JPS5843389B2 - Method for measuring collagenase activity using peptide nodule and peptide derivative - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel peptide derivative and a method for measuring collagenase activity using the same.

More specifically, the present invention relates to a novel octapeptide derivative useful as a specific substrate for collagenase, and provides a useful peptide derivative of the present invention and a method for measuring collagenase activity using the same.

In recent years, research on collagen and the enzyme collagenase that decomposes it has been actively conducted, and it has been used to treat diseases involving connective tissue lesions, such as various skin diseases, wounds, systemic erythema lupus, gastric mucosa of gastric ulcers, corneal ulcers, liver cirrhosis, and fibrosis. Tissues such as sarcoma,
It has been discovered that collagenase contained in the synovial fluid of rheumatoid arthritis, as well as granular leukocytes of patients with essential hyperemia, arteriosclerosis, diabetes, etc., fluctuates significantly.

In addition, research by the discoverers has revealed that collagenase activity in the serum of some patients with liver cirrhosis and other liver diseases is increased.

Therefore, in diagnosing the above-mentioned diseases and related diseases, it is of great significance to measure collagenase activity in living organisms.

Conventional collagenase activity measurements are performed using natural collagen,
For example, a collagenase preparation to be measured is applied to collagen extracted from animal skin, keys, etc. as a substrate,
This method has been used to determine the decomposition rate.

In this method, (1) the molecular weight of natural collagen is extremely large and has a wide molecular weight distribution; it is extremely difficult to obtain collagen with a constant molecular weight; and the extraction operation is extremely complicated.

■ The properties of collagen are not constant depending on the type and site of the animal from which collagen is extracted. Furthermore, ■ When measuring in vivo collagenase activity, serum protein a.
Although contamination with 2-macroglobulin cannot be avoided, this a2-macroglobulin acts as an inhibitor when measuring collagenase activity using natural collagen as a substrate. It was nothing.

In order to avoid the third drawback mentioned above, collagenase a2-
Attempts have been made to restore collagenase activity by pre-treating inhibitory substance conjugates such as macroglobulin with rhodan sodium, etc., but side reactions occur and the measured values fluctuate greatly, making it difficult to achieve practical accuracy. hard.

Therefore, there has been a long-awaited establishment of a simple and accurate method for measuring collagenase activity.

The present inventors believe that the shortcomings of conventional methods can be overcome by using an appropriate peptide as a substrate or as a model substance of natural collagen. We conducted extensive research on the specificity between the two substrates.

As a result, we have completed a novel octapeptide derivative that is a collagen model substance that has a high degree of substrate specificity and is particularly advantageous in terms of operation in measuring collagenase activity.

Furthermore, this octapeptide derivative has the above-mentioned serum protein a.
It was recognized that it has the excellent feature of being able to express collagenase activity without being inhibited by 2-macroglobulin at all.

Moreover, the present octapeptide derivative, especially its color-forming group, is stable and can be stored for a long period of time without being decomposed by light.

That is, the present invention has the following formula: Pro, Gin, Gly.

11e, Ala and Arg represent proline, glutamine, glycine, inleucine, alanine and arginine units, respectively.

)) and a method for measuring collagenase activity using the same.

When the above-mentioned octapeptide derivative is treated with collagenase, it becomes a tripeptide derivative having hydrophobicity;
-Pro-L-Gln-Gly-OH is released.

Since this tripeptide derivative is hydrophobic, it can be extracted easily and sufficiently with a suitable organic solvent such as ethyl acetate, ether, n-butanol, etc. Furthermore, since it has a chromophore group, the extract can be extracted using conventional colorimetric methods. can be accurately quantified using optical means.

During extraction, it is important for the accuracy of collagenase activity measurement to extract only this tripeptide derivative and not to extract other substances in the collagenase-containing sample that have absorption in the colorimetric region. and ethyl acetate is preferred.

This tripeptide derivative can be sufficiently extracted with suitable ethyl acetate.

Therefore, by using this octapeptide derivative as a substrate, collagenase activity can be determined accurately and extremely easily.

