A kind of preparation method of no carbonic anhydrase hemoglobin
Technical field
The present invention relates to a kind of preparation methods of hemoglobin, more particularly to a kind of system of no carbonic anhydrase hemoglobin
Preparation Method.
Background technique
As medicinal is increased with blood rigid demand with the speed of 10-15%, and number relative growth of donating blood is slow, makes
Insufficiency of supply-demand is obtained to widen year by year.In addition in facing the emergency emergency event such as war, natural calamity, the importance of blood security
It highlights.Due to the particularity of blood itself, such as storage life is short, traffic condition is harsh, blood group cooperation, security risk, institute
Using blood substitute as drug, there is irreplaceable advantage.
Blood substitute refers to the artificial preparations with blood formed element and aeriform part function, can be divided into red blood cell generation
Articles, blood platelet substitute and blood substitutes etc..It is usually said in view of the special efficacy and important function of red blood cell substitute
Blood substitute be actually red blood cell substitute.It includes chemical synthesis fluorocarbons class and hemoglobin-based oxygen carrier
Class.Wherein, hemoglobin-based oxygen carrier is to carry out chemistry based on natural or recombinant hemoglobin to change structure or package and obtain
With a kind of product for taking/putting oxygen function, the i.e. red blood cell substitute of narrow sense, and domestic and international red blood cell substitute in decades
The hot spot of research.In particular with the clear understanding to the natural carrier of oxygen (i.e. hemoglobin) structure and performance, make it
The optimal candidate of blood substitute, and the hemoglobin for preparing high-purity is to guarantee that the research is successfully basic.
Hemoglobin is the main component of red blood cell, accounts for about the 32% of cell weight, each haemoglobin molecule is by 1 pearl
Albumen and 4 ferroheme (also known as ferroprotoporphyrin) compositions.Each ferroheme forms a ring, center by 4 pyrrole radicals again
For a ferrous ion.Globin has 4 polypeptide chains, and every polypeptide chain is connected with 1 ferroheme, constitutes the list of hemoglobin
Body or subunit.Hemoglobin is the tetramer being made of 4 monomers.Haemoglobin molecule is under normal conditions with dimer and four
Aggressiveness equilibrium state exists, and the molecular weight of the tetramer is about 68Kd, and the molecular weight of dimer is about 34Kd, theoretical isoelectric point (PI)
About 6.7-6.9;Carbonic anhydrase (EC 4.2.1.1) is a kind of zinc enzyme, and have that catalysis carbonic acid resolves into carbon dioxide and water can
Back reaction, is one of major protein components of red blood cell, and status is only second to hemoglobin.Molecular weight is about 30kD, by single
Peptide chain composition includes about 260 amino acid residues, and each enzyme molecule includes a zinc ion, and theoretical isoelectric point (PI) is about
6.3-6.5.With isoelectric point, the dimer molecule amount of hemoglobin etc. all very close to being difficult general with molecular sieve, ion exchange etc.
Method separates them, simultaneously because the interference of high maternal hemoglobin concentrations, is also difficult to reach using simple affinitive layer purification mode
To ideal separating effect.Therefore, it is badly in need of researching and developing a kind of preparation of high-purity hemoglobin that no carbonic anhydrase can be separated
Method.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of preparation methods of no carbonic anhydrase hemoglobin, to remove blood
Carbonic anhydrase in hemoglobin solution, and can have the higher rate of recovery, while the hemoglobin for obtaining high-purity also
The carbonic anhydrase of high-purity has been arrived, two has been obtained at one stroke, dual income.
In order to solve the above technical problems, the present invention provides a kind of preparation method of no carbonic anhydrase hemoglobin, packet
Include following steps:
A, fresh anticoagulant ox blood, with 0.9%NaCl washed blood, with water lysed erythrocyte, centrifugation removal cell fragment and
Undissolved cell obtains supernatant hemoglobin solutions to be purified;
B, it obtains that filler Na is added in hemoglobin solutions to be purified in step a2SO4, obtain hemoglobin to be purified
Solution example;
C, before loading, with equilibrium liquid 0.1-0.3M Na2SO4Jie of/25-50mM Tris-HCl balance affinity chromatography chromatography
Matter, the hemoglobin solutions sample loading for then obtaining step b obtain flowing through liquid, hemoglobin as after purification.
