CN102911253A

CN102911253A - New dipeptide derivative and salt for detecting enzyme activity

Info

Publication number: CN102911253A
Application number: CN2012104986542A
Authority: CN
Inventors: 孟琛
Original assignee: CO-HEALTH (BEIJING) LABORATORIES Co Ltd
Current assignee: CO-HEALTH (BEIJING) LABORATORIES Co Ltd
Priority date: 2012-11-30
Filing date: 2012-11-30
Publication date: 2013-02-06

Abstract

The invention relates to a substrate for synthesis of an X-L-proline-Y chemical structure, which is used for determining the activity of an X-prolyl-depeptidyl aminopeptidase, wherein X represents an amino acid residue, and Y represents para-nitroaniline, para-aminoazobenzene or 4-benzeneazo-1-naphthylamine. The compound meets the various conditions required for the substrate, for example, easiness in determination (by enzymatic hydrolysis of the substrate), stability, and linear relation between the quantity of the substrate and the content of an enzyme, as well as between the hydrolysis quantity of the substrate and the incubating time. A new dipeptide derivative and a salt are used as the substrate for detecting the activity of the X-prolyl-depeptidyl aminopeptidase, and the substrate can be used for very effective auxiliary diagnosis for diseases, in particular to diagnosis of a quantity of malignant tumors.

Description

A kind of new dipeptidase derivant and salt for detection of enzymic activity

Technical field

The present invention relates to technological field of biochemistry, be specifically related to a kind of new dipeptidase derivant and salt,, can be used for enzymic activity and detect as substrate with this dipeptidase derivant and salt.

Background technology

In order to illustrate the relation of enzymic activity and disease in the animal body, done a large amount of correlative studys both at home and abroad.The research report is arranged, and there are significant correlation in enzymic activity and disease, so enzymic activity detects the supplementary means that can be used as a kind of disease surveillance.For example, detect active differentiation obstructive jaundice and the hepatocellular jaundice of being used for of leucine aminopeptidase, glutamic-oxal(o)acetic transaminase and lactate dehydrogenase activity detect the diagnosis that is widely used in hepatopathy.With respect to healthy population, the sample of numerous disease disease all can cause these enzymic activitys higher.Therefore, for the detection of specific disease, enzymic activity detects, and does not have very high confidence level, needs in conjunction with other clinical detection data, judges.The shortcomings such as the substrate stability that exists in the conventional method for detecting enzymatic activity is bad, poor repeatability, difficult mensuration, the invention provides a kind of new dipeptidase derivant and salt, can be used for enzymic activity and detect, for the auxiliary diagnosis of disease provides a kind of more credible, safer, simpler diagnostic method.

Summary of the invention

The present invention relates to a kind of new dipeptidase derivant and salt, as substrate, for detection of enzymic activity, more credible, more stable, simpler with this dipeptidase derivant.

Technical scheme of the present invention is as follows:

At first synthesized and contained X-L-proline(Pro)-Y (X: amino-acid residue.Y: whether p-Nitroaniline to dipeptidase derivant and the salt thereof of phenylazo amine or 4-benzeneazo-naphthalidine structure, exists some endonuclease capables to be hydrolyzed these mixtures in the detection animal body.At last, we find a kind of simple enzyme by name: X-prolyl-two acyltransferase polypeptides-aminopeptidase.This kind of enzyme can be hydrolyzed to X-L-proline(Pro) and Y-H to X-L-proline(Pro)-Y, and (that is: p-Nitroaniline is to phenylazo amine or 4-benzeneazo-naphthalidine.)。In various animals (the comprising the people) body this kind of enzyme is arranged, in the human serum, in the saliva and sialisterium and different reticular tissue, for example: all contain this kind of enzyme in ox or people's dental pulp, tooth hair follicle, gums and the rat granuloma.We do substrate with these dipeptidase derivants and salt; detected the activity of X-prolyl-two acyltransferase polypeptides-aminopeptidase in the normal human serum; find that this kind of enzyme enzyme activity level in normal population is almost identical; but there is significant difference in detected result in young man and young women, also has significant difference in middle and aged women and young woman.Liver and gall diseases is arranged simultaneously (such as acute or chronic hepatitis, liver cirrhosis) this enzyme activity level significantly raises among the patients serum, and essential hypertension and malignant tumour are arranged, and such as: noumenal tumour (for example: cancer of the stomach, carcinoma of the pancreas, lung cancer) and leukemia and this enzymic activity of sarcomata patient significantly reduce.Therefore, we confirm, as substrate, detection X-prolyl-two acyltransferase polypeptides-aminopeptidase activity can be used as a kind of very effective disease aided diagnosis method with this new dipeptidase derivant and salt, especially for the diagnosis of some malignant tumours.

