CN106986929A - A kind of cromoci preparation method - Google Patents
A kind of cromoci preparation method Download PDFInfo
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- CN106986929A CN106986929A CN201710281017.2A CN201710281017A CN106986929A CN 106986929 A CN106986929 A CN 106986929A CN 201710281017 A CN201710281017 A CN 201710281017A CN 106986929 A CN106986929 A CN 106986929A
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- cromoci
- feed liquid
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- zeolite
- ammonium sulfate
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
- C07K14/80—Cytochromes
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- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of preparation method of cromoci, using animal cardiac muscle as raw material, extracted using sulfuric acid, prepared by zeolite adsorption and hydrophobic chromatography process for purification.The present invention has that cost is low, step is simple, extraction conditions are gentle, suitable industrialized production the characteristics of.
Description
Technical field
The invention belongs to biological chemical field, particularly with regard to a kind of preparation method of cromoci.
Background technology
Cromoci (Cytochrome C) is a conjugated protein, and peptide chain is in most of species by 104 amino
Acid composition, prothetic group is iron-protoporphyrin IX (ferroheme).Prosthetic heme group is by the vinyl on porphyrin ring, and peptide chain Cys-14,
The cysteine residues of Cys-17 two are covalently attached with thioether bond.The center iron atom of ferroheme the 5th, 6 two coordinate bonds with
The sulphur atom coordination of His-18 nitrogen-atoms and Met-80.Peptide chain has an about 40% a- spiral fragments, and remainder includes β-turn
Angle, random coil and stretching, extension fragment.
Cromoci is distributed widely in the microorganisms such as animal, plant and yeast, is positioned at the appearance of mitochondrial inner membrane
Face.It is positively charged in physiological pH environment because there is multiple basic amino acids (particularly Lys) residue in molecule, electrostatic phase can be passed through
Interaction is mutually associated with membrane phospholipid matter, can be by H+Substitution is replaced with metal ion from film to get off.
Existing cromoci extracting method is sulfuric acid solution extraction, zeolite trapping, ammonium sulfate fractionation, trichloroacetic acid precipitation
And the five big steps such as ion-exchange chromatogram purification, have that extraction conditions are more violent, time-consuming, and yield it is relatively low the shortcomings of,
Its Control of Impurities has not reached the quality requirement of current medicine.Traditional handicraft application trichloroacetic acid is precipitated carefully under high salt concn
Cytochrome C, can generate the cromoci of modified, polymerization and deamination, function of the influence cromoci product in a variety of enzyme systems
Activity, influences safe and effective for medication.The application of trichloroacetic acid also results in noxious emission.
Also there is the report of cromoci separating and purifying technology both at home and abroad, such as modern Separation Research institute of Northwest University《With dredge
Water colour spectrometry one step from Pigs Hearts quickly prepares high-purity cromoci》Report claim, compared with classical way, this law purifying
Technique is simple, and the operating time is short, and mass recovery and purity can be improved to 98.5% and more than 99% respectively.But hydrophobic chromatography pair
The clarity and coherence request of process conditions especially feed liquid are higher, are only used for laboratory preparative-scale, extracting liquid volume
Less than 1 liter, post bed is small-sized, it is difficult to be put to business application.
The content of the invention
Zeolite component is lagoriolite, is a kind of cation exchange medium, because its surface charge density is big, to cytochromes
C trapping ability is strong, is eluted through zeolite adsorption, product can vast scale obtain concentration, material liquid volume reduces many times, can reduce
The pressure of later-period purification technique, is conducive to industry development and business to prepare.
Hydrophobic chromatography can realize that high-resolution is purified to cromoci.It is above-mentioned existing it is an object of the invention to improve
Technology deficiency and a kind of cromoci preparation method is provided, above-mentioned absorption and purification process are effectively combined, cost
Low, step is simple, extraction conditions are gentle, be adapted to industrialized production.
