CN100363381C

CN100363381C - Method for extracting purified high ferro myoglobins from cardiac muscle

Info

Publication number: CN100363381C
Application number: CNB200610037909XA
Authority: CN
Inventors: 金邦荃; 汤祥明; 刘兴余
Original assignee: Nanjing Normal University
Current assignee: Nanjing Normal University
Priority date: 2006-01-20
Filing date: 2006-01-20
Publication date: 2008-01-23
Anticipated expiration: 2026-01-20
Also published as: CN1800202A

Abstract

The present invention relates to an extractive and purified process for proteins, more specifically to a method for extracting and purifying high ferri-myoglobin from myocardium. In the specific method, fresh animal myocardium which is preprocessed is extracted to obtain crude extracts of tissue proteins; then, after being salted out by ammonium sulfate, the crude extracts are processed by a dialysis membrane whose molecular weight cutoff is 14000 to obtain dialysis samples; the dialysis samples are processed by anion exchange chromatography (DEAE-Sepharose Fast Flow) to collect brunneus eluent; the collected brunneus eluent are processed by gel filtration chromatography (SephacrylS-200HR) to collect brunneus eluent which is corresponding to an elution peak; thereby, the purified ferri-myoglobin is obtained. The method of the present invention has the advantages of high product purity, simple manufacturing process, low cost, etc.; the method not only can fill blank spaces for preparing biological reagents in China, but also can have superior economic value.

Description

From cardiac muscle, extract the method for purifying high ferro myohemoglobin

Technical field

The present invention relates to a kind of proteic extraction and purification process, specifically is the method for extracting purifying high ferro myohemoglobin from cardiac muscle.

Background technology

Myohaemoglobin is a kind of important reduced hematin in vertebrates skeletal muscle and the cardiac muscular tissue's endochylema, and myohaemoglobin has reversibly and oxygen bonded character, and its major function is transportation and store oxygen in the myocyte.Metmyoglobin (MetMb) is its oxidised form, it can transform with deoxidation myohaemoglobin and oxymyoglobin under the effect of certain enzyme system in animal body mutually, this has the important physical effect to finishing normal respiratory, will influence normally carrying out of respiratory if obstacle takes place this process.The metmyoglobin of purifying can be applied to the research of animal meat color stability etc., also can be used as the substrate of people's Keshan disease detection.Therefore, the metmyoglobin of purifying can be used as biological reagent and is applied to clinical detection and research.

The domestic also not relevant at present report of the separation and purification of metmyoglobin, once reported the method for purification oxymyoglobin from beef abroad in the document, but these methods need special equipment, as TSK-2000W gel high pressure chromatography column, high performance liquid chromatography (HPLC) etc., and required time is longer, be generally (Gotoh, T.andShikama, K. (1976) .Biochom.J. about 3 days, 80,397, Krzywicki, K. (1982) .Meat Sci., 7,29, Gatellier, Ph., Anton, M.and Renerre, M. (1993) .Meat Sci., 33,401, Renerre M, AntonM, Gatellier, Ph. (1992) .Meat Science, 32,331).Be unsuitable for suitability for industrialized production.

Summary of the invention

The purpose of this invention is to provide a kind of method of extracting purifying high ferro myohemoglobin, this method is selected DEAE-Sepharose Fast Flow and two kinds of chromatography medias of Sephacryl S-200HR for use, adopt chromatography from Pigs Hearts, to separate and purifying high ferro myohemoglobin, 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 from cardiac muscle may further comprise the steps:

1) the fresh animal cardiac muscle is minced, adds ice-cold myohaemoglobin extraction damping fluid, through homogenate, centrifugal, after filtering crude extract; Said myohaemoglobin extraction damping fluid is 10mM Tris-HCl, 1mM EDTA, pH8.0;

2) crude extract is got supernatant through the ammonium sulfate precipitation post precipitation of 70% saturation ratio, adds solid ammonium sulfate to 100% saturation ratio again, saltouts, and the precipitation that obtains is resuspended with level pad A, adds excessive K again ₃Fe (CN) ₆, get supernatant liquor after centrifugal to level pad A dialysis, the molecular weight cut-off of dialysis membrane is 14000, dialyzate; Said level pad A is 10mM Tris-HCl, 1mM EDTA, 10mM NaCl, pH8.5;

3) with sample on the dialyzate to DEAE-Sepharose Fast Flow anion-exchange chromatography post, the NaCl of the 10mM among the level pad A is transferred to 100mM, use this buffer solution elution again, collect the sorrel washing lotion of elution peak correspondence;

4) with the cool freeze-drying of elutriant of collecting is dry concentrate after, last sample is to Sephacryl S-200HR gel permeation chromatography post, usefulness level pad B wash-out, the corresponding sorrel elutriant of collection elution peak promptly obtains the metmyoglobin of purifying; Said level pad B is 100mM Tris-HCl, 100mM NaCl, pH7.5.