In addition, we confirm that present this new dipeptidase derivant and salt are safe mixtures, non-carcinogenesis, this mixture of while, satisfy the various conditions of substrate requirement, for example: it is linear easily to measure (by the enzymic hydrolysis substrate), stable, amount of substrate and enzyme content and substrate hydrolysis amount and incubation time.

(amino-acid residue (X) of the N-of X-L-proline(Pro)-Y) end comprises: amino-acid residue arbitrarily because this enzyme Main Function is in second amino-acid residue (L-PROLINE), rather than is directed to the amino-acid residue (X) of N-end to dipeptidase derivant.The amino-acid residue of N-end is often chosen from the amino acid that contains 15 above carbon atoms.For example, neutral amino acids, as: glycine, L-Ala, α-amino-isovaleric acid, leucine, Serine, Threonine, phenylalanine, tyrosine, tryptophane.Basic aminoacids, as: Methionin, arginine.If these amino acid contain asymmetric C atom, different stereochemical structures will appear, L-type is arranged, the D type, DL type space conformation generally is L-type amino acid.In addition, these dipeptidase derivants not only can exist with free form as substrate, and can the hydrochlorate form exist, and this form can inhibitory enzyme activity.For example: tosic acid, hydrochloric acid, Hydrogen bromide.Because the stability of salt is better, doing substrate with salt is better selection.

In X-L-proline(Pro)-Y, the Y part, should be from containing: p-Nitroaniline, select in the amino-acid residue to phenylazo amine or 4-benzeneazo-naphthalidine.In these several amino-acid residues, the residue that contains p-Nitroaniline is better selection, and is more stable because this residue has identical solubleness with dipeptides in water, easier to be synthetic.

Enzymic activity for X-L-proline(Pro)-Y depends on n terminal amino acid residue (X) difference.When X was neutrality or alkaline amino acid residue, dipeptidase derivant was the easiest of enzymic hydrolysis, compared not facile hydrolysis of acidic amino acid.Therefore, dipeptidase derivant X partly is neutrality or alkaline amino acid residue or hydrochlorate such as tosilate, be more suitable for as substrate, and the easy deliquescence of basic aminoacids.On the other hand, dipeptidase derivant X is glycine residue, under the environment of pH8.7, has the highest percent hydrolysis.X-L-proline(Pro)-Y derivative, more stable under the lucifuge condition, and water-soluble better.In addition, glycine and hydrochlorate thereof are compared some basic aminoacidss such as hydrochloric acid, tosic acid, are difficult for deliquescence, are easier to experimental implementation.In sum, among X-L-proline(Pro)-Y, X is glycine or its salt, during tosilate, is best substrate combination selection particularly.

Novel dipeptide has the such structure of X-L-proline(Pro)-Y, can adopt at an easy rate the method for knowing in the chemistry of peptides to synthesize.Obtain these dipeptides, both can adopt following method, with L-PROLINE-Y, corresponding to N end shelter-the N end of X part shelters-X amino acid, being coupled as the N end with the condensing agent of similar carbon diimide derivative shelters-X-L-proline(Pro)-Y, remove again and shelter group, also can adopt active ester or similar method.Be suitable for n terminal amino acid shelter group can from know group such as benzyloxy acyl group, uncle-Ding oxygen acyl group and uncle-penta oxygen acyl group select.If the amino acid that uses has the function side group, after the function side group was sheltered, this amino acid also can obtain to use.Such as, after side chain carboxyl group being converted to ester group (such as the benzyl ester), acidic amino acid can use.Guanidine radicals with general shelter group such as methylsulfonyl, to nitrobenzyl acyl group and 2-(isopropoxy acyl group)-3; 4; 5; after 6-tetrachlorobenzene formyl radical (especially front two kinds) is sheltered; can use; and the N of Methionin end-amino is sheltered with the amino group of sheltering of knowing usually, more usually be selected to N end-amino and shelter the identical group of sheltering.