The object of the present invention is achieved like this, a kind of cromoci preparation method, is the warp using animal cardiac muscle as raw material
Persulfuric acid is extracted, after zeolite adsorption and hydrophobic chromatography are refined, and desalination simultaneously lyophilized obtains cromoci powder.
Cardiac muscle is extracted after rubbing using sulfuric acid solution, extracts pH 3.5~4.5, temperature is room temperature, is extracted 2~3 hours, mistake
Slag is filtered out, extract solution ammoniacal liquor regulation pH 7.0~8.0 is obtained.
Adherent cell pigment C in feed liquid is added to using enough zeolites, 0.2% sodium chloride solution is used after the completion of absorption
Zeolite is washed, the ammonium sulfate for reusing enough 24~26% elutes cromoci from zeolite.It is thin in eluent
Cytochrome C concentration is relatively low, can be used the methods such as ultrafiltration that feed liquid is concentrated to after 1/10 volume, adds ammonium sulfate to saturation, filtering
Fall insoluble matter to obtain clarifying feed liquid.
Because by the absorption of zeolite, cromoci has obtained preliminary purifying, material liquid volume greatly reduces, and keeps away
Exempt from the problem apt to deteriorate of the feed liquid in large-scale production, next will become light using hydrophobic chromatography purifying process, and
And Linear Amplifer can be carried out to technique.
Suitable quantity of water is added in clarification feed liquid, ammonium sulfate concentrations therein is reduced to 12~13%, feed liquid is passed through into benzene
Base stationary phase hydrophobic chromatography post, collects the feed liquid flowed through, and now foreign protein is then adsorbed by hydrophobic chromatography post.To improve purifying effect
Really, hydrophobic chromatography post can be used in series.
Feed liquid after purification is lyophilized to produce cromoci powder after the method desalination such as ultrafiltration.
Specific embodiment
A kind of embodiment 1, cromoci preparation method, 5kg Pigs Hearts is rubbed, and adds appropriate concentration molten for 2% sulfuric acid
Liquid, pH value control is 3.5, and room temperature is extracted 2 hours, then through filtering, obtained extract solution adjusts pH 7.0 with ammoniacal liquor.Using enough
Zeolite is added to adherent cell pigment C in feed liquid, washs zeolite using 0.2% sodium chloride solution after the completion of absorption, reuses
Enough 24% ammonium sulfate elutes cromoci from zeolite.Feed liquid is concentrated to 1/10 using hyperfiltration process
After volume, ammonium sulfate is added to saturation, is filtered out insoluble matter and is obtained clarifying feed liquid.Suitable quantity of water is added in clarification feed liquid, makes it
In ammonium sulfate concentrations be reduced to 12%, by feed liquid by phenyl stationary phase hydrophobic chromatography post, collect the feed liquid flowed through.Feed liquid is passed through
Cross after hyperfiltration process desalination, it is lyophilized to produce cromoci powder 0.9g.
A kind of embodiment 2, cromoci preparation method, 30kg OX-heart is rubbed, and adds appropriate concentration molten for 2% sulfuric acid
Liquid, pH value control is 4.0, and room temperature is extracted 2.5 hours, then through filtering, obtained extract solution adjusts pH 7.5 with ammoniacal liquor.Use foot
Amount zeolite is added to adherent cell pigment C in feed liquid, washs zeolite using 0.2% sodium chloride solution after the completion of absorption, then make
Cromoci is eluted from boiling upper right with enough 25% ammonium sulfate.Feed liquid is concentrated to 1/ using hyperfiltration process
After 10 volumes, ammonium sulfate is added to saturation, is filtered out insoluble matter and is obtained clarifying feed liquid.Suitable quantity of water is added in clarification feed liquid, is made
Ammonium sulfate concentrations therein are reduced to 12.5%, by feed liquid by phenyl stationary phase hydrophobic chromatography post, collect the feed liquid flowed through.Material
Liquid is lyophilized to produce cromoci powder 5.9g after hyperfiltration process desalination.