According to the present invention, such novel peptide derivatives can be produced by methods well known in peptide chemistry.

That is, the general formula % formula % (However, Y represents a protecting group for the guanidino group of the arginine unit, and R represents a protective group for the carboxyl group of the arginine unit.

) can usually be obtained by deprotecting the protected peptide derivative shown in ().

As the protecting group Y for the guanidino group of the arginine unit, a hatosyl group (pt-luenesulfonyl group), a nitro group, a benzyloxycarbonyl group, a p-nitrobenzyloxycarbonyl group%2-(isopropyloxycarbonyl)-
Known protective groups such as 3,4,5,6-titrachlorobenzoyl group are commonly used, and the first three are generally preferred.

On the other hand, the protection of the carboxyl group of the arginine unit is carried out by converting it to esters commonly used in peptide chemistry.

That is, as the protecting group R, benzyl, t-butyl, p-nitrobenzyl, p-methoxybenzyl groups, particularly benzyl group and t-butyl group are preferably used.

Regarding the method of removing the protecting group, a known method may be selected as appropriate depending on the individual protecting group, but in this case, simultaneous removal or change of the constituent units such as the chromophore group X is not allowed.

Therefore, the elimination method that selectively eliminates both protecting groups is
It is important to select the protective group and chromophore group accordingly.

As the elimination method, hydrogen fluoride treatment is preferably used since both protecting groups can be eliminated at the same time.

When used for deprotection with hydrogen fluoride, it is generally used in an anhydrous state.

Although the reaction can be carried out in the presence or absence of a solvent, it is usually carried out using hydrogen fluoride as a solvent.

Further, in order to suppress side reactions, it is preferable to carry out the reaction in the presence of a small amount of anisole, phenol, etc.

The reaction temperature is -70° to 20°C, preferably -lO° to l
It is about 0°C.

After the reaction is completed, the target product can be obtained by means known per se.

The protected peptide derivative as a starting material can be synthesized by methods commonly used in peptide chemistry.

Specifically, protected amino acids such as L-proline modified between chromophore groups are sequentially peptide-condensed in a solid phase or a liquid phase (so-called Stepwise Elo
ngation), or a suitable fragment, such as L-I 1e-L-Ala-Gly-A〒g(Y
)-oR is synthesized in advance, and then each fragment is synthesized into a peptide using a condensing agent such as a water-soluble carbodiimide (ethyl N, N-dimethylaminopropylcarbodiimide or a salt thereof, etc.) or by other condensation methods such as an active esterification method. Derivatives can be obtained.

Although the chromophore group may be introduced into L-proline and subjected to the post-condensation reaction as described above, the N-terminus of L-proline may be substituted with a conventional amino protecting group, such as t-butyloxycarbonyl,
After protection with benzyloxycarbonyl, t-amyloxycarbonyl, etc. and subjecting to condensation reaction, the amino protecting group may be substituted with a chromophore group in the fragment stage during synthesis or in the final stage.

The method for measuring collagenase activity using the peptide derivative according to the present invention is the same as when using natural collagen as a substrate.

That is, first, an aqueous solution of the octapeptide derivative at a constant concentration is prepared.

This substrate solution is in the neutral region, more specifically at a pH of about 6~
9, particularly adjusted to pH 7 to 8, and subjected to measurement.

Among the present octapeptide derivatives, X-h' p -(4
-Hydroxy-1-naphthylazo)benzenesulfonyl groups may not have sufficient solubility.

In such cases, alcohol, such as ethyl alcohol, is added to promote dissolution.

In the measurement, a collagenase-containing sample was added to the substrate solution at 300%
After contacting and reacting at ~45°C, preferably 35°~40°C, and reacting for an appropriate time depending on the substrate and enzyme amount, the enzyme is deactivated by adding hydrochloric acid or the like.

X-L-Pro-L-Gl produced freely by enzymatic action
n-Gly-OH is released, which is extracted with a suitable organic solvent such as ethyl acetate, ether, n-butanol or a mixture thereof, using the most advantageous wavelength depending on the chromophore group used. For example, if X is p-(4-hydroxy-1
-Naphthylazo)benzenesulfonyl group is 389
nm.