The preparation method of above-mentioned no carbonic anhydrase hemoglobin, wherein in the step b, the filler Na2SO4Plus
Entering amount is that 1.4-4.3g Na is added in every 100ml hemoglobin solutions to be purified2SO4。
The preparation method of above-mentioned no carbonic anhydrase hemoglobin, wherein in the step b, the filler Na2SO4Plus
Entering amount is that 2.85g Na is added in every 100ml hemoglobin solutions to be purified2SO4。
The preparation method of above-mentioned no carbonic anhydrase hemoglobin, wherein the medium of the affinity chromatography chromatography is CM
Sephadex-Sulfanilamid。
The preparation method of above-mentioned no carbonic anhydrase hemoglobin, wherein in the step c, before loading, with equilibrium liquid 0.2M
Na2SO4The medium of/25mM Tris-HCl balance affinity chromatography chromatography.
The present invention also provides a kind of preparation methods of carbonic anhydrase comprising following steps:
A, fresh anticoagulant ox blood, with 0.9%NaCl washed blood, with water lysed erythrocyte, centrifugation removal cell fragment and
Undissolved cell obtains supernatant hemoglobin solutions to be purified;
B, it obtains that filler Na is added in hemoglobin solutions to be purified in step a2SO4, obtain hemoglobin to be purified
Solution example;
C, before loading, with equilibrium liquid 0.1-0.3M Na2SO4Jie of/25-50mM Tris-HCl balance affinity chromatography chromatography
Matter, the hemoglobin solutions sample loading for then obtaining step b obtain flowing through liquid, hemoglobin as after purification;
D, with affinity chromatography chromatography the first eluent 20-40mM Na2SO4/ 5-8mM Tris-HCl washes away non-specificity
The impurity of absorption;
E, continue to elute with the second eluent of affinity chromatography chromatography, which includes oxidized form
Ionic strength agent and CH3COONa, the concentration of the second eluent ionic strength agent are 0.4-0.8M, CH3The concentration of COONa is
0.2-0.3M washes away the carbonic anhydrase of Specific adsorption, the carbonic anhydrase purified.
The preparation method of above-mentioned carbonic anhydrase, wherein in the step e, the oxidized form ionic strength agent is NaClO3、
MgO2And NaClO4。
The preparation method of above-mentioned carbonic anhydrase, wherein in the step e, the oxidized form ionic strength agent is NaClO4。
The preparation method of above-mentioned carbonic anhydrase, wherein in the step e, second eluent is 0.6M NaClO4/
0.2M CH3COONa。
The preparation method of above-mentioned carbonic anhydrase, wherein in the step d, first eluent is 30mM Na2SO4/
5mM Tris-HCl。
Preparation method without carbonic anhydrase hemoglobin of the invention has the following beneficial effects:
1, the hemoglobin preparation method of no carbonic anhydrase of the invention is the base in specific affinity chromatography chromatographic isolation
On plinth, by optimizing alternate contact, to achieve the purpose that thoroughly to purify, by many experiments, find in blood red egg to be purified
The filler Na for optimizing alternate contact is added in white solution2SO4The absorption of carbonic anhydrase can be significantly improved, while also being reduced blood red
Nonspecific absorption of albumen can obtain the hemoglobin of high-purity, and purity can achieve 99.99%, while also obtain
The carbonic anhydrase of high-purity, purity can achieve 99.8% or more, kill two birds with one stone, dual income;
2, ionic strength agent NaClO is added in the preparation method without carbonic anhydrase hemoglobin of the invention, when elution4, can be with
Carbonic anhydrase is more thoroughly washed away, the yield of carbonic anhydrase can be obviously improved, yield is up to 95%, while also having reached medium
Good renaturation, shorten the filler renaturation manipulation time;
3, the preparation method without carbonic anhydrase hemoglobin of the invention is a kind of simple, removing that can amplify to pure blood red eggs
The new method of carbonic anhydrase in white, and medium renaturation is quick and easy, facilitates its recycling, reduces production cost, no
Need to put into complicated equipment, it is easy to promote and utilize.
Detailed description of the invention
Attached drawing 1 is the affinity chromatography 540nm spectrogram of the hemoglobin of the embodiment of the present invention 1;
Attached drawing 2 is that the affinity chromatography of the hemoglobin of the embodiment of the present invention 1 harvests the SDS-PAGE figure of product;
Attached drawing 3 is that the affinity chromatography of the hemoglobin of the embodiment of the present invention 1 harvests the Western blot figure of product.
Specific embodiment
The present invention is described in detail with reference to the accompanying drawings and examples.
