CN110128531B - Preparation method of carbonic anhydrase-free hemoglobin - Google Patents
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Abstract
The invention discloses a preparation method of carbonic anhydrase-free hemoglobin, which comprises the following steps: a. fresh anticoagulated bovine blood, washing the blood with 0.9% NaCl, dissolving red blood cells with water, centrifuging to remove cell debris and undissolved cells, and obtaining a supernatant to-be-purified hemoglobin solution; b. adding a filling agent Na into the hemoglobin solution to be purified obtained in the step a2SO4Obtaining a hemoglobin solution sample to be purified; c. before loading, use balance liquid 0.1-0.3M Na2SO4And c, 25-50mM Tris-HCl equilibrium affinity chromatography medium, then loading the hemoglobin solution sample obtained in the step b to obtain flow-through liquid, namely purified hemoglobin. The method can remove carbonic anhydrase in the hemoglobin solution, has higher recovery rate, obtains high-purity carbonic anhydrase while obtaining high-purity hemoglobin, and obtains two benefits at one stroke.
Description
Technical Field
The invention relates to a preparation method of hemoglobin, in particular to a preparation method of carbonic anhydrase-free hemoglobin.
Background
As the rigidity requirement of clinical blood increases at a speed of 10-15%, the number of blood donors is relatively slow to increase, so that the gap between supply and demand is increased year by year. In addition, the importance of blood guarantee is also highlighted in the face of emergency emergencies such as war, natural disasters and the like. Due to the particularities of blood itself, such as short shelf life, harsh transport conditions, blood type matching, safety risks, etc., blood substitutes have irreplaceable advantages as drugs.
The blood substitute is an artificial preparation having functions of blood formed components and blood non-formed components, and can be divided into red blood cell substitute, platelet substitute, blood plasma substitute, and the like. In view of the particular efficacy and important role of red blood cell substitutes, the so-called blood substitutes are actually red blood cell substitutes. It includes chemically synthesized fluorocarbons and hemoglobin oxygen carriers. Among them, hemoglobin oxygen carrier is a product with oxygen carrying/releasing function obtained by chemical modification or encapsulation based on natural or recombinant hemoglobin, namely a narrow-sense red blood cell substitute, and is also a hot spot of research on red blood cell substitutes at home and abroad for decades. Particularly, with clear knowledge of the structure and performance of natural oxygen carriers (i.e., hemoglobin), the natural oxygen carriers have become the best candidates for blood substitutes, and the preparation of high-purity hemoglobin is the basis for ensuring the success of the research.
Hemoglobin, the major component of red blood cells, accounts for approximately 32% of the cell weight, and consists of 1 globin and 4 heme (also known as ferroprotoporphyrins) per hemoglobin molecule. Each heme in turn consists of 4 pyrrole groups forming a ring, centered on a ferrous ion. Globin has 4 polypeptide chains, each of which is linked to 1 heme, constituting a monomer or subunit of hemoglobin. Hemoglobin is a tetramer of 4 monomers. Typically, hemoglobin molecules exist in an equilibrium state of dimers and tetramers, with the tetramers having a molecular weight of about 68Kd, the dimers having a molecular weight of about 34Kd, and a theoretical isoelectric Point (PI) of about 6.7 to 6.9; carbonic anhydrase (EC 4.2.1.1) is a zinc enzyme that has the reversible reaction of catalyzing the breakdown of carbonic acid into carbon dioxide and water, and is one of the major protein components of red blood cells, second only to hemoglobin. Has a molecular weight of about 30kD, consists of a single peptide chain, comprises about 260 amino acid residues, comprises one zinc ion per enzyme molecule, and has a theoretical isoelectric Point (PI) of about 6.3-6.5. The isoelectric point and the dimer molecular weight of the hemoglobin are very close, so that the hemoglobin is difficult to separate by using a general method such as molecular sieve and ion exchange, and simultaneously, due to the interference of high-concentration hemoglobin, an ideal separation effect is difficult to achieve by adopting a simple affinity chromatography purification mode. Therefore, it is urgently required to develop a method for preparing high-purity hemoglobin capable of separating carbonic anhydrase.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method of carbonic anhydrase-free hemoglobin, which is used for removing carbonic anhydrase in a hemoglobin solution, has higher recovery rate, obtains high-purity carbonic anhydrase while obtaining high-purity hemoglobin, and achieves double benefits by one action.
In order to solve the technical problem, the invention provides a preparation method of carbonic anhydrase-free hemoglobin, which comprises the following steps:
a. fresh anticoagulated bovine blood, washing the blood with 0.9% NaCl, dissolving red blood cells with water, centrifuging to remove cell debris and undissolved cells, and obtaining a supernatant to-be-purified hemoglobin solution;
b. adding a filling agent Na into the hemoglobin solution to be purified obtained in the step a2SO4Obtaining a hemoglobin solution sample to be purified;
c. before loading, use balance liquid 0.1-0.3M Na2SO4And c, 25-50mM Tris-HCl equilibrium affinity chromatography medium, then loading the hemoglobin solution sample obtained in the step b to obtain flow-through liquid, namely purified hemoglobin.