If it is a p-phenylazobenzoyl group, the absorbance is measured at 325 nm.

Since this octapeptide derivative has extremely high substrate specificity, collagenase activity can be determined with sufficient accuracy in ordinary collagenase-containing samples, such as blood sperm, by measurement using this octapeptide derivative as a substrate. However, in samples with particularly strong non-specific peptidase activity, such as collagenase samples from tissues such as liver and kidney, some of the octapeptide derivatives may be hydrolyzed, making it difficult to precisely measure collagenase activity. It turns out that there are cases where this is insufficient.

Therefore, in order to perform measurements with sufficient accuracy even in such cases, we conducted further research on peptide derivatives that are inactive to collagenase but undergo hydrolysis by other nonspecific peptidases that are often associated with collagenase-containing samples. As a result, it was found that a new hepatopeptidyl peptide derivative, which lacks a proline residue compared to the above-mentioned octapeptide derivative, satisfies these conditions.

In this case, the measurement can be performed as follows using a heptapeptide derivative lacking a proline residue.

That is, a control substrate solution of a heptapeptide derivative having the same chromophore group X as the octapeptide derivative used, the same molar concentration, and the same solvent is prepared.

After the same amount of sample is allowed to act on the substrate solution under exactly the same conditions, the released chromophore group-containing decomposition product is similarly extracted and the absorbance is measured at the same wavelength.

By subtracting the absorbance of the control experiment from the absorbance of the octapeptide derivative, the collagenase activity in the sample can be determined with high precision, without being influenced by other non-specific peptidases in the sample.

As is clear from the above description, the present invention provides a novel peptide derivative that can be extremely advantageously used for measuring collagenase activity, which has important significance in the field of medicine, especially diagnosis, and a method for measuring collagenase activity using the same. This is what we provide.

Hereinafter, the present invention will be explained in detail with reference to Examples.

Note that each abbreviation in the examples has the following meaning.

BOC: t-butyloxycarbonyl group, Z: benzyloxycarbonyl group, NABS: p-(hydroxy-■-naphthylazo)benzenesulfonyl group, PAB
: p-phenylazobenzoyl group, Bzl: benzyl group, Et: ethyl group, TosOH: p-toluenesulfonic acid, WSCI: ethyl-N,N-dimethylaminopropylcarbodiimide THF: Tehetrahydrofuran, DMF dimethylformamide, and The purified collagenase used in Examples 3 and 4 was Y
, Nagal et al.
m.

Biophys, Acta, 263,564 (
1972)).

Example I NABS-L-Pro-L-Gln-Gly-L-Il
e-Ala-Gly-L-Gln-D-Arg-O
Synthesis of H 1) Boc-L-Pro-L-Gin-Gly
-Synthesis of OBzl Boc-L-Gln-Gly-oBz
After adding trifluoroacetic acid (30-) to L (11,8,9,0,03 mol) under cooling, the mixture was stirred at room temperature for 2 hours.

After confirming the completion of the reaction by thin layer chromatography, trifluoroacetic acid was distilled off under reduced pressure, ether was added to the residue and kneaded to form a solid, and the ether was removed by decantation.

After repeating the same operation twice, the solid was dried under reduced pressure over sodium hydroxide.

This solid was dissolved in lO〇- THF and Boc-L-P
ro-OH (6.5 g, 0.03 mol), l
-Hydroxybent 1 assol (4.9g).

0.036 mol) was added and WSCI (0.036 mol) was added under cooling.
6.6 ml, 0.036 mol) was added thereto, and the mixture was stirred for 1 hour.

Thereafter, the mixture was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure, chloroform was added to the residue to dissolve it, and the chloroform layer was washed successively with water, IN hydrochloric acid, water, 5% aqueous sodium bicarbonate solution, water, and saturated brine, and then dried with sodium sulfate. did.

After removing Glauber's salt, chloroform was distilled off under reduced pressure, and hexane was added to the residue to obtain a solid.

It was recrystallized from methanol-ether.

