CN1313290A - Hydrolytic active substance of brain protein, its preparing process and its activity and sequence - Google Patents

Abstract

A process for separating and purifying active substance from the primary hydrolytic liquid of brain protein is disclosed. After activity, purity, molecular weight, amino acid sequence and terminal analysises to the obtained active substance, a high-purity active brain protein substance with single peak is found and synthesized, which has same components, structure and amino acid sequence as one separated from brain protein.

Description

Hydrolytic active substance of brain protein, its preparation method, activity and sequence

The present invention relates to a kind of hydrolytic active substance of brain protein, its preparation method with and activity and sequence, belong to pharmaceutical field.

The complication that causes of disease of brain such as presenile dementia, cerebral apoplexy, cerebral ischemia is perplexing the mankind always for a long time.The large quantities of medical workers in various countries actively throw oneself into this cause, have done a large amount of research and work, have also obtained many gratifying achievements.Many medicines are come out one after another.The cerebrolysin of Austria's EBEWE pharmaceutical factory exploitation is one of them.This passes through the R and D of several years surely, has developed cerebrolysin analogous products one brain protein hydrolyte.This achievement has been applied for national patent, the patent No. 94104516, and dropped into production, produced sizable social benefit.

The brain protein hydrolyte is organ specificity amino acid, the peptide complex that animal brain albumen produces through manually operated enzyme liberating, because it 85% is an amino acid, and its amino acid ratio to each other is similar to person in the normal brain activity, so still keep special " amino acid " property of its organ.These amino acid comprise: L-Ala, arginine, phenylalanine, proline(Pro), Serine, tryptophane, Xie Ansuan.Wherein 15% by molecular weight less than 1000d, do not have antigenic small-molecular peptides and form.

About the existing many article reports of this medicine, wherein most important effect is: it has provide protection to poisoning, anoxic, vagal stimulation.When hypoglycemic coma, it wakes effect up, and the maturation of brain cell is had promoter action.For the brain protein hydrolyte mechanism of action, existing at present a large amount of research.Phlorrhizen is poisoned to test and shown: the intravenous injection cerebrolysin is poisoned to animal provide protection.The clinical recovery and the electroencephalogram normalizing of poisoning hindbrain extract for treating group alive are very fast.Suffocate and anoxic experiment in, cerebrolysin can make it to recover better.The young animal that cerebrolysin is handled, the growth of brain capillary network better.Cerebrolysin has specific action to rrna, stimulates the synthetic of brain internal protein, stimulates diencephalon one hypothalamic hypophysial system growth hormone releasing, Triiodothyronine and quickens the g and D of animal.Austria has carried out relevant preclinical research, with the oxygen-consumption of polarogram commercial measurement brain homogenate.When adding cerebrolysin, its oxygen metabolism is obviously suppressed.Cerebrolysin can reduce the requirement of brain to oxygen, thereby causes brain that the anoxybiotic tolerance is increased.It also strengthens aerobic metabolism on the other hand, makes cell can finish energy dependence work, as: protein, neurotransmitter synthetic, iron consumes function.Experiment also shows: after handling animal with cerebrolysin, the animal learning achievement is better, and can keep several days.The research that this chamber is done has also proved this point.

In the brain protein hydrolyte, most amino acid plays very important trophism to the recovery of brain injury.In addition, peptide class wherein has tangible physiologically active.They act on some link of respiratory chain, and the oxygen metabolism of brain cell is played important regulatory role; Simultaneously, they also influence protein, polypeptide synthetic of brain cell, improve the physiological function of brain.The molecular weight of many brain peptides is little, and some fragment has whole or most of biological activitys of original peptide; Or some new intensive central action.These fragments in the brain protein hydrolyte are separated, study its biological activity, have certain theory and practical value.

About the research of brain protein hydrolyte, mostly concentrate on its physiologically active and pharmacological action aspect at home and abroad, do not see the report of separation and purification aspect.

The object of the present invention is to provide a kind of hydrolytic active substance of brain protein, this active constituent has very high purity.

Next purpose of the present invention is, adopts modern instrument, and the activeconstituents in the brain protein hydrolyte primary products is separated, and provides a kind of by separating and the method preparation composition of purifying is single, the method for the brain protein-active material of based on very high purity.

Another object of the present invention is by a series of mensuration, determines the purity and the sequence of brain protein-active material.

A further object of the present invention is to adopt the synthetic method, synthetic above-mentioned brain protein-active material, the pacing of going forward side by side fixed its purity, sequence and physicochemical constant.

To achieve these goals, the technical solution used in the present invention is as follows:

One. the separation of brain protein hydrolyte and purifying:

In the separating purification work of protein and polypeptide, the normal method that adopts has multiple, as liquid phase column chromatography, electrophoretic method, the precipitator method, centrifuging, thin-layer method etc.Wherein liquid column chromatography is because its separation condition gentleness, resolving power height, simple to operate, and obtained using widely.Liquid phase column chromatography chromatogram method generally is divided into two classes: classical liquid phase chromatography and high performance liquid chromatography.In the primary separation of protein and polypeptide, the classical ion exchange method and the method for gel filtration method Chang Zuowei first-selection.High performance liquid chromatography is owing to its high theoretical plate number is applied to the fine separation of sample, the strong instrument of Chang Zuowei later stage purifying.The present invention mainly adopts liquid chromatography that the active ingredient of brain protein hydrolyte is carried out separation and purification.Separate the method and the process that adopt and see Fig. 1.

At first, measure the activity of 5 groups of elementary brain protein products that Li Bao company provides, therefrom optimize active the byest force, carry out next step separation.

The protein-active measuring principle:

When cellular respiration, consume O ₂, emit CO simultaneously ₂The method that measurement is breathed has multiple, as chemical process, volume method, manometry, Carsesian cartesian diver method and oxygen electrode method.The present invention adopts the activity of manometry measure sample.The Wa Shi respirometer can be measured the gas that is taken place when tissue or microorganism carry out metabolism and change.O for example ₂Absorption and CO ₂Discharge.

Wa Shi respirometer manometry is under the condition of constant temperature and constant volume, represents to organize or the gas that taken place during cellular metabolism changes with the variation of pressure.Owing in respiratory, absorb CO ₂Carry out simultaneously with producing oxygen, so the viewed pressure change of pressure warning unit is oxygen absorption amount and CO ₂The overall result of generation.If in reaction flask, add alkali (as KOH), the CO that is produced when then breathing ₂Absorbed by alkali, can measure oxygen absorption by the variation of pressure thus.How much determine the bioactive height of trial-product with oxygen consumption in the present invention experiment.By surveying the slip-knot fruit as can be seen, the activity of the 3rd group of sample is the strongest, therefore mainly carries out separation, purifying and further analysis of sample III in the following example.

One) classical liquid phase chromatography

Although high performance liquid chromatography has obtained rapidly, application widely, be mainly used in the later stage purifying.Applied sample amount is big, the moving phase price is quite lower, the not high relatively plurality of advantages of the requirement of instrument, technology so the present invention are at first adopted classical liquid chromatography, and sample is carried out roughing out because classical liquid phase chromatography has; Also comprise ion exchange method and molecular sieve gel method in the classical liquid chromatography.