Example 1
1, high-purity hemoglobin is prepared
Fresh anticoagulant ox blood is taken, three times with 0.9%NaCl washed blood, with cold-water solution red blood cell.Under the conditions of 4 DEG C,
Centrifugation 30 minutes, removes cell fragment and undissolved cell, supernatant are hemoglobin solutions to be purified;To be purified
Hemoglobin solutions in be added filler Na2SO4, wait for that 2.85g filler Na is added in pure hemoglobin solution by 100ml2SO4,
Obtain hemoglobin samples to be purified;Before loading, with equilibrium liquid 0.2M Na2SO4/ 25mM Tris-HCl balances affinity chromatography color
The medium of spectrum, affinity chromatography used medium is CM Sephadex-Sulfanilamide, then to be purified blood red by what is prepared
Protein sample loading obtains flowing through liquid, the hemoglobin as purified until flushing with medium level.
2, high-purity carbonic anhydrase is prepared
With affinity chromatography chromatography the first eluent 30mM Na2SO4/ 5mM Tris-HCl washes off the impurity of non-specific adsorption
Substance;With the second eluent 0.6M NaClO4/0.2M CH3COONa continues to elute, and it is pure to get arriving to wash off the carbonic anhydrase of absorption
The carbonic anhydrase of change, while also renaturation medium;With equilibrium liquid 0.2M Na2SO4After/25mM Tris-HCl balance chromatographic column i.e.
Next purification cycle can be entered.
It is monitored in the present embodiment by Ultraviolet Detector and collects each peak albumen, specific spectral results are shown in Fig. 1;Pass through vim and vigour
Analysis-e/or determining hemoglobin concentration, and with the yield of this hemoglobin calculated for 95.2%;Pass through reproducibility SDS-PAGE
Method measures each protein ingredient and content for collecting peak, and referring to fig. 2, as the result is shown: the hemoglobin purity of collection can reach
99.99%, about 30kD protein band in hemoglobin solutions, disappears after purification before purification, occurs about in the second eluent
The single protein band of 30KD, and the molecular weight that this band is shown is consistent with carbonic anhydrase, preliminary judgement should be carbonic anhydrase,
Yield is 95.6%, purity 99.91%;Further pass through the production of Western blot method qualitative detection about 30kD band
Object, referring to Fig. 3, as the result is shown: about 30kD band before purification is carbonic anhydrase, the about 30kD item occurred in the second eluent
Band is also carbonic anhydrase.To sum up testing result shows: carbonic anhydrase and hemoglobin are obtaining thoroughly after affinity chromatography
Separation also obtained the carbonic anhydrase of high-purity while the hemoglobin for obtaining high-purity, at one stroke two, dual receipts
Benefit, effect are ideal.
Embodiment 2
1, high-purity hemoglobin is prepared
Fresh anticoagulant ox blood is taken, three times with 0.9%NaCl washed blood, with cold-water solution red blood cell.Under the conditions of 4 DEG C,
18000g is centrifuged 30 minutes, removes cell fragment and undissolved cell, supernatant are hemoglobin solutions to be purified;To
Filler Na is added in hemoglobin solutions to be purified2SO4, wait for that 1.40g filler is added in pure hemoglobin solution by 100ml
Na2SO4, obtain hemoglobin samples to be purified;Before loading, with equilibrium liquid 0.1M Na2SO4/ 25mM Tris-HCl balance is affine
The medium of thin layer chromatography, affinity chromatography used medium are CM Sephadex-Sulfanilamide, then will be prepared to pure
Change hemoglobin samples loading, until flushing with medium level, obtains flowing through liquid, the hemoglobin as purified.
2, high-purity carbonic anhydrase is prepared
With affinity chromatography chromatography the first eluent 20mM Na2SO4/ 5mM Tris-HCl washes off the impurity of non-specific adsorption
Substance;With the second eluent 0.4M NaClO4/0.2M CH3COONa continues to elute, and it is pure to get arriving to wash off the carbonic anhydrase of absorption
The carbonic anhydrase of change, while also renaturation medium;With equilibrium liquid 0.1M Na2SO4After/25mM Tris-HCl balance chromatographic column i.e.
Next purification cycle can be entered.
It is detected using method same as Example 1, hemoglobin yield is 92.3%, and the purity of hemoglobin is
99.92%;The purity of carbonic anhydrase is 99.87%, yield 94.3%.
Embodiment 3
1, high-purity hemoglobin is prepared
Fresh anticoagulant ox blood is taken, three times with 0.9%NaCl washed blood, with cold-water solution red blood cell.Under the conditions of 4 DEG C,
18000g is centrifuged 30 minutes, removes cell fragment and undissolved cell, supernatant are hemoglobin solutions to be purified;To
Filler Na is added in hemoglobin solutions to be purified2SO4, wait for that 4.30g filler is added in pure hemoglobin solution by 100ml
Na2SO4, obtain hemoglobin samples to be purified;Before loading, with equilibrium liquid 0.3M Na2SO4/ 50mM Tris-HCl balance is affine
The medium of thin layer chromatography, affinity chromatography used medium are CM Sephadex-Sulfanilamide, then will be prepared to pure
Change hemoglobin samples loading, until flushing with medium level, obtains flowing through liquid, the hemoglobin as purified.