2) Synthesis of Boc-L-Pro-L-Gln-Gly-OH oc-L-Pro-L-Gin-Gly-OBz
1 (4.0 El, 8.2 mmol) was dissolved in a 90% methanol aqueous solution (60 m), 1 tablespoon of Pd was added, and hydrogen gas was bubbled through for 6 hours.

After confirming the completion of the reaction by thin layer chromatography, the reaction solution was filtered to remove the catalyst, and the filtrate was concentrated to dryness under reduced pressure.

Ether was added to the residue to obtain a solid. Recrystallized from methanol-ether.

Yield 2.9g (91%) 3) D-krg (NO2) -0B z 1 ・
Synthesis of 2T osOH H, Gibian et al.
(Liebigs Ann.

D-Arg (NO2X 16.4'l,
75 mmol), p-toluenesulfonic acid (31,4
g, 165 mmol), benzyl alcohol (37 mA
The suspension was suspended in chloroform (150d) and refluxed for 5 hours to obtain a homogeneous solution.

Toluene (iooi) was added to the above homogeneous solution, and the mixture was concentrated under reduced EE.Chloroform (150 rrLl) was further added thereto, and the mixture was refluxed for 5 hours.

Next, toluene (100-x2) was added, and the mixture was concentrated under reduced EE. Dry ether was added to the residue, and the precipitated solid was collected by filtration.

The solid was washed with dry ether, recrystallized with methanol dry ether, and dried over phosphorous pentoxide at room temperature for 12 hours.

Yield: 42 g (86%) Melting point: 132° to 135°C Ca)'d -12,0 (C=3, pyridine) 4)
Boc-L-Gin-D-Arg(NO2)-0Bzl
For the synthesis of Hofmann et al.
, chem.

Soc, 87, 620 (1965)) Boc-L-GIn (5,09,20 mmol) and D -Arg (NO□) -0Bz 1 ・2Tos
OH (13.1g, 20mmol) in DMF-THF
(5TrLl to 20TLl), and after cooling to -10°C, triethylamine (2,F3rnl, 20 m
mol) 1-hydroxybenztriazole (2,
7g, 20mmol) WSCI (3,6rul, 2
0 mmol) was added sequentially and incubated at -10°C for 1 hour.
The mixture was then stirred at room temperature for 16 hours.

After the reaction, THF was distilled off under reduced pressure, and the residue was dissolved in chloroform (300rn0) and 1N hydrochloric acid (100TL1X
3), water (100 mO 110% aqueous sodium carbonate solution (100 x 3), water (io.

Washed with water (2).

Next, chloroform was distilled off under reduced pressure, ether was added to the residue, and the precipitated crystals were collected by filtration.

The crystals were washed with water and ether, recrystallized from methanol-ether, and dried under reduced pressure over phosphorus pentoxide at room temperature for 12 hours.

5) Boc-L-11e-L-Ala-Gly-OEt
Synthesis of M, Bergman et al (J, Bi
ol,Chem,.

109.325. (1935))
L-Ala-Gly-OEt(12.4g, 4
0 m mol) of 25% hydrogen bromide acetic acid solution under cooling.
The mixture was added vertically and stirred at room temperature for 1 hour.

When ether was added to the above solution, a solid precipitated.

The solid was washed with ether and dried under reduced pressure over sodium hydroxide.

Next, the obtained solid was dissolved in DMF30- and adjusted to pH 7 to 7.5 with triethylamine under cooling.
1) was added and stirred at room temperature for 24 hours.

200TrLl of water was added to the reaction solution, and ethyl acetate (200TrLl) was added to the reaction solution.
0TLl, 150ml! , 100 mO three times, and the organic layer was washed successively with 1N hydrochloric acid, water: 5% aqueous sodium bicarbonate solution, and dried over dehydrated magnesium sulfate.

Ethyl acetate was distilled off under reduced pressure, and ether was added to the residue to precipitate crystals. The crystals were collected by filtration, recrystallized from ethyl acetate-ether, and dried under reduced pressure in a room above phosphorus pentoxide for 2 hours.