1, ion exchange method is separated: ion-exchange chromatography is one of method that obtains in protein is purified at present widespread use.It utilizes proteinic alive part strong and weak different with the ion-exchanger absorption with opposite charges, adopts the moving phase of different eluting powers, respectively they is eluted.Ion Exchange Medium such as ion-exchange cellulose resin have open support skeleton, and macromole can enter and diffusion rapidly freely, thereby bigger to the loading capacity of sample.Cation exchange groups on the ion-exchange cellulose is arranged loose, and is not too firm to the absorption of albumen, polypeptide, makes and it can be eluted, the unlikely sex change that causes molecule with gentle condition.During the ion-exchanger sample separation, the main ionic strength or the change potential of hydrogen that increase buffered soln of relying on carried out wash-out.Because the charge density distribution difference of different proteins, the quantity of electric charge does not wait, the difference of iso-electric point and the difference of molecular size, thereby, utilize certain permutizer condition with different from bonding strength in exchanger, they can be separated one by one.The present invention utilizes the diethylin Mierocrystalline cellulose, and promptly 32 pairs of samples of DEAE Mierocrystalline cellulose carry out initial gross separation.By the determination of activity result of the sample of initial gross separation as can be seen, the isolated 1# of DEAE ion exchange method peak, promptly first group of sample DEAE1 has the most of active of raw sample, should further separate this sample.

2. gel filtration method is separated: molecular sieve refers to some hydrophilic porous media.When the mixture flow of the molecule that contains different sizes during through this medium, diameter can not enter numb glue inside greater than the molecule in aperture, just directly flow out from gel intergranular crack, and this is called full discharge.Can enter the medium internal void than small-molecule substance, take a devious route, the retention time in post is just long.Like this, eluted earlier than macromole, and than being eluted behind the small molecules, thereby reach isolating purpose.This effect is called as " molecular sieve " effect.Material with this effect is a lot, and wherein effect has dextrane gel preferably, and trade name Sephadex, polyacrylamide gel claim Bio-Gel P and sepharose Agrose etc. again.

Dextrane gel is the most frequently used a kind of gel, by bacterial dextran, claims dextran again, uses linking agent 1-chloro-2 between the long-chain of sugar, 3-propylene oxide (CH ₂-CH-CH ₂The Cl Epicholorohydrin) crosslinked forming; Its basic structure as shown in Figure 6; The simple principle of gel chromatography is seen Fig. 7; The sample liquid upper prop that at first will contain sized molecules among Fig. 3 a; The sample solution chromatography column of flowing through, small molecules enters in the gel micropore that draws by diffusion, macromole then by exclusion outside particle, therefore, the speed generation difference that sized molecules moves down, and sized molecules is separated; Add elutriant to the chromatography capital, the distance that sized molecules is separated increases, and the macromolecular substance stroke is shorter, has flowed out chromatography column, and small-molecule substance still among advancing, reaches isolating purpose thus.Among the present invention, adopt gel filtration method isolating ions exchange process to separate the sample DEAE1 that obtains, by measure and calculation result as can be seen, the sample DEX2 at No. 2 peaks has the most activity of raw sample, should further separate this sample.

Two) high pressure lipuid chromatography (HPLC) is separated:

Through the isolating sample of classical liquid phase chromatography, composition is simpler relatively than first extract, and constitutive property is very approaching, should adopt HPLC further to separate.Compare with classical liquid phase chromatography, high performance liquid chromatography has the separation efficiency height: now can reach every meter 100,000 theoretical plate number, at a high speed, high pressure, analysis time be short: be generally several minutes between dozens of minutes, the detection sensitivity height: as UV-detector, sensitivity is 10 ^-9The characteristics of g/ml.

1. high-effect ionic exchange liquid phase chromatography is separated (HPIEC)

Principle: the separation principle and the classical ion exchange chromatography principle of high-effect ionic exchange liquid chromatography are basic identical.Different is that the HPIEC filler can be high pressure resistant, has higher resolving power.With wetting ability superpolymer gel is IEC filler such as TSKGELDEAE-5Pw, SP-5PW, the CM-5PW etc. of matrix, is present most widely used product.The present invention can adopt above-mentioned various filler, preferably adopts ProGel-TSK DEAE-5PW post.Adopting high-effect ionic exchange liquid phase chromatography to separate adopts gel filtration method to separate the sample SEPHADEX II (being the DEX II) that obtains, by measure and calculation result as can be seen: the sample at TSK3 peak has the most activity of raw sample, should further separate this sample.

2, the efficient gel liquid phase chromatography is separated (HPGC)

The separation principle of efficient gel liquid phase chromatography is similar substantially to classical gel chromatography principle, and promptly under the moving phase of certain ionic strength, when sample passed through gel column, elution order was a molecular weight pre-preface from big to small.But owing to electric charge, the polar influence of silica gel in the HPGC filler and bonding phase, its separation has uniqueness again, has higher resolving power.Adopt Tianjin, island high-pressure liquid phase system among the present invention, sample separation TSK3, by measure and calculation result as can be seen: the sample at PAK2 peak has the most activity of raw sample, should further separate this sample.

3, high performance reversed phase chromatography separates (HPRPC) principle: high performance reversed phase chromatography claims non-polar linkage phase chromatogram again.The bonding phase surface all is the very little alkyl of polarity, as octadecyl, octyl, methyl, phenyl etc.The most frequently used is octadecylsilane chemically bonded silica, claims ODS again.And elutriant mostly adopts the damping fluid of strong polar solvent such as water, alcohol, acetonitrile or inorganic salt.In this bonded-phase chromatography, the compound elder generation wash-out (K ' little) that polarity is big, the compound that polarity is little are difficult for wash-out (K ' big).Reverse-phase chromatography has obtained using widely in the separation of protein, polypeptide, and its major cause is the simplicity and the handiness of this operating system.Adopt Tianjin, island high-pressure liquid phase system among the present invention, sample separation Pak2, by measure and calculation result as can be seen: the sample at ZBX4 peak has the most activity of raw sample.

Brief summary: five groups of samples are lived through surveying, and select beautiful precious III sample and be the powerhouse of activity.Through the separation of the exchange of DEAE cellulose ion, SePhadex gel-filtration and high performance liquid chromatography, isolated active result ZBX4, reached its intended purposes.Further it is further analyzed, measure its aminoacid sequence.

Two. purity and sequential analysis

One) purity testing:

The purpose of protein, polypeptide separation and purification obtains the single sample of composition exactly.In fact, also do not have a kind of method of complete purity testing, have only the impure or inhomogenous method of test sample.Purity finally depends on the type and the resolving power of used method.Method with low resolution proves pure sample, when using high-resolution method instead, just may prove that it is not pure, and each method can only be described sample character in one aspect.For example, measure with high-resolution SDS electrophoretic method, obtain a band, this can only interpret sample be a homogeneous aspect molecular weight.Therefore, best purity rubric is: set up various analysis, from different angles: the homogeneity of aspect working samples such as molecular weight, hydrophobicity, charging property.The present invention utilizes liquid phase chromatography multicolumn system and capillary electrophoresis to measure purity.

