. Fractions of the Ac0 month eluate, separated by treatment with trypsin to liberate 2-naphthylamine, were incubated in vacuo at 40°C.
It was concentrated and dried. This residue was placed in a vacuum desiccator for 60 minutes.
Amorphous compound 4C5,3, homogeneous in SSC as shown by TLC, dried over PO at °C,
(79.5% of the theoretical value). Using the elemental digit and the empirical formula C331 (43N604Br, the following value is obtained: n'fc: C = 59.18% (59,37%
), fl = 6.58% (6,49% l, N =
12.87 inches (12,59 inches) and l-1r=4.55
% (4,97%). 4d, 2HRr, H- (JIA-Arg-2-NA compound 4c 4.67y (7 mm!J mol) was deblocked from dK using 2N 14Br 28- in glacial acetic acid. Normal After treatment, the crude product was treated with MeO
Dissolve in H100 solution and add this bath solution to “Sephadex (5
ephadex) LiF-20" column. The fractions of the MeO)l eluate were purified by treatment with trypsin to liberate 2-naphthylamine. The fractions of the eluate were concentrated to dryness in vacuo at 30°C. The residue was dried over P2O5 at 40 °C in a vacuum desiccator to yield a homogeneous amorphous compound in SSC as shown by TLC = $d 3.95, (91.8% of theory). Elemental analysis and empirical formula C25t138N...02F
After modifying the calculation from 3r2, the following value was obtained.7t:
C=49.22% I4 8.8
7%), F(=6.30 (
6,23%), N=13.61% (13,6
8 mo) and Br=25.84% (26,01% I. Compound 4 d 3.07 y (5 mmol) Example 1e
Cbo-D-Vat-OpNP 2-05 y
The crude product obtained after the usual work-up was dissolved in 50% AcOH]QOO- and this solution was dissolved in "Sephadex"
) 0-15" column. The first main fraction of the AcOH eluate was digested by treatment with trypsin to liberate 2-naphthylamine at 40°C in total vacuum.
and concentrated to dryness in a vacuum desiccator at 60°C with P2O5.
The amorphous compound 4e 3.14 was dried on FC and thus homogeneously in SSC as shown by TLC.
y (81.9% of the theoretical value) was obtained fL 7'C0 After elemental analysis and calculation from the empirical formula C3B452N705Or, the following value was obtained: C = 58.92% I
59.52%), H=6.93% (6.84%
), N2 1 3.0 2% (12,79chi) and B
r = 10.18% (10.42%). 4f, 2) IF3r, 1(-D-Val-CIA, Ar
g-2-NA chemical compound 4 e 1-53 y (2mi')
Example 1 using Z N HBr 8- in glacial acetic acid
The fK construction was unlocked. The 1% crude product obtained after the usual treatment was dissolved in total MeOIl 40 tnl KtG and the solution was dissolved in 5 ephadex LH.
The main fraction of the MeOH eluate was concentrated to dryness in vacuo at 30 °C by treatment with trypsin under the formation of 2-naphthylamine. 'fr50%ΔcOH50-,
Add this solution to 1 cephadex (5 ephadex)
Purified with G-15# column. The main fraction of the AcOH eluate, which was resolved by treatment with trypsin with the formation of 2-naphthylamine, was concentrated to dryness in vacuo at 40 °C. This residue was heated to P2 at 40°C in a full vacuum desiccator.
Dried over O5 and labeled by TLC.
A homogeneous amorphous compound 4 f 1.05, (73.6% of the theoretical value) was obtained in C with nine elemental analysis and empirical formula C30
In the calculation from H47N703"2, the following value is obtained.
:C=50.16%(50,50%) ,)(,=
6.71% (6,64%), N=14.00% (13,
74% l and Br = 22.05% (22,40%)
. Amino acid analysis confirmed the presence of the desired amino acid in the correct ratio ζn: D-Val: 1.00-Arg: 0.98-C
HA: 0.97°1 (f value: 0.42° Example 5 Commercially available Cbo-Arg-4-MeO-2-NA,) IC
210,0, (20 mmol) 2 N in total glacial acid
Example 1b was deblocked using HBr 80-nfc obtained after normal treatment, and the crude product was dissolved in MeOH 150-nfc.