Yield 13.1 (84%) Melting point 148.5°-149°C (a "g"-50,9,' (C=2.2,
Ethanol) Elemental analysis CHN% Calculation directly (as C18H3□06N8) 55.7
98.5810.85 Analysis value 55
．． 548.7410.806) Boc-L-I 1e
Synthesis of -L-Ala-L-Gly-OH Boc-LI 1e-L-Ala-Gly-OEt (
3,99,10m mol) in dioxane 20rI
IN sodium hydroxide (ILmlSl 1 mmol) was added dropwise while stirring under cooling, and the mixture was further stirred at room temperature for 1 hour.

Next, the reaction solution was neutralized with 1N hydrochloric acid, and dioxane was distilled off under reduced pressure.

The aqueous layer was adjusted to pH 2 with 1N hydrochloric acid, and ethyl acetate (looTL
l.

Extracted twice at 50 mO, washed the organic layer with saturated saline,
It was dried with anhydrous sodium sulfate.

Ethyl acetate was distilled off under reduced pressure, and ether was added to the residue to precipitate crystals.

The crystals were collected by filtration, recrystallized from ethyl acetate-ether, and dried under reduced pressure over phosphorus pentoxide at room temperature for 12 hours.

7) Boc-LI le-L-Ala-Gly-L
-Gln-D-Arg (NO2) -0Bz l synthesis Boc-L-Gl n-D-Arg (NO2) -
Trifluoroacetic acid (20 mJ) was added to 0Bzl (5,59,10 mmol) under cooling, and the mixture was further stirred for 5 minutes under cooling and then for 45 minutes at room temperature.

Then, 6N hydrochloric acid/dioxane (1.7 m7!,
10 mmol) was added, excess trifluoroacetic acid was distilled off under reduced pressure, and ether was added to the residue to precipitate a solid.

The precipitated solid was washed with ether and dried over sodium hydroxide.

The obtained lettuce solid was dissolved in DMF-THF (10yd~30r
fLl), cooled to -10°C and adjusted to pH 5 with N-methylmorpholine.
la-Gly-OH (3.6 g, 10 mmol) l-hydroxybenztriazole (:l.

15mmol), WSCI (2.7ml, 15m
1 mol was added sequentially at -1O℃ for 1 hour, then at room temperature for 1 hour.
Stirred for 6 hours.

After the reaction, THF was distilled off under reduced pressure, and the residue was added with IN hydrochloric acid (t
The resulting solid was collected by filtration, washed with water, 5% sodium hydrogen carbonate, water, and ether, and then DMF-
It was suspended in methanol under heating.

Ethyl acetate was added, and the precipitated solid was collected by filtration and dried under reduced pressure over phosphorus pentoxide at room temperature for 12 hours.

8) Boc-L-Pro-L-Gin-Gly-L-1
1e-L-Ala-Gly-L-Gin-D-Arg
Synthesis of (NO2)-OBzl Boc-L-Ile-L-Ala-Gly-L-Gln
-D-Arg(NO2)-0Bzl (1,2El,
trifluoroacetic acid (1.5 mmol) under cooling.
5 mA was added, and the mixture was stirred at room temperature for 1 hour.

After confirming the completion of the reaction by thin layer chromatography, trifluoroacetic acid was distilled off under reduced pressure, ether was added to the residue and kneaded, and the ether was removed by decantation.

After repeating the same operation twice, the solid was dried under reduced pressure over sodium hydroxide.

The obtained solid was dissolved in 6TrLl DMF and Boc-
L-Pr.

-L-Gl n-G1 y-OH (0.6 g, 1.5
m mol) 1-hydroxybenztriazole (0
, :l.

2.25 mmol) was added.

WSCI (0,477171!, 2.25 under cooling)
m mol) was added thereto, and the mixture was stirred for 1 hour and then at room temperature overnight.

Ether was added to the reaction solution to precipitate a solid, which was collected by filtration.

The obtained solid was washed successively with water, IN-hydrochloric acid, water, and ethyl acetate.