Shelter the N end that group adopts the method for knowing to eliminate to obtain according to N end to shelter-X-L-proline(Pro)-Y shelter group, and the function side group that may exist shelter group.It is highly important that, in eliminating reaction, only eliminate and shelter group and not influential to other parts.

Also can shelter by N end-derivative N that X-L-proline(Pro) and Y-H at first are coupled synthesize the target dipeptides holds and shelters-X-L-proline(Pro)-Y, and group is sheltered in elimination again.

If wish to obtain the salt that dipeptidase derivant and acid form, then can be by in the media such as similar water, alcohol, alkali freely being contacted to prepare with acid.Can be sheltered group by what acid was eliminated like this if shelter group and be similar uncle-Ding oxygen acyl group, the derivative that the N end is sheltered contacts with identical acid, then can directly obtain the salt of expecting.

X-L-proline(Pro)-Y and salt can pass through chemical synthesis process, and is very easy to be synthetic.In various hydrochlorates, tosylate is optimal selection, and this is because benzene sulfonate can form and contain the stable crystal of a small amount of impurity, between this, can by the method for simple recrystallization, obtain the benzene sulfonate of higher degree.

Detecting enzymic activity with this dipeptidase derivant or salt as substrate is to be easy to very much.The aqueous solution of X-L-proline(Pro)-Y or salts solution at first should have a suitable concentration.Because need relatively long time dissolving substrate, therefore need to add some other suitable material hydrotropies, for example: nonionic detergent, acid, alcohol, perhaps by the substrate freeze-drying etc. method, allow substrate formation vesicular structure, to increase its solvability.The more convenient routine use of lyophilized powder.According to the substrate kind that adopts, the selection of the best pH of substrate solution is also different, but generally determines according to the experiment specific requirement.Best pH generally is selected between the 6-9, and suggestion is taken between the pH 7.5-8.9.PH is greater than 9.5, and substrate spontaneous hydrolysis, but substrate solution, particularly glycyl-L-PROLINE p-nitrophenyl amine aqueous solution is still more stable in greater than the above-mentioned best pH interval of mentioning.Can preserve more than the week, not degrade for 4 ℃.

The amount of Y-H is easy to by detecting specific wavelength, and the absorbancy of enzymatic reaction is calculated.Y-H, that is: p-Nitroaniline 385nm place is detected, and phenylazo amine 493nm is detected, and 4-benzeneazo-naphthalidine 532nm detects.This absorbance measurement method is very simple, convenient, accurate, is suitable for conventional enzymic activity and detects.

If Y-H is p-Nitroaniline, enzymic activity also can form diazonium salt by p-Nitroaniline under acidic conditions and the reaction of excessive nitrite sodium and detect.The amount of diazotizing p-Nitroaniline occuring, can calculate by the amount of the unreacted Sodium Nitrite of direct-detection, but generally detects by so-called diazo coupling method.Decompose Sodium Nitrite by thionamic acid, Ammonium sulfamate, urea or analogue, form a couplings with the reaction of p-nitrophenyl diazonium salt, then by detecting the suitable absorbance of this nitrogenous mixture, detect enzymic activity.This Indirect Detecting Method than direct mensuration absorbance method, complicated.It is high a lot of that but sensitivity is wanted, and particularly to some enzyme content sample seldom, is a kind of effectively detection method.

Embodiment

The preparation of embodiment 1:N-benzyloxy acyl group-L-PROLINE p-Nitraniline acid amides

Phosphorus oxychloride (50.6g, 0.33mol), under-10 ℃ of conditions, slowly stirring joins in tetrahydrofuran (THF) (400ml) solution that contains N-benzyloxy acyl group-L-PROLINE (74.8g, 0.3mol), p-Nitroaniline (41.4g, 0.3mol).Triethylamine (92.4mol, 0.66ml) dropwise joins in the above mixed solution, and internal temperature is controlled at-15 ℃.Behind triethylamine adjustment pH to 7.0, the reaction mixture temperature can allow to reach room temperature, then stirs 3 hours.

