CN101294148A - Method for extracting and purifying high ferro myohemoglobin reductase from myocardium - Google Patents
Method for extracting and purifying high ferro myohemoglobin reductase from myocardium Download PDFInfo
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- CN101294148A CN101294148A CNA2008101238401A CN200810123840A CN101294148A CN 101294148 A CN101294148 A CN 101294148A CN A2008101238401 A CNA2008101238401 A CN A2008101238401A CN 200810123840 A CN200810123840 A CN 200810123840A CN 101294148 A CN101294148 A CN 101294148A
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Abstract
The invention relates to a process for extracting and purifying protease, in particular to a method for extracting and purifying metmyoglobin reductase from cardiac muscle. The method comprises the following steps: obtaining crude extractive liquid from cardiac muscle of an animal, salting out, and separating and purifying metmyoglobin reductase from pig heart by chromatography adopting DEAE-Fast Flow and CM-Sephadex FF ion exchange medium and blue sepharose FF medium. The method has the advantages of high product purity, simple process, low cost, etc., and can fill the blank in bio-reagent preparation and achieve higher economic value.
Description
Technical field
The present invention relates to a kind of extraction and purification process of proteolytic enzyme, specifically is the method for extracting purifying high ferro myohemoglobin reductase from cardiac muscle.
Background technology
Yellowish pink is the important economical trait that meat product is produced, paid close attention to jointly by scientist and the producer, human consumer, yellowish pink scarletly be rich in gloss and show that meat is fresh, fine quality, and myohaemoglobin (Mb) and reductase enzyme system (Reductase) thereof in it and the muscle are closely related.Mb exists with three kinds of forms in vivo, is respectively the deoxidation myohaemoglobin, is red-purple; Oxymyoglobin (oxymyoglobin, MbO
2), be bright red; The two all can be oxidized to metmyoglobin, and (metmyoglobin MetMb), is dun.Exist the redox system that the three changes mutually in the muscle, phase co-conversion under the effect of enzyme, and maintain running balance and stable yellowish pink.After muscle exsomatizes, along with the active progressively forfeiture of high ferro myohemoglobin reductase (MetMbR), the brown stain gradually of muscle color, prompting muscle is begun to go bad by living contaminants in the air and muscle, or degradation under the skeletal muscle function.
Even in external (between fresh meat preservation term), utilize the generation that look mechanism can be intervened MetMb of protecting of self MetMbR redox enzyme system, maintain the freshness of muscle and good yellowish pink, embody fresh meat meat quality within a certain period of time; The high conversion of MetMbR activity means that cardiac muscle and skeletal muscle have good function of carrying oxygen etc.
Therefore, research MetMbR and MetMb interaction relationship just need obtain more satisfactory MetMbR biomaterial as standard substance, could improve the science studied, reliability etc.Yet MetMbR biomaterial desirable and purifying is difficult to preparation, and the experimental condition research of this respect is made slow progress, and does not also have the MetMbR biological products on the market and occurs.If can obtain the MetMbR of pure product, experiment condition and material will be provided for the research of meat, sports medical science and cardiovascular disorder.
Summary of the invention
The purpose of this invention is to provide a kind of method of extracting purifying high ferro myohemoglobin reductase, this method is selected DEAE-Fast Flow, CM-Sephadex FF ion exchange chromatography and three kinds of chromatography medias of blue-sepharose gel FF for use, adopt chromatography from Pigs Hearts, to separate and the method for purifying high ferro myohemoglobin reductase, can obtain electrophoretically pure metmyoglobin in the short period of time, and the yield height has solved problem effectively.