9) NABS-L-Pro-L-Gln-Gly-L-
11e-L-Al a-Gly-L-Gln -D-A
Synthesis of rg-OH Boc-L-Pro -L-Gl n-G1 y-L-
I le -L-Ala -Gly-L-Gin-D
-Arg (NO2) -OB z l (300m9
．． Anisole (0.5 ml) in 0.28 m mol
was added thereto, and then hydrogen fluoride (7 ml) was added at 0°C and the mixture was reacted for 1 hour.

Excess hydrogen fluoride was distilled off under reduced pressure, ether was added to the residue and kneaded to form a solid, and the ether was removed by decantation.

After repeating the same operation twice, it was dried under reduced pressure over sodium hydroxide.

The obtained solid was dissolved in a 50% aqueous acetic acid solution and 'D o
It was passed through a Wexl X 2 (acetate type) and flushed out with the same solvent.

The effluent was concentrated to dryness under reduced pressure, the residue was dissolved in 5TLl of water, and sodium hydrogen carbonate (110rn9.1.
3 mmol) and p-(4-hydroxy-
■-Naphthylaso)benzenesulfonyl chloride (
A solution of 240m9.0.7mmol) dissolved in THF5TL1 was added.

Thereafter, the mixture was stirred at room temperature for 3 hours.

The reaction solution was concentrated under reduced pressure to remove THF, and the aqueous layer was extracted with ethyl acetate to remove unreacted p-(4-hydroxy-l-naphthylazo)benzenesulfonyl chloride.

Next, the aqueous solution was diluted with a large amount of water, and then passed through a high porous resin (DIAION HP-20).

After washing the resin with a large amount of water, it was eluted with a 90% methanol aqueous solution.

The eluate was concentrated to dryness under reduced pressure, and the residue was dissolved in 50% acetic acid aqueous solution, passed through Dowex IX2 (acetate type), and eluted with 50% acetic acid aqueous solution.

The effluent was concentrated to dryness under reduced pressure, and ethyl acetate was added to the residue to obtain a red solid.

Yield 190■ (57%) Melting point (decomposition point) 230° to 239°C Example 2 PAB-L-Pro ro-L-Gln-Gly-L-I
1e-L-Ala -Gly -L-Gin -D-A
Synthesis of rg-OH 1) PAB-L-Pro-L-Gin
-Synthesis of Gly-OH L-Pro-L-GIn-Gly
-OH (400 μl, 1.3 mmol) was dissolved in 5 TLl of water, and sodium hydrogen carbonate (220 mtjl,
2.6 mmol) was added, and under cooling, p-phenylazobenzyl chloride (320 m9, 1.3 m mo
1) dissolved in 5 ml of THF was added, followed by stirring at room temperature for 3 hours.

After concentrating the reaction solution while reducing the volume and distilling off THF, IN-
The pH of the aqueous solution was adjusted to 2 with hydrochloric acid, and ethyl acetate was added for extraction.

The ethyl acetate layer was washed with saturated brine and dried over Glauber's salt.

After removing Glauber's salt, the mixture was concentrated to dryness under reduced volume, and the residue was reprecipitated from methanol-ether.

Yield 360■ (53□) Melting point (decomposition point) 1898-194℃ (a) 28 -2.0° (C=0.5.DMF) Elemental analysis CHN% Calculated value 55.605.8815.5
6 (as C25H280e No.7H20) Analysis value
55.615.4815.332
)PAB-L-Pro-L-Gln-Gly-L-11
e-L-Ala-Gly-L-Gin-D-Arg
(No. 2) Synthesis of -0Bzl oc -L-I 1e-L-Ala -Gly-L
-Gl n-D-Arg(NO2)-0Bzl (20
Trifluoroacetic acid was added to the solution (0771, 0.26 mmol) under cooling, and the mixture was stirred at room temperature for 1 hour.

After confirming the completion of the reaction by thin layer chromatography, excess trifluoroacetic acid was distilled off under reduced volume, ether was added to the residue and kneaded to form a solid, and the ether was removed by decantation.