Solvent exchanges with ethyl acetate (1.2L).Ethyl acetate is water (400ml), 1mol hydrochloric acid (300mlx5) successively, water (400ml), 5% sodium bicarbonate (300mlx3) and saturated salt solution (300ml) washing, then dried over mgso.Dry liquid is to form by low pressure is concentrated, and nubbin is crystallization, then product recrystallization in ethyl acetate (300ml) and methyl alcohol mixed liquor by adding ether.

Output: 56g

Fusing point: 159-161 ℃.

Specific rotatory power :-107.6 ⁰(C=1, methyl alcohol)

Ultimate analysis	C	H	N%
				Calculated value (C ₁₉H ₁₉O ₅N ₃）	61.77	5.19	11.38
Experimental value	62.07	5.20	11.62

The preparation of embodiment 2:L-proline(Pro) p-Nitraniline acylamino hydrogen bromate

N-benzyloxy acyl group-L-PROLINE p-Nitraniline acid amides (55.4g, 0.15mol) dissolves with 26% anhydrous hydrogen bromide solution, and 0 ℃ joins in the 200ml acetum, stirring at room two hours.Mixed solution stirs in dried ethyl ether (2L), to crystallization.Obtain product by clarification, ether washing, drying.

Product amount 47g.

Raw product is used for following example, does not pass through purifying.Analytic sample obtains by recrystallization in methyl alcohol.

Fusing point: 225-226 ℃.

Specific rotatory power :-35.60(C=1.0, methyl alcohol)

Ultimate analysis	C	H	N%
				Calculated value (C ₁₁H ₁₄O ₃N ₃Br）	41.79	4.46	13.29
Experimental value	41.75	4.34	13.30

The preparation of embodiment 3:N-benzyloxy acyl group-glycyl-L-PROLINE p-Nitraniline acid amides

N-benzyloxy acyl group-glycinate (48g; 0.16mol) in dimethyl formamide (100ml) mixed solution that joins the triethylamine solution (22ml) that contains L-PROLINE p-Nitraniline acylamino hydrogen bromate (47g, 0.15mol) and contain N-hydroxybenzotriazole (1g).0 ℃ of stirring and evenly mixing of whole mixed solution one hour is transferred pH with triethylamine in the blending process in the meantime, and pH is 8.0 in control, and room temperature continued stirring and evenly mixing 19 hours subsequently.

Solution evaporates through low pressure, desolventizing, residuum adds 350ml water, through ethyl acetate (500ml, 200ml) respectively extracting twice get product.After the extracted twice thing mixes, successively water (200ml), 1N hydrochloric acid (200mlx2), water (200ml), 5% sodium hydrogen carbonate solution (200mlx4) and water (200ml) washing are finally by dried over mgso.

Output: 67g

Embodiment 4: the preparation of glycyl-L-PROLINE p-Nitraniline acid amides tosylate

Glycyl-L-PROLINE p-Nitraniline acid amides (584mg) is by the ethanolic soln of tosic acid monohydrate neutralization (380mg, 10ml).Solution places refrigerator, and crystallization gets raw product.Raw product is by methyl alcohol ethyl ether recrystallization.

Output: 312mg

Fusing point: 223-225 ℃

Specific rotatory power :-81.00, (C=1.0, methyl alcohol)

Embodiment 5: the preparation of uncle-Ding oxygen acyl group-L-Ala-L-Pro p-Nitraniline acid amides

Uncle-Ding oxygen acyl group-ALANINE (5.7g; 0.03mol) and L-PROLINE p-Nitraniline acylamino hydrogen bromate (9.5g; 0.03mol) dissolve with chloroform (40ml); under 0 ℃ of condition; add triethylamine (4.2ml; 0.03mol) and N, the chloroformic solution of N'-dicyclohexylcarbodiimide (5ml).Mixed solution, stirring at room 44 hours.After adding several acetic acid, prolong again and stirred 2 hours.By the method for filtering, remove throw out, filtrate is concentrated through low pressure.The residuum water is resuspended, and product obtains through ethyl acetate extraction.Extract 1N hydrochloric acid, water, 5% sodium hydrogen carbonate solution, water, saturated nacl aqueous solution, then washing uses dried over mgso successively.Dry liquid is used ether dissolution through concentrated.Some insoluble products are removed after filtration.Concentrating filter liquor is ground through normal hexane and is solidified, and gets product.