1, high performance size exclusion chromatography method (HPGC)

Gel chromatography is based on molecular dimension and carries out isolating method, the present invention's homogeneity of gel filtration method test sample aspect molecular weight.The gel-filtration that obtains as a result figure and gel-filtration three dimensional chromatogram all only provide one unimodal, illustrate to obtain the single sample of purity.

2, high performance reversed phase chromatography (HPRPC)

High performance reversed phase chromatography is based on the hydrophobic effect between solute, polarity moving phase and non-polar stationary phase surface and a kind of chromatogram mode of setting up.The present invention utilizes its test sample polar homogeneity.Detected result shows that purity is good.

3, high performance capillary electrophoresis (HPCE)

Two) efficient gel chromatography determination molecular weight

Principle: the separation principle of efficient gel liquid phase chromatography is similar substantially to classical gel chromatography principle, and promptly under the moving phase of certain ionic strength, when sample passed through gel column, the pre-preface of wash-out was a molecular weight order from big to small.The molecular weight of the position at peak and this material has direct quantitative relationship during wash-out.In the gel column, the volume of the contained solution of freeboard is called void volume V between particle ₀Can not enter those macromole of gel, when elution volume is V _oThe time, elution peak appears.The cumulative volume of gel particle internal void is called inner volume Vi, can all penetrate those small molecules of gel, when elution volume is V ₀During+Ve, elution peak appears.

The molecular sieve partition ratio is defined as: K=Ve/Vi.If sample molecule be a sphere, the Stock radius is a, and the aperture of modern gel is the cone of homogeneous, the radius of bottom surface be r according to theoretic analysis, within the specific limits, K and Loga/r are in linear relation.K=-bLoga/r-c (b, c are constant).Because molecular weight and S tock radius have proportional relation, following formula also can be expressed as:

K=-b′logM+c′

Can make typical curve thus, the molecular weight of working sample.

According to the literature, adopt suitable column condition and moving phase, the albumen that can measure, the scope of polypeptide molecular weight can extend to 1000Dol.Measure by standard specimen among the present invention, obtain standard equation and be:

LogM=-0.20087097X+5.34795

Dispersion:0.1297979

Efficient gel color atlas in conjunction with sample obtains molecular weight analyte calculation result:

MN=10 ^{(-020087097×9.502+5.34795885)}=2749.68

Three) ultraviolet spectroscopy:

When the light Continuous irradiation molecule or the atomic time of certain wavelength region, there is the light of one or several wavelength to be absorbed, this spectrum is called absorption spectrum, and the light that is absorbed is in the ultraviolet region and then is called UV spectrum.

Material is decided by the distribution and the combination of electronics in its molecular structure, the molecule to the absorption of UV-light.From chemical bond, and mainly contain three kinds of the relevant electronics of absorption spectrum: form single bonded σDian Zi, the n electronics that forms the πDian Zi of multiple key and share.

Containing peptide bond in polypeptide and the protein, is a kind of conjugated system, and strong absorption is arranged near 210nm.Phenylalanine, tyrosine, tryptophane have absorption peak owing to contain phenyl ring near 280nm.This experiment utilizes water as solvent, the ultra-violet absorption spectrum of working sample.

Four) the N-terminal homogeneity is measured:

Principle: polymeric amide is class raw materials of chemical fibres, claims nylon again.It has adsorption to many polar compounds, has special resolution performance, can be used for column chromatography, thin-layer chromatography and membrane chromatographic.Polyamide layer is that polymeric amide is coated on the polyester flake, forms quality porous membrane closely uniformly, and chromatographic property is very good.

Because polymeric amide-C=O is basic to be reached-NH ₂Base can form hydrogen bond with separated material, thereby can adsorb acids, phenols, quinones, nitro and aminocompound etc.Because various materials are different with the ability that polymeric amide forms hydrogen bond, be the adsorptive power difference of polymeric amide to various materials, and in the chromatography process, exhibition layer solvent and separated material competitively form hydrogen bond at polyamide surface, therefore, select suitable exhibition layer solvent, can make various materials to be separated between polyamide surface and solvent, different partition ratios be arranged, through the attached exhibition layer process of adsorption and desorption, separate by certain order separately.

Fluorescent reagent Dansyl-Cl (dimethylamino naphthalic sulfonic chloride) can be combined into the Dansyl-amino acid that has fluorescence with amino acid, and reaction principle is seen Figure 24.

Reaction product DNS monoamino-acid has the intensive green fluorescence under ultraviolet ray, detection sensitivity can reach 10 ^-9--10 ^-10Mole, higher 100 times than dinitrofluorobenzene method, only need 1 nmole sample just can analyze.Have 70% destroyed, tryptophane and the DNS derivative completely destroy thereof except that DNS one proline(Pro) spends the night in 110 ℃ of hydrolysis through 6MHCl, DNS amino acid is more steady.

Five) sequencing

The amino-acid sequence of phthalein is measured the main Edman of use chemical degradation method, also has enzymolysis process, enzymolysis-peptide spectrum overlay method in addition, and gas chromatography/mass spectrometry coupling method etc.Edma n chemical degradation method is the sequence measurement of using always.

Principle: Edman chemical degradation method (Edman Degradation) is that P.Edman at first put forward in nineteen fifty.Be used for the N-terminal analysis at first, i.e. phenylthioisocyanate fat (PITC) method.Edman degraded reagent (PITC) and the amino effect of the free-end of polypeptide chain, DeR can be divided into for three steps.

The first step is a linked reaction:

Be reflected in the weak alkaline medium and carry out, PITC can only work with unprotonated amino.

Second step was the cyclisation cleavage reaction:

The PTC-peptide that forms in the first step reaction is that the peptide bond of close PTC base will rupture in anhydrous strong acid media such as trifluoroacetic acid, and the PTC-amino-acid residue is cyclized into thiazolinone aniline (aniline thiazolinine) derivative.Reaction needs to carry out under anhydrous condition, otherwise other peptide bond is with hydrolysis.

The 3rd step was conversion reaction:

The ATZ instability that generates in the second step reaction is difficult to be used for identifying this amino acid, therefore it is changed into PTH-amino acid in acidic aqueous solution.Conversion reaction reality was divided into for two steps again carries out, and at first hydrolysis generates PTC-amino acid, is cyclized into PTH-amino acid then.This is a highly stable compound.PTH-amino acid has strong absorption in the ultraviolet region, obtained the maximum absorption is at the 268nm place.PTH-amino acid can utilize various chromatographic techniques to separate.Because PITC with after the free α terminal amino group of peptide chain combine, just weakens the peptide bond of the terminal residue carboxyl side of adjacent PTC base, therefore under anhydrous condition acid effect, only downcut and that amino-acid residue that PITC reacts.At this moment Sheng Xia minimizing the peptide bond of a residue just expose a new free N-terminal at its N-terminal, can participate in second again and take turns reaction.And then downcut second residue, circulate with this.If the n wheel reacts like this, just can measure the order of n residue.

Utilize Edman degraded, once can measure the order of the peptide section of 60-70 residue continuously, also have report once to measure 90-100 the pre-preface of residue I.