The technical solution adopted for the present invention to solve the technical problems is:
A kind of method of extracting purifying high ferro myohemoglobin reductase from cardiac muscle may further comprise the steps:
1) the fresh pig cardiac muscle is minced, add the water homogenized, thick homogenate 2N NH
4OH transfers to 7.5 to pH, leave standstill centrifugal, after filtering crude extract;
2) crude extract is through the 40%-70% ammonium sulfate precipitation, and the precipitation that obtains is resuspended with level pad, gets supernatant liquor after centrifugal level pad is dialysed, and the molecular weight cut-off of dialysis membrane is 14 000, dialyzate; Said level pad is 0.02M, pH6.0 phosphate buffered saline buffer;
3) sample on the dialyzate is arrived buffer A equilibrated DEAE-Sepharose Fast Flow anion-exchange chromatography post, Xian takes off foreign protein with buffer A, buffer A and buffer B are carried out gradient elution again, collect to merge the enzymic activity part, concentrate dialysis: said buffer A is 0.002M, pH7.0, contain 0.001M EDTA phosphate buffered saline buffer; Buffer B is 0.2M, pH7.0, contains 0.001M EDTA phosphate buffered saline buffer;
4) with the CM-Sephadex Fast Flow cation-exchange chromatography post of sample on the dialyzate that obtains to damping fluid C equilibrate overnight; With damping fluid C wash-out foreign protein, after wash-out is intact,, collect merging enzymic activity part with damping fluid D wash-out, dialysis concentrates: said damping fluid C is 0.02M, pH6.0 phosphate buffered saline buffer, and said damping fluid D is 0.2M, pH6.0 phosphate buffered saline buffer;
5) will have enzyme lives after the dialyzate lyophilize of component, last sample is to using damping fluid E equilibrated blue-sepharose gel FF affinity chromatography, with damping fluid E wash-out foreign protein, at last with damping fluid E with contain 1.0M NaCl damping fluid E and carry out linear linear gradient, collection has the elutriant of enzyme active component, promptly obtain the high ferro myohemoglobin reductase of purifying: said damping fluid E is 0.03M, pH6.0, contains 0.001M EDTA phosphate buffered saline buffer.
For the ease of preserving, also can be after dialysis, lyophilize with the above-mentioned high ferro myohemoglobin reductase liquid that obtains ,-80 ℃ of packing are preserved.
Similar to common proteolytic enzyme extraction purifying, wherein the 2nd～5 step was preferably under 4 ℃ and carries out.
Said animal cardiac muscle in the inventive method, preferred pig myocardium is because its source is wide, cost is low; The cardiac muscle of other animals such as ox, sheep etc. equally can be as starting material.For those skilled in the art, be appreciated that except that cardiac muscle other are rich in the animal muscle such as the skeletal muscle of high ferro myohemoglobin reductase, equally can be with reference to step of the present invention, the preparation high ferro myohemoglobin reductase.The inventive method has the product purity height, and technology is simple, low cost and other advantages, both can fill up the blank of this biological reagent preparation of China, has higher economic value again.
Description of drawings
The present invention is further described below in conjunction with drawings and Examples.
Fig. 1 is a DEAE-Sepharose Fast Flow chromatography collection of illustrative plates
Fig. 2 is a CM-Sephadex Fast Flow chromatography collection of illustrative plates
Fig. 3 is blue-sepharose Fast Flow affinity chromatography figure
Fig. 4 is SDS-PAGE and PAGE electrophorogram
M. albumen marker 1. crude extracts 2. thing 3. anionresin things 4. cationic exchange thing 5. affinity chromatography things, 6. affinity chromatography things (PAGE electrophoresis) of saltouing wherein
Fig. 5 is the SDS-PAGE electrophorogram
M. albumen marker 1. affinity chromatography things wherein
Fig. 6 is a gel scintigram behind the electrophoresis
Wherein A is a gel scintigram behind the crude extract sample electrophoresis
B is a gel scintigram behind the anion-exchange chromatography sample electrophoresis
C is a gel scintigram behind the cation-exchange chromatography sample electrophoresis
D is a gel scintigram behind the sample electrophoresis after the affinity chromatography
Embodiment
Employed in the present invention term unless other explanation is arranged, generally has the implication of those of ordinary skills' common sense.