After repeating the same operation twice, the solid was dried under reduced pressure over sodium hydroxide.

The obtained solid was dissolved in 311 Ll of DMF and PAB-
L-Pro-L-Gln-Gly-OH (130m1i
l, 0.26 mmol) l-hydroxybenztriazole (70 ■.

WSCI (0.52 mmol) was added under cooling.
, 1 ml, 0.52 mmol) and stirred for 1 hour.

Thereafter, the mixture was stirred at room temperature for one night.

Ether was added to the reaction solution, and the solid was collected by filtration and washed successively with water, 1N aqueous hydrochloric acid, and ethyl acetate.

Yield 230Tn9 (75%) 3) PAB-L-Pro-L-Gin-Gly-L-1
1e-L-Ala-Gly-L-Gl n-D-Arg
Synthesis of -OH PAB-L-Pro-L-Gln-L-I 1e-L-
AlaGly-L-Gln-D-Arg (NO2)
-0Bz l (2001119, 0,17m
mol) was added to 0.5 ml of anisole, and the mixture was reacted with 7 TIL of hydrogen fluoride for 1 hour at o'c.

Excess hydrogen fluoride was distilled off under reduced pressure, and ether was added to the residue to make it solid, and the ether was removed by decantation.

The same operation was repeated twice, and the obtained solid was dried under reduced pressure over sodium hydroxide.

This solid was dissolved in 50% acetic acid aqueous solution, -, Dowe
xl x 2 (acetate type) and eluted with 50% acetic acid aqueous solution.

The effluent was concentrated to dryness under reduced pressure, and ethyl acetate was added to the residue to obtain an orange solid.

Yield 1501n9 (80%) Melting point (decomposition point) 1968-205°C (a) Back -34.9° (C = 0.4, 50% acetic acid aqueous solution Example 3 NABS-L-Pro-L- as substrate Ala-Gly
-L-11e-L-Ala-Gly-L-Gln-D-
Using Arg-OH, 20% ethanol-0.05M Tris-HCl buffer (pH 7.8, 0.15M NaCl, 5mM Ca
C12fi).

Purified collagenase (17.4 units 7ml) diluted with buffer in 0.511L of the above substrate solution 0.1
- was added to start the reaction.

After 60 minutes, 0.1 rfull of 5N hydrochloric acid was added to the reaction solution to stop the enzyme reaction.

Next, 1 ml of ethyl acetate was added to extract the NABS-containing hydrophobic decomposition product.

3,000-5.000 rpm using a centrifuge 10
After centrifugation at room temperature for minutes, the organic layer was separated into a 0.5-cell and the absorbance at 389 nm was measured to determine the enzyme activity.As a result, the activity was OD 389/hr/mA'=0
．． It was 100.

Example 4 Substrate binding PAB-L-Pro-L-Ala-C1y-L
-Lle-L-Ala-Gly-L-Gln-D-Ar
Using g-OH, the substrate concentration was 5 x 10-'M.
．． 05M Tris-HCl buffer (pH 7.8, 0.15M
NaC1.

(containing 5mM CaCl2).

0.1 ml of purified collagenase diluted with a buffer solution was added to 0.1 ml of the above substrate solution to start the reaction.

After 60 minutes, 0.5 rrLl of IN hydrochloric acid was added to the reaction solution to stop the reaction.

Next, after adding sodium chloride to saturation, the PAB-containing hydrophobic decomposition product was extracted with 1 TIL of ethyl acetate, and thereafter treated in the same manner as in Example 3, and the absorbance at 325 nm was measured.

Claims (1)

[Claims] 1 General formula % Formula % (wherein, Pro, Gln. Gly, Ile, Ala and Arg are respectively proline, glutamine, glycine, isoleucine,
Alanine and arginine residues are shown. ) Peptide derivatives shown. 2 General formula % Formula % ((However, , Gln. Gly, Ile, Ala and Arg represent proline, glutamine, glycine, isoleucine, alanine and arginine residues, respectively. 1. A method for measuring collagenase activity, which comprises contacting and then quantifying XL-Pro-L-Gln-Gly-OH that is released.