Through clinical proof, brain proteolysis ripple has the obvious treatment effect to cerebral ischemia, brain injury.5 groups of samples that Li Bao company provides are to get the Asia crude product through the gel-filtration initial gross separation.Its retention time increases successively.According to surveying slip-knot really, wherein the 3rd group, the 5th group sample has activity.First group, second group, the 4th group sample do not have activity.This illustrates that contained active substance is different in these two groups of samples, promptly contains many group active substances in the brain protein hydrolyte.Obtained confirmation in this experiment afterwards.In 4 groups of components that are separated to ion exchange method, there are two groups to have activity.When separating, also there are two groups of components to show activity with ultra-fine gel-filtration.This all illustrates, contains the various active composition in this medicine.Active power and whether have synergy each other between these components still needs further to inquire into.For negative, may be because sample itself has the effect that suppresses breathing as for some activity in other group.

In experiment, find each active ingredient effect and existence relation between the time.The 3rd group of sample is after 1.5 hours, and the Wa Shi respirometer is measured and found that activity is significantly increased.The 5th group of sample then do not have obvious variation.Other 3 groups with control group do not have notable difference.This illustrates that the 5th group of sample influences cell activity with a kind of direct mode of action, and the 3rd group of sample also exists a kind of profound level, the indirect mode of action simultaneously.It may promote certain product of cell expressing.This product can promote the anti-hypoxia reaction of cell, and has stronger activity.If this inference is correct, this material should be hormones or other gene expression regulation factor, and its mechanism of action is quite complicated, relates to multiple vital process.Sample can promote the utilization of cell to oxygen, and is smooth for the vital vital process of cell to guarantee; It also suppresses the oxygen consumption that the existence of pair cell has the vital process of secondary role in the cell simultaneously, to save nutrient, guarantees that born of the same parents can be survived under emergency rating.Therefore, there are two kinds of effects in this material, and the one, just to regulate and control, the necessary protein of cells survival under activation or the synthetic emergency rating of promotion cell is the oxygen consumption increase of cell from performance.The 2nd, the negative regulation effect, sample is directly or by active substance in the activating cells or promotion cell synthetic active substance, the nonessential vital process of inhibition cell is to the consumption of oxygen.This is a kind of general mechanism in the gene expression regulation.Promptly by one or more factors, at different molecular levels, the behavior of regulating cell is to conform.

In dextran gel filtration, because Sephadex has certain adsorption, so it is necessary adding the salt of suitable ionic strength, and the pH value of moving phase is also influential to separating effect, and in experiment, we adopt the phosphate buffered saline buffer of 0.05M, promptly can keep the stability of moving phase pH value, make the ionic strength of moving phase obtain increasing again, avoid the absorption of gel matrix, in order to avoid influence the separating effect of sample to sample.

In reverse-phase chromatography, when moving phase was water, the sample retention time was shorter, and each sample can not separate fully.So we have added KH ₂PO ₄Increase the ionic strength of moving phase, the surface tension of moving phase is increased, produce and dredge solvation effect, thereby increase the reservation of sample, but peak spreading is obvious.Because organic solvent can improve peak shape and retention time, therefore have to have added 5% methyl alcohol.Peak shape is good.

The mensuration of polypeptide molecular weight, for macro-molecular protein, as long as keep certain ionic strength, separating effect is just more satisfactory, and retention time has linear relationship.But, also need to add SDS or Guanidinium hydrochloride for polypeptide small molecule.When adding Guanidinium hydrochloride, the viscosity ratio of solvent is bigger, and flow velocity can not be too high.In experiment, when not adding SDS, there is not linear relationship in polypeptide standard appearance time.Can't carry out estimating of molecular weight.According to bibliographical information, we have added 0.2% SDS, good separation.

Three. the synthetic of brain protein-active material

Adopt the synthetic above-mentioned brain protein-active material that obtains through separation of synthetic method, measure its physicochemical constant and sequential structure, prove identical with above-mentioned brain protein-active material.

The invention discloses in the brain protein hydrolyte and to separate and the method for purification active substance, and gained activity, purity, molecular weight, aminoacid sequence and terminal analysis have been carried out to active substance, obtained a kind of single, high-purity brain protein-active material, further adopt the synthetic method, synthesized above-mentioned brain protein-active material, prove by analysis, its composition, structure and aminoacid sequence are with to separate the brain protein-active material that obtains identical, for the research and development of brain protein new drug, significant.

Describe the present invention in detail below in conjunction with the drawings and specific embodiments.

Description of drawings is as follows:

Fig. 1. be the flow process of proteic separation of the present invention and purifying

Fig. 2. for being the activity figure of 5 groups of samples of Li Bao company

Fig. 3. be gradient elution device figure

Fig. 4. separate the separate colors spectrogram of the 3rd group of sample eluent of Li Bao company for the DEAE ion exchange method

Fig. 5. separate the activity figure of four groups of samples that the 3rd group of sample of Li Bao company obtain for the DEAE ion exchange method

Fig. 6. the basic block diagram of dextrane gel

Fig. 7. the simple principle of gel chromatography

Fig. 8. the separate colors spectrogram of gel-filtration sample separation DEAEI elutriant

Fig. 9. the activity figure of four groups of samples that gel-filtration sample separation DEAEI obtains

Figure 10. high-effect ionic exchange liquid phase chromatography is separated the separate colors spectrogram of SEPHADEX II elutriant

Figure 11. high-effect ionic exchange liquid phase chromatography is separated the activity figure of SEPHADEX II elution peak

Figure 12. the separate colors spectrogram of efficient gel liquid phase chromatography sample separation TSK3 effluent liquid

Figure 13. efficient gel liquid phase chromatography sample separation TSK3 flows out the activity figure of sample

Figure 14. the separate colors spectrogram of high performance reversed phase chromatography sample separation Pak2 effluent liquid

Figure 15. high performance reversed phase chromatography sample separation Pak2 flows out the activity figure of sample

Figure 16. the gel-filtration of sample ZBX4 is figure as a result

Figure 17. the gel-filtration three dimensional chromatogram of sample ZBX4

Figure 18. efficient reverse-phase chromatography figure

Figure 19. high performance capillary electrophoresis is measured the spectrogram of purity

Figure 20. the efficient gel color atlas of standard polypeptide sample

Figure 21. the polypeptide molecular weight typical curve

Figure 22. the efficient gel color atlas of sample of the present invention

Figure 23. the UV spectrum of sample of the present invention

Figure 24. the terminal measurement result figure of the N-of standard amino acid

Figure 25. the terminal measurement result figure of the N-of sample

Embodiment one. 5 groups of sample determinations of activity of the elementary brain protein product of Li Bao company

Adopt the Wa Shi respirometer in the experiment: Warburg system 12062 types, produce by Braun-Melsungen; The no side arm reaction flask of marking; YQ-3 type high-speed homogenization machine; Cleaning level male guinea pig 250 ± 10; Reagent adopts 10% potassium hydroxide solution, 0.2M phosphoric acid buffer: pH7.4.

Sample: get Li Bao company sampling I, II, III, IV, V, respectively take by weighing 50mg, be dissolved in 0.5ml distilled water, the vibration mixing.Put to death cavy rapidly, get liver, weighing hepatic tissue 4g adds the 24ml phosphoric acid buffer, and 6000rpm homogenate 30 seconds is filtered.Weighing Sodium cholic acid 5g, sodium-chlor 23g dissolve the back respectively and merge.Add Evans Blue 100mg, add water and be settled to 500ml, add several anticorrosion Brodie liquid that get of vanilla phenol spirituous solution again.