Below in conjunction with specific embodiment, and the present invention is described in further detail with reference to the accompanying drawing data.Should be understood that these embodiment and accompanying drawing just in order to demonstrate the invention, but not limit the scope of the invention by any way.
In following embodiment, various processes of Xiang Ximiaoshuing and method are not ordinary methods as known in the art.The source of agents useful for same, trade(brand)name and be necessary to list its moiety person indicate when occurring first that all used thereafter identical reagent if no special instructions, and is all identical with the content of indicating first.
Embodiment 1: the preparation of high ferro myohemoglobin reductase
Material
Fresh Pigs Hearts, the anion-exchange chromatography material (DEAE-Sepharose Fast Flow, Pharmacia), cation-exchange chromatography material (CM-Sephadex Fast Flow, Pharmacia), blue-sepharose FastFlow affinity chromatography gel (Affi-Gel Blue Fast Flow, Pharmacia), (pig myocardium myohaemoglobin (this laboratory self-control), EDTA (Amresco company), NADH (purity 98%, Amresco company), phosphoric acid salt, K
4Fe (CN)
6Deng being homemade analytical pure, dialysis tubing (1 4000CoMW), polyoxyethylene glycol, the Xylene Brilliant Cyanine G test kit, other reagent is analytical pure.
Key instrument
Spectrumlab54 ultraviolet-visible pectrophotometer (Shanghai), 722 spectrophotometers (Shanghai), FJ-200 high speed dispersion homogenizer (Shanghai), the accurate pH meter (Shanghai) of PHS-3C type, JB22 type constant temperature blender with magnetic force, thermostatic water-circulator bath pot (Shanghai), biochemical temperature refrigerator (REVCO, USA), LGR10-4.2 high-speed refrigerated centrifuge (Beijing), Ultralow Temperature Freezer (REVCO, USA), CXG-1 computer thermostat layer analysis system (Shanghai), DYY-12 type electrophoresis apparatus and electrophoresis chamber, CS-9000 Dual-wavelength Flying-spot scanner (SHIMADZU, JAPAN)
Method
(1) the thick extraction
Remove the fresh Pigs Hearts of fat and reticular tissue, be cut into small pieces,, remove residual blood with ice-cold normal saline flushing.Mince, every gram meat sample adds 2mL cold water again, under condition of ice bath, with FJ-200 high speed dispersion clarifixator homogenate 60～90s, thick homogenate 2N NH
4OH transfers to 7.5 with the pH value, leaves standstill about 30min, and the centrifugal 20min of 13700g gets supernatant liquor.
(2) saltout and dialyse
Supernatant liquor adds solid ammonium sulfate to 40% saturation ratio, and pH value transfers to 7.5, stirring 30min, leave standstill 2～3h under 4 ℃ after, the centrifugal 20min of 13700g abandons precipitation.Supernatant liquor adds solid ammonium sulfate to 70% saturation ratio, and pH value transfers to 7.5, stirring 30min, leave standstill 2～3h under 4 ℃ after, the centrifugal 20min of 13700g.Throw out after the dialysis, is concentrated into certain volume earlier in water, (pH6.0) fully dialysis in the 0.002M phosphoric acid buffer, and the centrifugal 30min of 22 000g abandons precipitation, keeps supernatant liquor.
(3) anion-exchange chromatography (DEAE-Sepharose Fast Flow)
Sample on the dialyzate is arrived with 0.002M phosphate buffered saline buffer (pH7.0, contain 0.001M EDTA) equilibrated DEAE-Sepharose Fast Flow anion-exchange chromatography post (3 * 20cm), take off foreign protein with same damping fluid Xian, flow velocity is 30ml/h, and the 2ml/ pipe is used 0.002M phosphate buffered saline buffer (pH7.0 again, contain 0.001M EDTA) and 0.2M phosphate buffered saline buffer (pH7.0, contain 0.001M EDTA) carry out gradient elution, flow velocity is 20ml/h, the 3ml/ pipe.Chromatography collection of illustrative plates such as Fig. 1 collect merging enzymic activity part, concentrate.