For the ease of preserving, can be with packing after the product lyophilize that obtains, in-80 ℃ of preservations.

Similar to common proteins extraction purifying, wherein the 2nd～4 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 metmyoglobin, equally can be with reference to step of the present invention, the preparation metmyoglobin.

At present a lot of hospitals of China and scientific research institution are in the research of doing aspect the myohaemoglobin, and the metmyoglobin standard substance all need to buy from Sigma company, cost an arm and a leg.The price of Sigma company metmyoglobin is about 150 dollars/gram, and purity can only reach 〉=and 95%, and from cardiac muscle, purify the metmyoglobin purity that obtains greater than Sigma company by the inventive method, and it is pure that purity reaches electrophoresis, and yield reaches 14.1%.And starting material such as Pigs Hearts can be directly from the slaughterhouse buying, and are with low cost.Therefore 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 Sephacryl S-200HR sieve chromatography collection of illustrative plates

Fig. 3 is the SDS-PAGE electrophorogram

M. albumen marker wherein; 1. crude extract; 2. sample after dialysing; 3. sample after ion-exchange; 4. sample behind the gel chromatography

Fig. 4 A is a gel scintigram behind the crude extract sample electrophoresis

Fig. 4 B is a gel scintigram behind the ammonium sulfate precipitation sample electrophoresis

Fig. 4 C is a gel scintigram behind the ion exchange chromatography sample electrophoresis

Fig. 4 D is a gel scintigram behind the sample electrophoresis behind the gel chromatography

Fig. 5 is the spectrophotometer scintigram of MetMb

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 metmyoglobin

Material

Fresh Pigs Hearts, anion-exchange chromatography material (DEAE-Sepharose Fast Flow, Pharmacia), gel permeation chromatography material (Sephacryl S-200HR, Amersham-Pharmacia), horse cardiac muscle metmyoglobin standard substance (Sigma, purity＞90%), the Xylene Brilliant Cyanine G test kit, other reagent is analytical pure.Myohaemoglobin extraction damping fluid: 10mM Tris-HCl, 1mM EDTA, pH8.0; Level pad A:10mM Tris-HCl, 1mMEDTA, 10mM NaCl, pH8.5; Level pad B:100mM Tris-HCl, 100mM NaCl, pH7.5.Key instrument and reagent

DS-1 high-speed tissue mashing machine, FJ-200 high speed dispersion homogenizer, JB-2 type constant temperature blender with magnetic force, the accurate pH meter of PHS-3C type, BioLogic LP chromatographic system, Gel Doc2000 imaging system, JeDa801 gel analysis software, Spectrumlab54 ultraviolet-visible pectrophotometer, the LGJ1.5 freeze drier, the LGR10-4.2 high-speed refrigerated centrifuge, the high scheming of the desk-top freezing high speed of Eppendorf 5415R, DYY-12 type electrophoresis apparatus and electrophoresis chamber.

Method

(1) the thick extraction

Remove about 220 grams of fresh Pigs Hearts of fat and reticular tissue, be cut into small pieces,, remove residual blood with ice-cold normal saline flushing.Mince again, add myohaemoglobin extraction damping fluid 440ml, smash to pieces with DS-1 high-speed tissue mashing machine in 1: 2 ratio (V/M).Use FJ-200 high speed dispersion homogenizer with 20000rpm homogenate 100s again, 4 ℃ leave standstill extraction 1h after, with 5000rpm, 4 ℃ of following centrifugal 10min, supernatant liquor carries out coarse filtration with the middling speed qualitative filter paper, must crude extract 350ml.