Output: 12.9g

Embodiment 6:L-alanyl-L-Pro p-Nitraniline amide hydrochloride

The dioxy hexane solution (20ml) of uncle-Ding oxygen acyl group-L-Ala-L-Pro p-Nitraniline acid amides (12.9g) 6N anhydrous hydrogen chloride dioxy hexane solution (45ml) room temperature treatment 55min.Reaction mixture is concentrated through low pressure, from the alcohol crystal thing, grinds through ether and solidifies, and gets product.

Output: 6.4g

Fusing point: 130-140 ℃

Specific rotatory power :-103.40(C=1, methyl alcohol)

Ultimate analysis	C	H	N%
				Calculated value (C ₁₄H ₁₉O ₄N ₄Cl）	46.03	5.93	15.34
Experimental value	46.12	6.09	15.50

Embodiment 7:L-aspartyl L-PROLINE p-Nitraniline acid amides

Uncle-Ding oxygen acyl-beta-benzyl-L-aspartyl-L-PROLINE p-Nitraniline acid amides (10.3g) is dissolved in methyl-phenoxide (8ml) solution, abundant mixing, and solution was processed 1 hour for 0 ℃ with anhydrous hydrogen fluoride (50ml).By the method for distillation, remove hydrogen fluoride, then add ether, make the product precipitation.Throw out ether washed twice is separated by decantation, and is then dry.

Raw product is dissolved in the acetic acid of 0.1M, and solution fully washs with ether, then crosses post (strong anion-exchange resin).Collect washes, low pressure is concentrated, and product is crystallization in water.

Output: 5.2g

Fusing point: 144-145 ℃

Specific rotatory power :-100.30(C=1,1N-HCL)

Ultimate analysis	C	H	N%
				Calculated value (C ₁₅H ₁₈O ₆N _4.3/2H ₂O）	47.74	5.61	14.85
Experimental value	48.03	5.63	14.16

Embodiment 8: about substrate hydrolysis amount and incubation time experiment.

Use respectively that glycyl-L-PROLINE p-Nitraniline acid amides is as substrate, human serum is as proenzyme.

The substrate hydrolysis amount is by following direct spectrphotometric method for measuring.

Glycyl-L-PROLINE p-Nitraniline acid amides tosylate is dissolved in 2% the nonionic detergent aqueous solution, and concentration of substrate is 3mM.Need to prepare following 4 reaction tubess: 1) developmental tube: contain 0.5ml, glycine-sodium hydrate buffer solution of the pH8.7 of 0.15M, 0.5ml substrate solution, 0.05ml human serum.

2) blank tube: contain 0.5ml, glycine-sodium hydrate buffer solution of the pH8.7 of 0.15M, 0.5ml substrate solution, 0.05ml water.

3) standard pipe: 0.5ml damping fluid, 0.5ml substrate solution, and 0.05ml 3mM p-nitrophenyl amine aqueous solution.

4) control tube: 0.5ml damping fluid, 0.5ml substrate.

4 pipes are pressed the listed incubation time of table 1, hatch through 37 ℃.By adding 3ml, 1M sodium Diacetate (pH4.2) termination reaction.Control tube after termination reaction, adds the human serum of 0.05ml.Making the blank tube absorbancy by adjustment is 0, reads developmental tube (E), control tube (C), standard pipe (S), the absorbancy of 385nm (1cm optical path).By the enzyme effect, liberate p-nitroaniline is by the amount of following formula calculating p-Nitroaniline.

E-C/S?x?150(nmol)

Test-results is as follows:

Table 1

Incubation time (dividing)	The p-Nitroaniline amount (nmol) that generates
		15	18.11
30	35.43
		60	71.65
90	100.79
		120	135.43

Confirm that according to above testing data enzymatic reaction and incubation time grow up to linear relationship.