(4) cation-exchange chromatography (CM-Sephadex Fast Flow)
The enzyme liquid that concentrates certain volume is dialysed in 0.02M phosphoric acid buffer (pH6.0), behind the centrifugal 30min of 22 000g, sample on the dialyzate is arrived the CM-Sephadex Fast Flow cation-exchange chromatography post (2 * 20cm) of using 0.02M phosphoric acid buffer (pH6.0) equilibrate overnight; With same buffer solution elution foreign protein, flow velocity is 20ml/h, treat that wash-out is intact after, with 0.2M phosphoric acid buffer (pH6.0) wash-out, flow velocity is 30ml/h, the 3ml/ pipe.Chromatography collection of illustrative plates such as Fig. 2 collect merging enzymic activity part, concentrate.
(5) blue-sepharose FastFlow affinity chromatography (Affi-Gel Blue Fast Flow)
With the dialysis in 0.03M phosphate buffered saline buffer (pH6.0 contains 0.001M EDTA) of spissated above-mentioned enzyme liquid, behind the centrifugal 30min of 22000g, sample on the supernatant liquor is arrived with same damping fluid balance affinity column (0.4 * 2cm post); With 0.03M phosphate buffered saline buffer (pH6.0, contain 0.001M EDTA) the wash-out foreign protein, flow velocity is 12ml/h, after treating that wash-out is intact, with 0.03M phosphoric acid buffer (pH6.0, contain 0.001M EDTA) and same damping fluid (containing 1.0M NaCl) carry out linear gradient elution, flow velocity is 18ml/h, 2ml/ pipe.Chromatography collection of illustrative plates such as Fig. 3 collect merging enzymic activity part.
(6) the pure product that will obtain dialysis in 0.002M phosphate buffered saline buffer (pH7.0 contains 0.001M EDTA) is after the lyophilize, in-80 ℃ of preservations.
Embodiment 2: the qualitative and quantitative of high ferro myohemoglobin reductase
The product that slightly get sample that (1)-(5) step obtains among the embodiment 1, through the ammonium sulfate precipitation sample, through zwitterion displacement chromatography sample with handle sample through affinity chromatography and carry out SDS-PAGE and the test of PAGE electrophoresis, electrophorogram is seen Fig. 4.With the CS-9000 gel analysis software (λ=595nm of gel behind the SDS-PAGE electrophoresis; W * H:0.05 * 2.0mm) carry out band scanning quantitative analysis the results are shown in Figure 6.As can be seen from Figure 6, slightly get sample product and the albumen kind that after 40%～70% ammonium sulfate segmentation is saltoutd, contains more, and can not be fine between the albumen separately; Behind the zwitterion displacement chromatography, roughly can see 2 bands, band is also more clear, and the corresponding band concentration of MetMbR institute is the highest, and the relative content of MetMb has reached 68.01% through scanning as can be known; After after the affinity chromatography, all obtain single proteolytic enzyme band at last through SDS-PAGE and two kinds of detections of PAGE, it is pure to show that high ferro myohemoglobin reductase has reached electrophoresis, and content is greater than 98% (Fig. 6), this proteic molecular weight about 46KDa (Fig. 5).With the enzyme liquid that obtains after the affinity chromatography, be the 580nm place by spectrophotometer at wavelength, sweep measuring enzymic activity (table 1) judges that thus this albumen is pure high ferro myohemoglobin reductase.