Determination step: switch power supply, transfer stirring velocity, keeping bath temperature is 37 ± 0.1 ℃.Prepare the reaction flask of blank, reference substance and trial-product; Add solution by table 1 order.Open pressure warning unit kapillary piston, Erlenmeyer flask is connected on the corresponding pressure warning unit; Erlenmeyer flask is immersed water-bath fully, open the vibration motor, the frequency modulation rate is 100 rev/mins; Reaction flask carries out the temperature and pressure benefit and tastes; After 20 minutes, utilizing knob to regulate each pressure warning unit right side pipe is 150 scale places, record A1.-airtight total system was reacted 15 minutes, and record left side pipe reading is E1; Open the pressure warning unit piston, readjustment pressure warning unit right side pipe is 150 scale places, record A2; Shut the pressure warning unit piston, restart device, past tense 5 minutes, record E2.-open the pressure warning unit piston, readjustment pressure warning unit right side pipe is 150 scale places, record A3; Shut the pressure warning unit piston, restart device, past tense 5 minutes, record E3.

Table 1

	Blank	Sample
			Damping fluid (ml)	????1.3	????1.1
Trial-product (ml)		????0.2
			10% potassium hydroxide (ml)	????0.2	????0.2
Liver homogenate thing (ml)	????1.0	????1.0

The result calculates:

1, principle: read liquid level by pressure warning unit left side kapillary, calculate the pressure difference of representing with mm by the liquid level difference, and then draw the O under the normal condition of representing with μ l ₂Consumption, the numerical value that multiply by Erlenmeyer flask constant gained is Δ O ₂

2, formula

ΔO ₂=(D1+D2+D3)×K

A1=initial value (mm)

E2=scala media segment value (mm)

D1=A1-E1(mm)

Initial value (mm) after the A2=intermediate stage is readjusted

E2=scala media segment value (mm)

D2=A2-E2(mm)

It is surplus that the rest may be inferred

K: Erlenmeyer flask constant

3, vigor calculates: $\frac{\mathrm{Rs}-B}{B}\×100\%$

Rs=sample Δ O wherein ₂The blank Δ O of B= ₂

By aforementioned calculation, draw the vigor result, the vigor soprano of selecting carries out next step liquid phase separation.

Conclusion: table 2 has been listed the determination of activity of 5 groups of samples of Li Bao company; Table 3. has been listed active calculation result.

Table 2

	Blank	The sample I	The sample II	The sample III	The sample IV	The sample V
							????K	?0.83	????0.86	????0.93	?0.8	????0.8	????0.99
????A1 ????E1 ????D1 ????A2 ????E2 ????D2 ????A3 ????E3 ????D3 ?D1+D2+D3 ??K×∑D	?150 ?102 ?48 ?150 ?101 ?49 ?150 ?99 ?51 ?148 ?122.84	????150 ????102 ????48 ????150 ????99 ????51 ????150 ????99 ????51 ????150 ????129	????150 ????106 ????44 ????150 ????107 ????43 ????150 ????108 ????42 ????134 ???124.62	?150 ?52 ?98 ?150 ?49 ?101 ?150 ?50 ?100 ?351 ?280.8	????150 ????98 ????52 ????150 ????97 ????53 ????150 ????97 ????53 ????158 ???126.48	????150 ????89 ????61 ????150 ????90 ????60 ????150 ????88 ????62 ????183 ???181.17

Table 3

The sample I	The sample II	The sample III	The sample IV	The sample V
					?5.49％	1.45％	128.59％	2.96％	47.48％

Fig. 2 is the activity figure of 5 groups of samples of Li Bao company.

Embodiment two. and classical liquid phase chromatography is separated the brain protein hydrolyte

Experiment material: take by weighing the brain protein hydrolyte dried frozen aquatic products sample III 500mg that Li Bao company provides, be dissolved in 10ml distilled water; Glass column size: 14mm (ID) * 95mm.Reagent A liquid: pH9.0 disodium phosphate soln; B liquid: get A liquid 500ml, add NaCl14.6g.

Experimental technique: get the 30g gel, be suspended in the 500ml water, spend the night; The supernatant suspension that inclines 3 times so repeatedly, obtained testing gel in ultrasonic 20 minutes.In glass column, slowly add the gel suspension; Post lower end flow rate control is 1ml/min.When gel coat arrives apart from upper end 3cm, cover the surface of gel bed with a thick cloth; Connect peristaltic pump, detector and totalizing instrument; With 1ml/min pure water detergent gel post 2 hours; Changing moving phase is the NaOH of 0.5M; Washed 2 hours, be washed with water to neutrality after, again with the HCl of 0.5M washing balanced gel post 1 hour; Washing is spent the night; With phosphate buffered saline buffer balance columns 3 hours; Last sample 0.5ml is with the current gradient wash-out of 1ml/min; Detect wavelength 280nm; Gradient elution; Fig. 3 is a gradient elution device, and this device is made up of two containers, and one is reservoir, and one is hybrid bottle.Two bottles utilize siphon principle to communicate by the upper end pipe connecting; The hybrid bottle exit links to each other with peristaltic pump 2, and electromagnetic mixing apparatus 1 is arranged in the hybrid bottle, and B liquid is housed in the reservoir, and A liquid is housed in the hybrid bottle, and liquid volume equates that liquid level is equal in two bottles.

Press elution profile, collect each component peaks successively; Then each sample is passed through the desalination of SeghadexG10 post, freeze-drying, weighing 150mg, be dissolved in 1ml water, the separate colors spectrogram of sample is seen Fig. 4; Adopt the method for front to measure activity, four peaks of sample are surveyed slip-knot and are really seen Table 4-5, and the active figure of the isolated four groups of samples of DEAE ion exchange method sees Fig. 5.By the isolated 1# of the visible DEAE ion exchange method of said determination peak, promptly first group of sample DEAEI has the most of active of raw sample.

The isolated four groups of samples of table 4 DEAE ion exchange method are surveyed the slip-knot fruit

	Blank	Blank	????DEAE1	????DEAE2	????DEAE3	????DEAE4
							????k	????0.83	????0.86	????0.93	????0.8	????0.8	????0.99
????A1 ????E1 ????D1 ????A2 ????E2 ????D2 ????A3 ????E3 ????D3 ??D1+D2+D3 ??k×∑D	????150 ????115 ????35 ????150 ????112 ????38 ????150 ????120 ????30 ????103 ????85.49	????150 ????116 ????34 ????150 ????111 ????39 ????150 ????119 ????31 ????104 ???89.44	????150 ????82 ????68 ????150 ????75 ????75 ????150 ????79 ????71 ????214 ????199.02	????150 ????116 ????34 ????150 ????115 ????35 ????150 ????118 ????32 ????101 ????80.8	????150 ????108 ????42 ????150 ????112 ????38 ????150 ????110 ????40 ????120 ????96	????150 ????110 ????40 ????150 ????108 ????42 ????150 ????112 ????38 ????120 ????118.8

The active calculation result of the isolated four groups of samples of table 5 DEAE ion exchange method

????DEAE1	????DEAE2	????DEAE3	????DEAE4
				????127％	????-7.6％	????9.8％	????36％

Embodiment three. and the molecular sieve gel method is separated DEAEI

Adopt ion-exchange elution samples DEAEI in the experiment, glass column size: 20mm (ID) * 90cm; 0.05mol/L, pH7.4 phosphate buffered saline buffer, 0.05% NaN ₃The aqueous solution; 0.2% blue glucan aqueous solution; Take by weighing this product 3g, being dissolved in is as the moving phase of protein hydrolyte in the 15ml water.