A kind of embodiment 3, cromoci preparation method rubs the 100kg horses heart, adds appropriate concentration molten for 2% sulfuric acid
Liquid, pH value control is 4.5, and room temperature is extracted 3 hours, then through filtering, obtained extract solution adjusts pH 8.0 with ammoniacal liquor.Using enough
Zeolite is added to adherent cell pigment C in feed liquid, washs zeolite using 0.2% sodium chloride solution after the completion of absorption, reuses
Enough 26% ammonium sulfate elutes cromoci from zeolite.Feed liquid is concentrated to 1/10 using hyperfiltration process
After volume, ammonium sulfate is added to saturation, is filtered out insoluble matter and is obtained clarifying feed liquid.Suitable quantity of water is added in clarification feed liquid, makes it
In ammonium sulfate concentrations be reduced to 13%, by feed liquid by phenyl stationary phase hydrophobic chromatography post, collect the feed liquid flowed through.Feed liquid is passed through
Cross after hyperfiltration process desalination, it is lyophilized to produce cromoci powder 18.5g.
| Detection | Detection method | Embodiment 1 | Embodiment 2 | Embodiment 3 |
| Content >=95.0% | Liquid phase process | 99.0% | 99.6% | 99.4% |
| Enzyme activity >=95.0% | Standards of pharmacopoeia | 95.9% | 97.0% | 96.5% |
| Iron content 0.4~0.46% | Standards of pharmacopoeia | 0.45% | 0.46% | 0.46% |
Claims (4)
1. a kind of cromoci preparation method, it is characterized in that using animal cardiac muscle as raw material, extracted through persulfuric acid, zeolite adsorption and
After hydrophobic chromatography is refined, desalination simultaneously lyophilized obtains cromoci powder.
2. a kind of cromoci preparation method according to claim 1, it is characterized in that cardiac muscle is carried after rubbing using sulfuric acid
Take, extract pH3.5~4.5, temperature is room temperature, extract 2~3 hours, filter cleaner, obtain extract solution ammoniacal liquor regulation pH7.0
~8.0.
3. a kind of cromoci preparation method according to claim 2, it is characterized in that being inhaled at room temperature using enough zeolites
Cromoci in attached extract solution, after the completion of absorption using 0.2% sodium chloride solution washing zeolite, reuse enough 24~
26% ammonium sulfate elutes cromoci from zeolite, and ammonium sulfate is added after eluent concentration to saturation, filtering
Fall insoluble matter to obtain clarifying feed liquid.
4. a kind of cromoci preparation method according to claim 3, it is characterized in that being added in clarification feed liquid appropriate
Water, makes ammonium sulfate concentrations therein be reduced to 12~13%, by feed liquid by phenyl stationary phase hydrophobic chromatography post, collects what is flowed through
Feed liquid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710281017.2A CN106986929A (en) | 2017-04-19 | 2017-04-19 | A kind of cromoci preparation method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710281017.2A CN106986929A (en) | 2017-04-19 | 2017-04-19 | A kind of cromoci preparation method |
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| Publication Number | Publication Date |
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| CN106986929A true CN106986929A (en) | 2017-07-28 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201710281017.2A Pending CN106986929A (en) | 2017-04-19 | 2017-04-19 | A kind of cromoci preparation method |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102295699A (en) * | 2011-09-13 | 2011-12-28 | 西北大学 | Process for purifying cytochrome C |
| CN103520123A (en) * | 2013-10-31 | 2014-01-22 | 成都天台山制药有限公司 | Cytochrome C freeze-dried powder injection solution |
-
2017
- 2017-04-19 CN CN201710281017.2A patent/CN106986929A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102295699A (en) * | 2011-09-13 | 2011-12-28 | 西北大学 | Process for purifying cytochrome C |
| CN103520123A (en) * | 2013-10-31 | 2014-01-22 | 成都天台山制药有限公司 | Cytochrome C freeze-dried powder injection solution |
Non-Patent Citations (1)
| Title |
|---|
| 高小娥: "猪心中细胞色素C的提取与鉴定 ", 《南方农机》 * |
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Application publication date: 20170728 |
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