(2) saltout and dialyse

Filtrate is regulated pH value to 8.0 with the cold ammonium water of 2M, under 4 ℃, and the centrifugal 20min of 10000rpm.The beaker of containing supernatant liquor is put on the JB-2 type constant temperature blender with magnetic force, slowly added solid (NH ₄) ₂SO ₄To 70% saturation ratio, regulate pH value to 8.0 with the cold ammonium water of 2M, leave standstill 30min after, with the centrifugal 20min of 10000rpm, abandon precipitation under 4 ℃.Supernatant liquor slowly adds solid (NH again ₄) ₂SO ₄To 100% saturation ratio, regulate pH value to 8.0 with the cold ammonium water of 2M.Leave standstill 30min, the centrifugal 25min of 10000rpm abandons supernatant, precipitates resuspended with cold level pad A.Add excessive K ₃Fe (CN) ₆, the centrifugal 10min of 10000rpm gets supernatant level pad A dialysis (14000MWcut-off) is spent the night, and changes liquid therebetween 3 times, gets dialyzate 102ml.

(3) ion exchange chromatography

The dialyzate that obtains arrives the DEAE-Sepharose Fast Flow anion-exchange chromatography post of using level pad A equilibrate overnight with sample on the flow velocity of 0.4ml/min, with the unconjugated albumen of same damping fluid flush away, carry out wash-out with the same damping fluid that contains 100mM NaCl again, elution speed is 1.25ml/min.280nm detects down and record automatically, collects the elutriant of elution peak correspondence automatically.The chromatography collection of illustrative plates as shown in Figure 1, the effluent liquid of elution peak correspondence is a sorrel.

(4) gel-filtration

With the elutriant of above-mentioned collection after the lyophilize of LGJ1.5 freeze drier concentrates, last sample is to using the good Sephacryl S-200HR gel permeation chromatography post of level pad B balance, use same buffer solution elution, elution speed is 1ml/min, collect the sorrel elutriant, promptly obtain metmyoglobin.The chromatography collection of illustrative plates has 3 peaks as shown in Figure 2 on the curve, first peak correspondence be colourless liquid, second peak correspondence be sorrel liquid, the 3rd peak correspondence also be colourless liquid.

(5) with after the product lyophilize that obtains, in-80 ℃ of preservations.

Embodiment 2: the qualitative and quantitative of metmyoglobin

To implement the product that slightly get sample that (1) in 1-(4) step obtains, dialyzate sample, through the ion exchange chromatography sample with handle sample through gel-filtration and carry out the test of SDS-PAGE electrophoresis, the result is as shown in Figure 3.After gel is taken pictures with Gel Doc2000 imaging system behind the electrophoresis, carry out band scanning quantitative analysis, result such as Fig. 4 through JeDa801 gel analysis software.

From Fig. 3 and Fig. 4 analysis as can be known, the albumen kind that contains in the product that slightly get sample is more, and can not fine separately (Fig. 4 A) between the albumen; After the segmentation of 70%-100% ammonium sulfate is saltoutd and is dialysed, roughly can see nine bands, promptly mainly constitute, and gel scans as can be known behind the electrophoresis by nine kinds of protein, the relative content of MetMb is 39.29% (Fig. 4 B) in the extracting solution after the dialysis; Behind ion exchange chromatography, roughly be made up of five bands, band is also more clear again, and the corresponding band concentration of MetMb institute is the highest, and the relative content of MetMb has reached 88.12% (Fig. 4 C) through scanning as can be known; After after the gel-filtration, only stay next band, show the purified electrophoretically pure albumen that obtained, content is greater than 98% (Fig. 4 D), and this proteic molecular weight is about 17000.

Second peak correspondence of collecting after the gel-filtration carried out length scanning for sorrel liquid and horse cardiac muscle metmyoglobin standard substance, by spectrophotometer scanning spectra (Fig. 5), as seen they are that 505nm and 630nm place have charateristic avsorption band respectively at wavelength all.Judge that thus this albumen is exactly metmyoglobin.

Claims

1. method of extracting purifying high ferro myohemoglobin from cardiac muscle may further comprise the steps:

1) the fresh animal cardiac muscle is minced, adds ice-cold myohaemoglobin extraction damping fluid, through homogenate, centrifugal, after filtering crude extract; Said myohaemoglobin extraction damping fluid is 10mM Tris-HCl, 1mM EDTA, and pH8.0:

2. according to the said method of from cardiac muscle, extracting purifying high ferro myohemoglobin of claim 1: it is characterized in that wherein the 2nd～4 step carried out under 4 ℃.

3. according to claim 1 or the 2 said methods of from cardiac muscle, extracting purifying high ferro myohemoglobin, it is characterized in that said animal cardiac muscle is a pig myocardium.

4. according to the said method of from cardiac muscle, extracting purifying high ferro myohemoglobin of claim 3, it is characterized in that also have step 5): the metmyoglobin liquid that obtains is through lyophilize, in-80 ℃ of preservations.