Table 1 high ferro myohemoglobin reductase purification result
Claims (2)
1, a kind of method of extracting purifying high ferro myohemoglobin reductase from cardiac muscle may further comprise the steps:
1) the fresh animal cardiac muscle is minced, add the water homogenized, thick homogenate 2N NH
4OH transfers to 7.5 to pH, leave standstill centrifugal, after filtering crude extract;
2) crude extract is through the 40%-70% ammonium sulfate precipitation, and the precipitation that obtains is resuspended with level pad, gets supernatant liquor after centrifugal level pad is dialysed, and the molecular weight cut-off of dialysis membrane is 14 000, dialyzate; Said level pad is the 0.02M phosphoric acid buffer, pH6.0;
3) sample on the dialyzate is arrived with buffer A equilibrated DEAE-Sepharose Fast Flow anion-exchange chromatography post, Xian takes off foreign protein with buffer A, carries out gradient elution with buffer A and buffer B again, collects to merge the enzymic activity part, concentrates and dialyses; Said buffer A is 0.002M, pH7.0, contain the phosphate buffered saline buffer of 0.001M EDTA; Buffer B J is 0.2M, pH7.0, contain 0.001M EDTA phosphate buffered saline buffer;
4) sample on the dialyzate that obtains is arrived the CM-Sephadex Fast Flow cation-exchange chromatography post of using damping fluid C equilibrate overnight; With damping fluid C wash-out foreign protein, after wash-out is intact,, collect merging enzymic activity part with damping fluid D wash-out, dialysis concentrates; Said damping fluid C is 0.02M, pH6.0 phosphoric acid buffer, and said damping fluid D is 0.2M, pH6.0 phosphoric acid buffer;
5) will have enzyme lives after the elutriant lyophilize of component, last sample is to using damping fluid E equilibrated blue-sepharose gel FF affinity chromatography, with damping fluid E wash-out foreign protein, at last with the linear gradient elution of damping fluid E with the damping fluid E composition that contains 1.0M NaCl, collection has the elutriant of enzyme component alive, promptly obtains the high ferro myohemoglobin reductase of purifying; Said damping fluid E is 0.03M, pH6.0, contain 0.001M EDTA phosphate buffered saline buffer.
2, according to the said method of from cardiac muscle, extracting purifying high ferro myohemoglobin reductase of claim 1, it is characterized in that also having step 6): the high ferro myohemoglobin reductase liquid that obtains after dialysis, lyophilize ,-80 ℃ of packing are preserved.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060294A (en) * | 2012-12-28 | 2013-04-24 | 青岛九龙生物医药有限公司 | Preparation method for urokinase capable of removing pyrogens and viruses |
| CN105399817A (en) * | 2015-10-22 | 2016-03-16 | 浙江海洋学院 | Extraction and purification method of tuna myoglobins |
| CN106404480A (en) * | 2016-08-30 | 2017-02-15 | 宁波美康生物科技股份有限公司 | Method for extracting myoglobin in pig hearts and human total antioxidant status kit containing myoglobin |
| CN114773454A (en) * | 2022-04-27 | 2022-07-22 | 广州蕊特生物科技有限公司 | Extraction and purification method for extracting myoglobin from horse heart |
-
2008
- 2008-06-06 CN CN2008101238401A patent/CN101294148B/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060294A (en) * | 2012-12-28 | 2013-04-24 | 青岛九龙生物医药有限公司 | Preparation method for urokinase capable of removing pyrogens and viruses |
| CN103865909A (en) * | 2012-12-28 | 2014-06-18 | 青岛九龙生物医药有限公司 | Preparation method of urokinase capable of removing pyrogen and virus |
| CN103865909B (en) * | 2012-12-28 | 2016-04-13 | 青岛九龙生物医药有限公司 | A kind of preparation method that can remove the urokinase of pyrogen and virus |
| CN105399817A (en) * | 2015-10-22 | 2016-03-16 | 浙江海洋学院 | Extraction and purification method of tuna myoglobins |
| CN106404480A (en) * | 2016-08-30 | 2017-02-15 | 宁波美康生物科技股份有限公司 | Method for extracting myoglobin in pig hearts and human total antioxidant status kit containing myoglobin |
| CN114773454A (en) * | 2022-04-27 | 2022-07-22 | 广州蕊特生物科技有限公司 | Extraction and purification method for extracting myoglobin from horse heart |
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