Get SephadexG25 gel 50g, gel is suspended in 2000ml water, spend the night; The supernatant suspension that inclines, 3 times so repeatedly.Ultrasonic 20 minutes, in the glass column of packing into, post lower end flow rate control was 1ml/min; When gel coat arrives apart from master's end 3cm, cover the surface of gel bed with a thick cloth; Connect peristaltic pump, detector and totalizing instrument; With 1ml/min pure water detergent gel 12 hours, be replaced by the 0.05M phosphate buffered saline buffer, balanced gel post 2 hours, the chromatography bed should be even, and no lines, bubble add blue dextran, and chromatographic band should be even, smooth; Sample 0.5ml on chromatographic column is with the flow velocity wash-out of 1ml/min; Detect wavelength 280nm, collect elutriant respectively and be followed successively by DEX1, DEX2, DEX3, DEX4 in order, the sample separation color atlas is seen Fig. 8, repeats sample, respectively gets 2ml and lives with the same method survey, the results are shown in Table 6-6a and Fig. 9.By measure and calculation result as can be seen,

The survey slip-knot fruit of four groups of samples that table 6 gel-filtration sample separation DEAEI obtains

	Blank	Blank	????DEX1	????DEX2	????DEX3	????DEX4
							????k	????0.83	????0.86	????0.93	????0.8	????0.8	????0.99
????A1 ????E1 ????D1 ????A2 ????E2 ????D2 ????A3 ????E3 ????D3 ?D1+D2+D3 ???k∑D	????150 ????120 ????30 ????150 ????118 ????32 ????150 ????119 ????31 ????93 ???77.19	????150 ????119 ????31 ????150 ????118 ????32 ????150 ????119 ????31 ????92 ???79.12	????150 ????114 ????36 ????150 ????115 ????35 ????150 ????115.5 ????34.5 ????105.5 ????98.115	????150 ????87 ????63 ????150 ????90 ????60 ????150 ????88 ????62 ????185 ????148	????150 ????117 ????33 ????150 ????116 ????34 ????150 ????117 ????33 ????100 ????80	????150 ????123 ????27 ????150 ????124 ????26 ????150 ????122 ????28 ????81 ???80.19

The active calculation result of four groups of samples that table 6a gel-filtration sample separation DEAEI obtains

DEX1	?DEX2	????DEX3	????DEX4
				?29.3％	?89％	????2.3％	????2.6％

Embodiment four. and high-effect ionic exchange liquid phase chromatography is separated Sephadex gel-filtration elution samples SEPHADEX II (being the DEX II); A liquid: 0.1MTris-HCl damping fluid; B liquid: get A liquid 500ml, add sodium-chlor 14.6g, two liquid are all used 0.45 μ m membrane filtration.

Instrument adopts Tianjin, island high-pressure liquid phase system, comprises the LC-1OAT pump, SPD-MXA detector, CTO-1OA post case, FRC10A collector, Progel TSKDEAE-5PW post [4.6mm (ID) * 25cm 5 μ m]

The gradient program:

A??????B：

0min??????????????100％???0

30min?????????????0??????100％

Flow velocity 1ml/min, linear gradient.

During separation, with A liquid balance columns 30min, sample introduction 10 μ l, collect elutriant, repeat sample introduction, color atlas is seen Figure 10, distribute and collect elutriant, be followed successively by TSK1, TSK2, TSK3, TSK4, TSK5, TSK6, TSK7, TSK8, TSK9, TSK10 in order, elutriant Sephdex G10 post desalination, freeze-drying; Respectively get the 10mg dried frozen aquatic products again, be dissolved in 0.5ml water, method is lived with before measurement, the results are shown in Table 7-8 and Figure 11, and by above-mentioned table and figure as can be seen: the sample at TSK3 peak has the most activity of raw sample.

Table 7 high-effect ionic exchange liquid phase chromatography is separated the survey slip-knot fruit of SEPHADEX II gained sample

	Blank	???TSK1	???TSK2	????TSK8	TSK4	???TSK5	?TSK6	???TSK7	?TSK8	?TSK9	?TSK10
												????k	????0.8	???0.86	???0.93	????0.8	0.8	???0.99	?0.86	???0.83	?0.86	?0.93	0.8
????A1 ????E1 ????D1 ????A2 ????E2 ????D2 ????A3 ????E3 ????D3 ?D1+D2+D3 ???k∑D	????150 ????112 ????38 ????150 ????113 ????37 ????150 ????112 ????38 ????113 ???90.4	????150 ????110 ????40 ????150 ????109 ????41 ????150 ????112 ????38 ????119 ???102.34	????150 ????115 ????35 ????150 ????116 ????34 ????150 ????114 ????36 ????105 ????97.65	????150 ????70 ????80 ????150 ????71 ????79 ????150 ????75 ????75 ????234 ??187.2	150 108 ?42 150 109 ?41 150 111 ?39 121 96.8	????150 ????113 ????37 ????150 ????115 ????35 ????150 ????114 ????36 ????108 ??106.92	?150 ?113 ?37 ?150 ?112 ?38 ?150 ?110 ?40 ?115 ?98.9	????150 ????110 ????40 ????150 ????111 ????39 ????150 ????109 ????41 ????120 ????99.6	?150 ?111 ?39 ?150 ?111 ?39 ?150 ?110 ?40 ?118 101.48	?150 ?116 ?34 ?150 ?117 ?33 ?150 ?113 ?37 ?104 96.72	150 114 ?36 150 121 ?29 150 110 ?40 115 ?92

The active calculation result that table 8 high-effect ionic exchange liquid phase chromatography is separated SEPHADEX II gained sample

TSK2	TSK1	?TSK3	?TSK4	?TSK5	?TSK6	?TSK7	?TSK8	?TSK9	?TSK10
										?7.3％	13.2％	122.61％	?7.08％	18.3％	0.55％	?10.1％	?4.42％	?6.99％	?1.77％

Embodiment five. and the efficient gel liquid phase chromatography is separated (HPGC)

Adopt Tianjin, island high-pressure liquid phase system, comprise the LC-1OAT pump, SPD-MXA detector, CTO-1OA post case, FRC1 OA collector, Protein-pak 60 posts [7.8mm (ID) * 300mm); Moving phase is pH6.5, concentration 0.05M, the phosphate buffered saline buffer of crossing 0.45 μ m film; Sample is dissolved in 3ml water for taking by weighing TSK3 60mg.

Use moving phase balance columns 40min during separation, sample introduction 50 μ l, sample introduction is collected elution peak step by step repeatedly, is followed successively by PAK1, PAK2, PAK3, and the separate colors spectrogram is seen Figure 12, respectively gets the 2ml method and lives with before measurement, the results are shown in Table 9-10 and Figure 13; By above-mentioned table and figure as can be seen: the sample at PAK2 peak has the most activity of raw sample.