-, dissolve this solution in total 1 cephadex (5 epha
dex) Purified with LH-20= column. The main fraction of the MeOH eluate, cleaved by treatment with trypsin to liberate 4-methoxy-2-naphthylamine, was concentrated to dryness in vacuo at 30°C. After drying the residue over P2O5 at 40 DEG C. in a vacuum desiccator, a homogeneous amorphous compound 5b in SSC, designated as 'I'LCK, was obtained with a theoretical value of 91 ．．
Using elemental analysis and experiments to calculate from the formula C17H2sN5C]zRr2, the following values were obtained: C=41.22% (41,57%)
, 1 (=5.19% (5,13%), N=1
4.40% (14,26% 'I and Hr = 32
．． 01% (32,53%). Compound 5b4.91r (10 mmol) was prepared in Example 1C and reacted with Cbo-CHA-OpNP 4.69 y (4 mmol). The entire crude product obtained after the usual work-up was dissolved in 50 ml of AcOE-450 and this solution was purified on a column of 1 ephadex G-15#. The first main fraction of the eluate was concentrated to dryness in vacuo at 40° C. by treatment with trypsin to liberate 4-methoxy-2-naphthylamine. After drying over P2O5 at 60 °C in a vacuum dessicator, the process was shown by LC.
A homogeneous amorphous compound 5C5,36SF (76.8% of theory) was obtained in c. Elemental analysis and empirical formula C34
After calculating from 445N605Br, the following value was obtained.
L hole: C=58.85 ratio (58,53%□), H=
6.59% (6,50%), N=4.91% (12,0
5%) and 13r = 4.32% (4,45chi). Compound 5c4-88y (1 mmol) was deblocked by dK using 2N+18r28- in glacial acetic acid. The crude product f' obtained after conventional treatment is M
The solution was purified on a column of Sephadex LH-201.
The main fraction of the eluate was concentrated to dryness in vacuo at 30°C. This residue was dried over P2O5 at 40 °C in a full vacuum desiccator and then sifted on LC to give an amorphous compound homogeneous in SSC as shown in IL (91.3% of theory). ) was obtained, then elemental analysis and empirical formula C26
The following values were obtained by calculating from H4oN603Br2: γL7j: C=48.92chi (48,46%), H
= 6.36chi (6.26%), N=12.84% (1
3,04%) and Br = 24,33chi (24,80%
). Compound 5d 3.22 , (5 mmol) in Example 1e
Cbo-D-Val-OpNP 2.05 y
(5-5 mmol). The product obtained after conventional processing is dissolved in 50% Ac0)-425-,
Add this solution to 1 cephadex (5 ephadex)
Purification was performed using a G-15= column. The first main fraction of the AcOH effluent, which was decomposed by treatment with trypsin to liberate 4-methoxy-2-naphthylamine, was concentrated to dryness in vacuo at 40°C. This residue is placed in a vacuum desiccator.
The amorphous compound 5e is homogeneous in SSC as shown by TLC after drying over P2O5 at 60 °C in
3-151 (79.1% of theory) was obtained. After elemental analysis and calculation from the empirical formula C3, t154N706Rr, the following value was obtained: A7j2C=58.35% (
58,79%), ■(= 6.78% (6,83%
), N = 12.68% < 12.31%) and 13r = 9.82% I 10.03%). 5f, 2HRr, II-D-Va 1-C
I-1Δ-Arg-4-MeO-2-NA compound 5el
-59y (2 mmol) in 2 N HHr in glacial acid
Using 8-, it was unblocked by example f. The crude product obtained after the usual work-up was dissolved in MeOH40- and the solution was dissolved in "Sephadex" L
Preliminary purification was carried out using a column of H-20=. 4-methoxy-2
The main fraction of the eluate was concentrated in vacuo at 30°C. This pre-prepared product was dissolved in 50% AcOH and 60% of this solution was purified on a 50% Sephadex G-15 column. 1 was degraded by treatment with trypsin with the formation of 4-methoxy-2-naphthylamine.
The main fraction of the cOH eluate was concentrated to dryness in vacuo at 40°C. This residue was heated to P2O5 at 40°C in a vacuum desiccator.
After drying on the surface, the SSC will be shown as TLCK.
A homogeneous amorphous compound 5 f 1.09 (73.3% of theory) was obtained. Elemental analysis and empirical formula C
31H49N7041-1r2, the following value was obtained: rL7'c: C = 49.63% (50,07%
), H=6.70% (6,64%), N=13.42%
(13,19%) and Sr=21.22% (21,
49%). The presence of the desired amino acid in the correct ratio was confirmed in the amino acid analysis: D-Val: 1.00-Arg: 1.