2, high-purity carbonic anhydrase is prepared
With affinity chromatography chromatography the first eluent 40mM Na2SO4/ 8mM Tris-HCl washes off the impurity of non-specific adsorption
Substance;With the second eluent 0.8M NaClO4/0.3M CH3COONa continues to elute, and it is pure to get arriving to wash off the carbonic anhydrase of absorption
The carbonic anhydrase of change, while also renaturation medium;With equilibrium liquid 0.3M Na2SO4After/50mM Tris-HCl balance chromatographic column i.e.
Next purification cycle can be entered.
It is detected using method same as Example 1, hemoglobin yield is 94.7%, and the purity of hemoglobin is
99.89%;The purity of carbonic anhydrase is 99.85%, yield 95.2%.
Embodiment 4
It is identical as the method for preparing high-purity hemoglobin and high-purity carbonic anhydrase of embodiment 1, the difference is that,
When preparing high-purity carbonic anhydrase, the oxidized form ionic strength agent used in the second eluent is different, uses in the present embodiment
Oxidized form ionic strength agent be NaClO3, carbonic anhydrase purity obtained and yield are shown in Table 1.
Embodiment 5
It is identical as the method for preparing high-purity hemoglobin and high-purity carbonic anhydrase of embodiment 1, the difference is that,
When preparing high-purity carbonic anhydrase, the oxidized form ionic strength agent used in the second eluent is different, uses in the present embodiment
Oxidized form ionic strength agent be MgO2, carbonic anhydrase purity obtained and yield are shown in Table 1.
Table 1
By table 1 it is found that influence of the different ionic strength agent to carbonic anhydrase purity is little, but from the influence to yield
On see, select NaClO4The yield of carbonic anhydrase can be obviously improved as ionic strength agent.
Comparative example 1
It is identical as the method that embodiment 1 prepares high-purity hemoglobin and high-purity carbonic anhydrase, the difference is that,
The filler being added in hemoglobin solutions and equilibrium liquid to be purified is CaSO4, the yield and purity of hemoglobin be shown in Table 2.
Comparative example 2
It is identical as the method that embodiment 1 prepares high-purity hemoglobin and high-purity carbonic anhydrase, the difference is that,
The filler being added in hemoglobin solutions and equilibrium liquid to be purified is CaHPO4, the yield and purity of hemoglobin be shown in Table 2.
Comparative example 3
It is identical as the method that embodiment 1 prepares high-purity hemoglobin and high-purity carbonic anhydrase, the difference is that,
Filler is added without in hemoglobin solutions to be purified, the yield and purity of hemoglobin are shown in Table 2.
Comparative example 4
It is identical as the method that embodiment 1 prepares high-purity hemoglobin and high-purity carbonic anhydrase, the difference is that,
Filler Na is added without in hemoglobin solutions to be purified2SO4, and filler Na is added without in equilibrium liquid2SO4, blood red egg
White yield and purity is shown in Table 2.
Table 2
By table 2 it is found that using CaSO in comparative example 1 and comparative example 24And CaHPO4As filler, and embodiment 1
Using Na2SO4As filler, the hemoglobin yield obtained using different fillers is not much different, but uses Na2SO4Make
For filler, it is obviously improved the purity of hemoglobin, the hemoglobin that available purity is 99.99%.In comparative example 3
Filler is not added in hemoglobin samples to be purified, compared with Example 1, yield is not much different, but filler is added
Na2SO4It has been obviously improved the purity of hemoglobin afterwards.Filling is not added in comparative example 4 in hemoglobin samples to be purified
Agent, and filler is not also added in equilibrium liquid, compared with Example 1, yield is declined slightly, but filler is added
Na2SO4Afterwards, the hemoglobin purity that embodiment 1 obtains is obviously improved, and can achieve 99.99%.Comparative example 3 and comparison
Example 4 is compared, and filler is added in equilibrium liquid and slightly improves the yield and purity of hemoglobin, and embodiment 1 further to
Filler Na is added in purified hemoglobin sample2SO4, it has been obviously improved the purity of hemoglobin.Therefore, pass through above-mentioned comparison
Experiment, the present invention select Na2SO4As the filler of loading hemoglobin samples to be purified and equilibrium liquid, blood can be effectively improved
The purity of Lactoferrin.