Table 9 efficient gel liquid phase chromatography sample separation TSK3 flows out sample and surveys the slip-knot fruit

	Blank	???PAK1	????PAK2	???PAK3
					????k ????A1 ????E1 ????D1 ????A2 ????E2 ????D2 ????A3 ????E3 ????D3 ????D1+D2+D3 ????k∑D	????0.8 ????150 ????109 ????41 ????150 ????110 ????40 ????150 ????108 ????42 ????123 ????98.4	????0.83 ????150 ????110 ????40 ????150 ????108 ????42 ????150 ????111 ????39 ????121 ????100.43	????0.86 ????150 ????75 ????75 ????150 ????77 ????73 ????150 ????76 ????74 ????222 ????190.92	????0.93 ????150 ????113 ????37 ????150 ????114 ????36 ????150 ????112 ????38 ????111 ????103.23

Table 10 efficient gel liquid phase chromatography sample separation TSK3 flows out the active calculation result of sample

????PAK1	?PAK2	????PAK3
			????2.06％	?93.08％	????4.9％

Embodiment six. and high performance reversed phase chromatography separates (HPRPC)

Adopt Tianjin, island high-pressure liquid phase system among the present invention, comprise the LC-1OAT pump, SPD-MXA detector, CTO-1OA post case, FRC1 OA collector, sample: Pak2, post: Zorbax300SB-C18[4.6mm (ID) * 150mm), reagent 0.05MKH ₂PO ₄, 0.1% trichoroacetic acid(TCA), 5% methanol-water mixing solutions, ultrasonic 20min, 0.45 μ m membrane filtration.

With moving phase balance columns 40min, treat that baseline walks steadily, the beginning sample introduction, sample size 100 μ l collect elution peak, and the separate colors spectrogram is seen Figure 14, and sample introduction is collected elutriant repeatedly, is followed successively by ZBX1, ZBX2, ZBX3, ZBX4, ZBX5 in order; Sample freeze-drying with collecting respectively is dissolved in 1ml water, gets the 0.5ml method and lives with before measurement, the results are shown in Table 11-12 and Figure 15; By above-mentioned table and figure as can be seen: the sample at ZBX4 peak has the most activity of raw sample.

Table 11 high performance reversed phase chromatography sample separation Pak2 flows out sample and surveys the slip-knot fruit

	Blank	ZBX1	?ZBX2	???ZBX3	?ZBX4	?ZBX5
							?k	?0.83	?0.86	???0.8	????0.8	?0.93	?0.99
?A1 ?E1 ?D1 ?A2 ?E2 ?D2 ?A3 ?E3 ?D3 ?D1+D2+D3 ?k∑D	?150 ?119 ?31 ?150 ?118 ?32 ?150 ?120 ?30 ?93 ?77.17	?150 ?120 ?30 ?150 ?121 ?29 ?150 ?121 ?29 ?88 ?75.68	????150 ????123 ????27 ????150 ????124 ????26 ????150 ????122 ????28 ????81 ????64.8	????150 ????117 ????33 ????150 ????117 ????33 ????150 ????118 ????32 ????98 ????78.4	?150 ?94 ?56 ?150 ?90 ?60 ?150 ?92 ?58 ?174 ?161.82	?150 ?125 ?25 ?150 ?123 ?27 ?150 ?125 ?25 ?77 ?76.23

Table 12 high performance reversed phase chromatography sample separation Pak2 flows out the active calculation result of sample

ZBX1	?????ZBX2	?ZBX3	?ZBX4	?ZBX5
					-2％	????-12.3％	1.21％	109.7％	?-0.93％

Embodiment seven. and high performance size exclusion chromatography method (HPGC) is measured purity

Instrument adopts Tianjin, island high pressure wave phase system, comprises the LC-1OAT pump, SPD-MXA detector, CTO-1OA post case; Moving phase is 0.1M phosphate buffered saline buffer (pH7.0); Post is Waters Protein-pak 60[7.8mm (ID) * 300mm]; Sample: ZBX4, flow velocity: 1ml/min, sample size: 20 μ l; The results are shown in Figure 16-17, all have only among the figure one unimodal, illustrate to obtain the single sample of purity.

Embodiment eight. and high performance reversed phase chromatography (HPRPC) is measured purity

Instrument adopts Tianjin, island high-pressure liquid phase system, comprises the LC-1OAT pump, the SPD-MXA detector, CTO-1OA post case, post: YWG-C181[4.6mm (ID) * 250mm] north, Beijing branch technical device company, moving phase: 5% methyl alcohol, column temperature: 35 ℃, flow velocity: 1ml/min, sample size: 20 μ l; Detected result is seen Figure 18, and except that a small peak was arranged in the early stage, the sample peak was that 6.976MIN occurs in retention time mainly, proves that purity is good.

Embodiment nine. and high performance capillary electrophoresis is measured purity

Instrument adopts Waters Quanta 4000E capillary electrophoresis apparatus, post: CEleet-P175 75 μ m (ID) * 57.5cmSupelco, moving phase: 0.1M SODIUM PHOSPHATE, MONOBASIC one phosphoric acid buffer, pH2.5, detect wavelength: 214nm, applied sample amount: 10 μ l the results are shown in Figure 19.

Embodiment ten. efficient gel chromatography (HPGC) determining molecular weight

Instrument adopts Tianjin, island high pressure and phase system, comprise the LC-IOAT pump, the SPD-MXA detector, CTO one 1OA post case, the phase of surging: 0.1M phosphate buffered saline buffer 0.2%SDS (pH7.0), post: TSK-GEL 2000SW[7.5mm (ID) * 300mm), flow velocity: 1ml/min, polypeptide standard: get polypeptide standard 1 μ l, dilute 20 times, last sample 4 μ l; Sample: go up sample 10 μ l.According to polypeptide standard substance peak, see Figure 20, obtain the typical curve table, see Table 13, drawing standard curve Figure 21 in conjunction with efficient gel stratographic measurement result, sees Figure 22 again, the calculation sample molecular weight is 2749.68.

Table 13

	????RT(min)	??Moiecular?Weight
			????1 ????2 ????3 ????4 ????5	????5.120 ????5.636 ????6.667 ????9.801 ????10.486	????26.625 ????16.950 ????6.512 ????3.496 ????1.423

Embodiment 11. ultraviolet spectroscopy

Utilize water as solvent, adopt the ultra-violet absorption spectrum of Hitachi's 3201 ultraviolet spectrophotometer working samples.The results are shown in Figure 23.

The terminal homogeneity of embodiment 12 .N is measured

1. the Dansylization of sample: sample thief 0.1ml, add DNS-Cl 0.6ml (2.5mg/ml), be transferred to ph9.0-9+.1 with triethylamine, mixing is filled in, and DNSization is 2 hours in 40 ℃ of baking ovens.Take out the reaction back, takes out acetone in vacuum drier.