Replace human serum with the enzyme that extracts purifying from people's submaxillary gland, do revision test by above experimental procedure.The result confirms that also enzymatic reaction and incubation time grow up to linear relationship.

Embodiment 9: the amount of enzyme and substrate hydrolysis the relationship between quantities.

Do substrate with glycyl-L-PROLINE p-Nitraniline acid amides tosylate, human serum is as proenzyme.The substrate hydrolysis amount detects by following diazo coupling method.

Need to prepare following reaction tubes:

1) developmental tube contains glycine-sodium hydrate buffer solution (pH8.7) of 1.0ml 0.15M, and the 1ml substrate solution (process by the method for pressing embodiment 8.), 0.01ml serum.

2) control tube contains glycine-sodium hydrate buffer solution (pH8.7) of 1.0ml, and the 1ml substrate solution (process by the method for pressing embodiment 8.）

3) developmental tube and control tube equal 37 ℃ hatch 30 minutes after, control tube adds 0.1ml serum.By adding high chloro acid solution's termination reaction of 0.4ml30%.Centrifugal 10 minutes of 3000rpm.Every pipe is got supernatant 0.5ml and is transferred to another pipe.In addition, add in the standard pipe: 30% the perchloric acid of the p-Nitroaniline of the 0.5M of 0.1ml and 2.4ml.Blank tube adds the perchloric acid solution of 2.5ml.

Under 4 ℃ of conditions, add respectively the sodium nitrite solution of 0.5ml 0.2% in 4 pipes, kept 10 minutes for 4 ℃.Subsequently, the thionamic acid aqueous ammonium 0.5ml of adding 0.5%.After 2 minutes, add 0.05% N-(1-naphthyl) ethylenediamine solution of 1ml, 37 ℃ of mixed solutions, lucifuge, temperature was bathed 30 minutes.

The blank tube absorbance is adjusted into 0, reads developmental tube (E), control tube (C), and the absorbance of standard pipe (S) 548nm.

The p-Nitroaniline growing amount, calculate by following formula:

(E-C)x500A/S?x0.5

Wherein, A: the consumption (ml) of 0.5mM p-nitrophenyl amine aqueous solution in the standard pipe.

Table 2

Serum amount (ml)	P-Nitroaniline growing amount (nmol)
		0.01	14.24
0.02	32.91
		0.03	45.89
0.04	62.50
		0.05	82.44
0.10	155.06

According to above-mentioned data acknowledgement, enzymatic reaction and enzyme amount are linear.

Embodiment 10 :

Glycyl-L-PROLINE p-Nitraniline acid amides is dissolved in 2% the nonionic detergent aqueous solution, the substrate solution of preparation 3mM.Different derivative or salt can be used as substrate, have measured the relative activity of different substrates, and enzyme is to percent hydrolysis and the optimum response pH of substrate.Table 3 is test-results.

Note:

A. the enzyme assay damping fluid is pH 5.6-8.8, glycine-sodium hydrate buffer solution of the 0.15M of 0.5M Tris-maleate damping fluid and pH 8.2-9.4.For substrate L-alanyl L-PROLINE p-Nitraniline acid amides, the damping fluid of usefulness is the amidiol damping fluid, rather than glycine buffer, because glycine buffer has interference to absorbancy between pH 9-10.

B. people's submaxillary gland enzyme of purifying at pH7.0, is measured under the Tris-maleate buffer conditions.The result gets the mean value of twice replication.

C. enzymic activity is measured at Tris or the 0.15M glycine-sodium hydrate buffer solution of the 0.15M of pH 8.7.The result gets the mean value of twice replication.

Embodiment 11: detected the enzymic activity of X-prolyl-two acyltransferase polypeptides-aminopeptidase with the method for embodiment 8, done substrate with glycyl-L-PROLINE p-Nitraniline acid amides, 88 routine normal human serum samples are as proenzyme.Detected result such as table 4.