0l-D-Cl4: 0.98°Rf value: 0.4
A series of other tripeptide derivatives were prepared using the methods described in the previous examples. These one tripeptide derivatives are
4. summarized in Table 1 below. ing. The tripeptide intermediates used in the preparation of the tripeptide derivatives shown in Table 1 are listed in Table 2. 7・'/' Example 12 2HRr, HD-Vat-CHA-Arg-pNA
(Prepared as in Example 38) 7.09 (10 mmol) was dissolved in an aqueous solution of 604Me01 (75).
e) '(registered trademark) JFLA-401 column was packed. This column'f! : After stripping with a 60% MeOH aqueous solution, tIRr was replaced with AcOH by ion conversion. The eluate was concentrated to dryness in vacuo at 40°C. After drying the residue in a vacuum desiccator at 40°C over P2O5, the bromide-free 2A, C0Il
. )1-D-Vat-CHA-Arg-pNA 6.33
, (98.5% of theory) was obtained. In some cases, according to the above method, other salts with organic acids, such as m-acids, propionic acid, oxalic acid, oxalic acid, citric acid, lactic acid, benzoic acid, chlorbenzoic acid, salicylic acid or phthalic acid, are used to prepare the aforementioned tripezotide. It can be manufactured from derivatives. Ion exchangers, such as “Amberlite” (Am
berlite)" JRA-401, which converts the ion exchanger to the basic OH form by treatment with caustic soda solution and then converts the basic ion exchanger into the basic OH form k 60 '16 MeOH. The tripeptide substrate according to the present invention can be converted to the desired acid salt form by treatment with a solution of a 1:1 mixture of an organic acid and its sodium salt in a water bath solution. Quantitative analysis of the enzyme can be carried out as follows: 1. Urine kallikrein analysis TRI5-imidazole slow vli with pH 7,9 and ionic strength 1.0' in urine 1d at 37 °C Incubate for 5 minutes and then centrifuge to remove the precipitate. 1.4 m/ml of distilled water heated to 37°C and 0.4 m/ml of centrifuged water.
Mix thoroughly in a plastic cuvette. To this mixture was added 2
Add all and mix ingredients quickly. This mixture is kept at 37° C. for exactly 15 minutes. This reaction mixture f6: is then mixed with 0.2-ml of glacial acetic acid to stop the cumulative reaction. For the color total measurement, a raw sample is used consisting of the same components but with 1 glacial acetic acid added before the addition of the prescribed substrate to inhibit the enzymatic reaction. The amount of colored compound R-Nl2 formed is then measured photometrically or spectrophotometrically at 405 nm from the difference between the raw sample and the test sample. From the obtained n;b value, the activity of urinary kallikrein in urine is calculated by the following formula: Add 1i4: 15 minutes
Predetermined conversion factor F to convert to U f mU = urine (2)
Dilution factor V = Volume of sample = 0.4 m ε = Extinction coefficient divided by 1000 = 10.4 The total group kallikrein content in urine g is the amount of IL that is formed - NH,2 < e.g. p-nitroaniline)? Continuous measurement can also be carried out. The application of this method to the analysis of glandular kallikrein in whole tract sputum will be described later. Besides urinary kallikrein, urine also contains urokinase as a proteolytic oxygen which can also degrade the substrate according to the invention, albeit in small amounts. In the above analysis method, the total activity of urinary kallikrein and urokinase is measured. To obtain an accurate value for urinary kallikrein activity, urokinase activity must be subtracted. This urokinase activity was determined to be 1 ml of buffer in order to completely suppress urinary kallikrein activity in a comparative test. per trypsin inhibitor (trypsin inhibitor from bovine lung 10.0
It can be determined by adding 75 units and measuring the total urokinase activity alone. 21! Analysis of glandular kallikrein in i8 sputum: approximately 0.5
ml of TI shellfish S-imidazole buffer (ionic strength 1.
0+2vj! This mixture is pre-incubated at 37° C. for 5 minutes and the incubate is centrifuged. The test cuvette is filled with 1.5 mm of distilled water at 37 DEG C. and 0.25 mm of centrifuge is added thereto. Mix all these ingredients thoroughly. Furthermore, 2XTO-3 mol of substrate aqueous solution 0
．． 2m1. Add to. Next, the change in absorbance at 405 nm is continuously tracked for 5 to 10 minutes using the entire recording device ff1. From the measured value of Δ00 per minute, the kallikrein activity per minute is calculated using the following formula: VX ε F=5 V=1.95 V=0.25 1[J (Unit 1 = p+4, an acetic acid solution capable of decomposing 1 micromole of substrate in 1 minute under optimal conditions of ionic strength, temperature, and substrate concentration or under other specified conditions. In pancreatic juice, pancreatic kallikrein is After activation of prekallikrein, which exists mainly in the form of prekallikrein and can be analyzed only after activation, e.g. using trypsin, trypsin, soybean trypsin inhibitor (5BTI
) is suppressed by I. The kallikrein content in this activated mixture can be determined using one of the methods described above. 3. Plasmin analysis: Mix at 37°C with 0.1 - of the plasmin solution in Tl Is-imidazole buffer (pH 7.5, Io 7 strong 1f 0.24.7 dk 25 tiglycerol), and add this mixture to 3
Incubate at 7°C for 1 minute. This mixture was heated 2x at 37°C.