2.DNS the acid hydrolysis of a sample: the hydrochloric acid soln that adds 0.5ml 5.7M in the sample of above-mentioned DNSization makes it dissolving, transfers to the hydrolysis Guan Zhongyong flame tube sealing of hard glass again, 110 ℃ of hydrolysis 18 hours.Remove after the hydrolysis and seal, hydrolyzed solution is shifted out, be put in the 5ml tubule, extract hydrochloric acid out, add small amount of hydrochloric acid again, extract hydrochloric acid again out to doing.

3. add the 0.5ml water dissolution, add the extraction of 1ml ethyl acetate again, extract 2-3 time, combining extraction liquid boils off ethyl acetate, adds the 0.1ml acetone solution then, and is standby.

4. the DNSization of standard amino acid: take by weighing amino acid standard substance 1ml (18 seed amino acid), be dissolved in the 0.5ml0.2M sodium hydrogen carbonate solution.Add the 0.5mIDNS-Cl acetone soln, DNSization is 1 hour in 40 ℃ of baking ovens, extracts acetone in vacuum drier out.Then, with the 1M hydrochloric acid soln amino acid of DNSization is acidified to PH2-3, with ethyl acetate extraction 2-3 time, uses 1ml more at every turn, combining extraction liquid boils off ethyl acetate, adding 0.1ml acetone solution.

5. cutting out of polymeric amide pellicle: polyamide layer is cut into two of 7 centimetres of sizes of 7 cm x.Each standardized straight line of 0.5 centimeters apart from both sides, the standardized intersection cross at the point sample place.

6. point sample: DNS one standard amino acid, sample hydrolyzed solution are respectively with kapillary sampling, point sample on polyamide membrane.

7. exhibition layer: with the light of polyamide layer towards outside, polymeric amide bind round a nylon strip circle towards interior, places the interior little culture dish of little moisture eliminator, fills chromatography solvent I [85% formic acid: water=1.5: 100 (V: V)) in the culture dish.When treating that the solvent front will reach film edge, take out, dry up with blower.Then film is changed 90 ° of directions, in the molten Ji II of exhibition layer [benzene: open up layer in the glacial acetic acid=9: 1 (V: V)), when treating that the solvent front will reach film edge, take out, dry up.

8. detect: observe down in the ultraviolet detection lamp.The DNS monoamino-acid is green, and the DNS-CI product is a blue-fluorescence.With pencil with under each fluorescence zoning.Determine the number and the amino acid kind of sample N-terminal, the N one terminal measurement result figure of standard amino acid sees Figure 25, and the measurement result of sample is seen Figure 26, according to test result, can think that the gained sample has only a N-end, is arginine.

Embodiment 13. sequencing

Instrument: LF3000 Protein Sequencer Beckman.

Embodiment 14. the chemosynthesis of brain protein-active material

Adopt Peptide synthesizer,, and measure the physicochemical constant of the synthetic brain protein-active material of institute and sequence step by step according to the progressively synthetic brain protein-active material of the sequence of measuring.

Embodiment 15. the chemosynthesis of brain protein-active material

Use the synthetic method, synthetic above-mentioned brain protein-active material, and measure the physicochemical constant of the synthetic brain protein-active material of institute and sequence step by step, the active substance that obtains is basic identical with separating.

Claims (9)

1. hydrolytic active substance of brain protein is characterized in that:

(1) activity of the elementary brain proteolysis sample of mensuration;

(2) get the highest active sample DEAE ion exchange method separation in the elementary brain proteolysis sample, obtain the highest active sample DEAEI;

(3) separate DEAEI with the molecular sieve gel method, obtain the highest active sample DEX2;

(4) separate DEX2 with high-effect ionic exchange liquid phase chromatography, obtain the highest active sample TSK3;

(5) with efficient gel liquid phase chromatography sample separation TSK3, obtain the highest active sample P AK2;

(6) separate PAK2 with high performance reversed phase chromatography, obtaining having the most of active sample ZBX4 of raw sample is hydrolytic active substance of brain protein.

2. hydrolytic active substance of brain protein according to claim 1 is characterized in that one of the demonstration in the spectrogram of high performance size exclusion chromatography, the three-dimensional chromatogram of gel-filtration, efficient reverse-phase chromatography, HPCE spectrogram and efficient gel chromatography determination molecular weight of described hydrolytic active substance of brain protein is unimodal.

3. hydrolytic active substance of brain protein according to claim 2, it is characterized in that the absorption peak of described hydrolytic active substance of brain protein in high performance size exclusion chromatography, efficient reverse-phase chromatography, HPCE spectrogram is respectively: t=9.886,6.976 closes 24; Molecular weight is 2749.68.

4. hydrolytic active substance of brain protein according to claim 1 is characterized in that only N-end of described hydrolytic active substance of brain protein, is arginine.

5. hydrolytic active substance of brain protein according to claim 1 is characterized in that described hydrolytic active substance of brain protein has specific absorption and has specific sequence on UV spectrum.

6. hydrolytic active substance of brain protein according to claim 1 is characterized in that described hydrolytic active substance of brain protein is a pure protein.

7. method for preparing hydrolytic active substance of brain protein is characterized in that:

(1) activity of the elementary brain proteolysis sample of mensuration;

(2) get the highest active sample DEAE ion exchange method separation in the elementary brain proteolysis sample, obtain the highest active sample DEAEI;

(3) separate DEAEI with the molecular sieve gel method, obtain the highest active sample DEX2;

(4) separate DEX2 with high-effect ionic exchange liquid phase chromatography, obtain the highest active sample TSK3;

(5) with efficient gel liquid phase chromatography sample separation TSK3, obtain the highest active sample P AK2;

(6) separate PAK2 with high performance reversed phase chromatography, obtaining having the most of active sample ZBX4 of raw sample is hydrolytic active substance of brain protein.

8. the method for preparing hydrolytic active substance of brain protein according to claim 7 is characterized in that:

(1) measures the activity of elementary brain proteolysis sample with Wa Shi respirometer manometry;

(2) the highest active sample in the elementary brain proteolysis sample is got in the separation of DEAE ion exchange method, and last sample 0.5ml is with the current gradient wash-out of 1ml/min; Detect wavelength 280nm;

(3) adopt the SephadexG25 gel to separate DEAEI with the molecular sieve gel method, last sample 0.5ml is with the flow velocity wash-out of 1ml/min; Detect wavelength 280nm, obtain the highest active sample DEX2;

(4) with Progel TSK DEAE-5PW post high-pressure liquid phase system, the high-effect ionic exchange liquid phase chromatography of sample introduction 10 μ l is separated DEX2, obtains the highest active sample TSK3;

(5) with efficient gel liquid phase chromatography Protein-pak60 post high-pressure liquid phase system, moving phase be pH6.5, concentration 0.05M, phosphate buffered saline buffer, sample separation TSK3 obtains the highest active sample P AK2;

(6) use high performance reversed phase chromatography, Zorbax 300SB-C18 post high-pressure liquid phase system, 0.05MKH ₂PO ₄, 0.1% trichoroacetic acid(TCA), 5% methanol-water mixing solutions separate PAK2, obtaining having the most of active sample ZBX4 of raw sample is hydrolytic active substance of brain protein.

9. a method for preparing hydrolytic active substance of brain protein is characterized in that: adopt the synthetic hydrolytic active substance of brain protein of synthetic method.

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