Table 4

The sample group	Sample number	Age	Enzymic activity (umol/min/1serm ± S.E)
				Total sample	88	15-81	54.88±1.50
The male sex	53	15-81	56.30±1.90
				The women	35	20-76	52.49±2.47
Young	42	15-40	54.76±2.07
				The male sex	24	15-40	60.10±2.84 ^a
The women	18	20-40	47.59±2.07
				Person in middle and old age	46	41-81	55.04±2.23
The male sex	29	41-81	53.18±2.43
				The women	17	48-76	58.16±4.37 ^b

Note:

The detected value of a young man (less than 40 years old) is significantly higher than young women, test of significance P＜0.001

B middle-aging male detected value is significantly higher than the young woman, test of significance P＜0.05

Embodiment 12: detected the enzymic activity of X-prolyl-two acyltransferase polypeptides-aminopeptidase with the method for embodiment 8, done substrate with glycyl-L-PROLINE p-Nitraniline acid amides, 88 routine pathology serum samples are as proenzyme.Detected result such as table 5.

Table 5

The sample group	Sample number	Enzymic activity (umol/min/1serm ± S.E)	With check sample test of significance relatively
				Normal (contrast) sample	88	54.9±1.5 P＜0.01	P＜0.01
The hepatitis B sample	23	75.8±28.5	P＜0.001
				Early stage primary hypertension sample	20	80.94±3,87	P＜0.001
The primary hypertension sample	17	89.29±3.69	P＜0.001
				The cancer of the stomach sample	37	38.4±2.3	P＜0.001
The lung cancer sample	17	39.8±2.6	P＜0.001
				The acute lymphoblastic leukemia sample	22	38.8±11,9	P＜0.01
The lymphosarcoma sample	11	36.3±5.6	P＜0.01
				The Hodgkin's disease sample	15	31.0±9.5	P＜0.01

Embodiment 13:

Detected the enzymic activity of X-prolyl-two acyltransferase polypeptides-aminopeptidase with the method for embodiment 8, different amino-acid residues is as substrate, and patients serum's sample is as enzyme source.Detected result such as table 6.

Note: Gly, L-Ala, L-Asp, L-Glu, L-Arg represent the amino-acid residue X in the X-proline(Pro) p-Nitroaniline.

Claims

1. dipeptidase derivant is following structure: X-L-proline(Pro)--Y, wherein X can choose from following amino acid whose residue: glycine, L-Ala, α-amino-isovaleric acid, leucine, Serine, Threonine, phenylalanine,, tyrosine, tryptophane, L-glutamic acid, aspartic acid, Methionin, arginine; Y can choose from following compound: p-Nitroaniline, to the bisalt of phenylazo amine, 4-benzeneazo-naphthalidine and dipeptidase derivant.

2. described according to claim 1, it is characterized in that, X be glycine, L-Ala, α-amino-isovaleric acid, leucine, Serine, Threonine, phenylalanine,, tyrosine, tryptophane, L-glutamic acid, aspartic acid, Methionin, arginic amino-acid residue, hydrochlorate is following several: tosilate, hydrochloride, hydrobromate.

3. described according to claim 1, wherein Y is the p-Nitroaniline residue, and X then is the L-type amino-acid residue for other amino-acid residue X except glycine residue.

4. described according to claim 1, it is characterized in that when X was glycine residue, X can be glycyl-L-PROLINE p-Nitraniline acid amides.

5. described according to claim 1, it is characterized in that when X was glycine residue, X can be glycyl-L-PROLINE p-Nitraniline acid amides tosylate.

6. described according to claim 1, when X was the L-type amino-acid residue, X-L-proline(Pro)-Y can be, L-lysyl L-PROLINE p-Nitraniline acid amides.

7. described according to claim 1, when X is the L-type amino-acid residue, X-L-proline(Pro)-Y can be, L-arginyl L-PROLINE p-Nitraniline acid amides.

According to claim 1 described when X be the L-type amino-acid residue, X-L-proline(Pro)-Y can be, L-alanyl L-PROLINE p-Nitraniline acid amides.

9. described according to claim 1, when X is the L-type amino-acid residue, X-L-proline(Pro)-Y can be, L-glutamy L-PROLINE p-Nitraniline acid amides.

10. described according to claim 1, when X is the L-type amino-acid residue, X-L-proline(Pro)-Y can be, L-aspartyl L-PROLINE p-Nitraniline acid amides.