Add 0.2-mole of the 10-M aqueous substrate solution and mix the ingredients quickly. Then, I separated from the substrate per unit time
Continuously measure the crude gold content of the decomposition product NH2. The plasmin activity per sample is calculated in mU from the values measured per minute using the following formula: ΔE = number of decomposition products separated per minute = total volume of the test mixture. Volume V - Extinction coefficient divided by sample volume ε = 1000 4 Antizolazmin analysis in human plasma: γ plasma 0.1 - diluted with 'l'RIs-imidazole buffer in a ratio of 1:20, 25 Tiglycerose K] human plasmin (A13 Kabi (Stock
olm. (Sweden)) 1.25 CU and hirudin (Pentapharm A, (L (Ra5le)
, 5w1tzerland 1) 50 AT
[Mix with the solution of J O,02-. This mixture is 37
Incubate at ℃ for 90 seconds. To this incubate was mixed 1.7 rnl of 'I'RIS-imidazole buffer Q (pH 7.5, ionic strength 0.2) at 37°C, and an additional 2 x 10-3 molar substrate water bath P & 0.2 mi. Mix. Next, the crude gold decomposition product R-Nl2 separated from the substrate per unit time is continuously measured. The residual plasmin activity is calculated using the method described above from the measured values. In the blank test, the plasma was replaced with an equivalent amount of buffer, but otherwise the test was carried out as described above. measured 1
Shiru plasmin activity! , corresponds to the starting zolazmine amount. The antiplasmin activity is calculated from the difference between the plasmin activity measured in the nitrogen test and the residual plasmin activity measured in the plasma test using the following formula: (Δ” '2RR plasma test H)/"", xVxFxlooO
v × ε ml of plasma [J/volume F = dilution factor of plasma (20). One of the substrates according to the present invention is obtained by modifying the f of plasmin, which is converted and formed using urokinase or streptokinase in the zlazminodiene 'kMfj system present in plasma, into the above-mentioned zolazmin analysis method. It can also be used to analyze plasminogen in human plasma by measuring it using The amount of plasminodiene naturally present in plasma is derived from measurements for plasmin, since one molecule of plasmin is formed from one molecule of plasminodiene under activation. Table 4 below shows the sensitivity of some substrates according to the invention to organ or vascular kallikrein, plasmin and thrombin. 7. ...L Degradation product F formed during enzymatic hydrolysis of the substrate
When measuring L-NH2, this decomposition product must have an entire UV spectrum that is different from the UV spectrum of the substrate! ll
Based on the premise of moving towards higher wavelengths. 4
The absorption of the substrate at 0.05 nm is practically absent. p-2rroaniline u, 380 n as a decomposition product
It shows an absorption maximum at m and a molar extinction coefficient of 13.200. However, the extinction coefficient decreases slightly at 405 nm.
In other words, it becomes 9650. The amount of p-nitroaniline separated is determined by enzymatic hydrolysis of the substrate, which is proportional to the amount of p-nitroaniline.
Kj can be measured using a spectrophotometric method at 5 nm. Measurements at 405 nm are not affected even in the presence of excess substrate.

Claims (1)

[Scope of Claims] 1 Contains a proteolytic enzyme in the medium of arginine or lysine, or degrades the natural peptide chain on the carboxyl side chain of arginine or lysine that forms or consumes the proteolytic oxygen in the medium. Proteolytic enzymes, especially enzyme reconstituted E. C. 3.4.2.1. In the method for quantitatively analyzing an enzyme of the general formula I: H-D-X-Y-Arg-R I [wherein, Y represents a cyclohexylalanyl group or a cyclohexyltyrosyl group, R represents a p-nitrophenylamino group, a 2-naphthylamino group, a 4-methoxy-2-naphthylamino group, 4 -methyl-coumaryl-7-amino or 1,3-di(
methoxycarbonyl)-phenyl-5-amino group] and the colored or fluorescent decomposition product R-NH_2 formed by the hydrolytic action of the enzyme on the tripeptide derivative. A method for quantitatively analyzing proteolytic oxygen, characterized in that the amount is measured by photometry, spectrophotometry, fluorescence spectrophotometry, or an electrochemical method. 2. The method according to claim 1, which analyzes organs or glands kallikrein in human body fluids, such as bed, pancreatic juice, intestinal mucus, mammary gland secretions, sweat gland secretions, sputum, or blood. 3. The method according to claim 1, which analyzes plasmin in blood or plasma. 4. The method according to claim 1, which analyzes thrombin in blood or plasma.