The method for producing carbonic anhydrase-free hemoglobin, wherein in the step b, the filler Na is added2SO4In an amount of 1.4-4.3g Na per 100ml of the hemoglobin solution to be purified2SO4。
The method for producing carbonic anhydrase-free hemoglobin, wherein in the step b, the filler Na is added2SO4In an amount of 2.85g Na per 100ml of the hemoglobin solution to be purified2SO4。
The preparation method of the carbonic anhydrase-free hemoglobin, wherein the medium of affinity chromatography is CM Sephadex-Sulfanilamid.
In the above method for preparing carbonic anhydrase hemoglobin, in step c, a balance solution of 0.2M Na is used before loading2SO425mM Tris-HCl equilibrationMedia for affinity chromatography.
The invention also provides a preparation method of carbonic anhydrase, which comprises the following steps:
a. fresh anticoagulated bovine blood, washing the blood with 0.9% NaCl, dissolving red blood cells with water, centrifuging to remove cell debris and undissolved cells, and obtaining a supernatant to-be-purified hemoglobin solution;
b. adding a filling agent Na into the hemoglobin solution to be purified obtained in the step a2SO4Obtaining a hemoglobin solution sample to be purified;
c. before loading, use balance liquid 0.1-0.3M Na2SO425-50mM Tris-HCl equilibrium affinity chromatography medium, then loading the hemoglobin solution sample obtained in the step b to obtain flow-through liquid, namely purified hemoglobin;
d. subjecting the first eluate to affinity chromatography to obtain 20-40mM Na2SO4Eluting non-specifically adsorbed impurities by using 5-8mM Tris-HCl;
e. eluting with affinity chromatography second eluent comprising oxidized ionic strength agent and CH3COONa, the concentration of ionic strength agent in the second eluent is 0.4-0.8M, CH3The concentration of COONa is 0.2-0.3M, and the carbonic anhydrase specifically adsorbed is eluted to obtain purified carbonic anhydrase.
In the above method for producing carbonic anhydrase, in the step e, the oxidation type ionic strength agent is NaClO3、MgO2And NaClO4。
In the above method for producing carbonic anhydrase, in the step e, the oxidation type ionic strength agent is NaClO4。
In the above method for producing carbonic anhydrase, in step e, the second eluent is 0.6M NaClO4/0.2M CH3COONa。
In the above method for producing carbonic anhydrase, in step d, the first eluent is 30mM Na2SO4/5mM Tris-HCl。
The preparation method of the carbonic anhydrase-free hemoglobin has the following beneficial effects:
1. the method for preparing the carbonic anhydrase-free hemoglobin is characterized in that the aim of thorough purification is achieved by optimizing the interphase contact on the basis of the separation of the specific affinity chromatography, and a large number of experiments show that a filler Na for optimizing the interphase contact is added into a hemoglobin solution to be purified2SO4The adsorption of carbonic anhydrase can be obviously improved, the non-specific adsorption of hemoglobin is also reduced, high-purity hemoglobin can be obtained, the purity can reach 99.99%, and simultaneously, high-purity carbonic anhydrase is also obtained, the purity can reach more than 99.8%, and double benefits are achieved;
2. the preparation method of the carbonic anhydrase-free hemoglobin is characterized in that an ionic strength agent NaClO is added during elution4The carbonic anhydrase can be more thoroughly eluted, the yield of the carbonic anhydrase can be obviously improved, the yield reaches 95 percent, simultaneously, the good renaturation of a medium is also achieved, and the renaturation operation time of the filler is shortened;
3. the preparation method of the carbonic anhydrase-free hemoglobin is a simple and enlargeable new method for removing carbonic anhydrase in hemoglobin to be purified, and the medium renaturation is quick and simple, so that the reuse of the hemoglobin is facilitated, the production cost is reduced, no complex equipment is required to be invested, and the popularization and the application are facilitated.
Drawings
FIG. 1 is a 540nm spectrum of affinity chromatography of hemoglobin of example 1 of the present invention;
FIG. 2 is an SDS-PAGE pattern of an affinity chromatography harvest of hemoglobin according to example 1 of the present invention;
FIG. 3 is a Western blot chart of the hemoglobin-derived affinity chromatography of example 1 of the present invention.
Detailed Description
The present invention is described in detail below with reference to the drawings and examples.
Example 1
1. Preparation of high purity hemoglobin
Taking fresh anticoagulated bovine blood, washing the blood with 0.9% NaClThree times, the red blood cells were lysed with cold water. Centrifuging for 30 minutes at 4 ℃ to remove cell debris and undissolved cells, wherein the supernatant is the hemoglobin solution to be purified; adding filler Na to the hemoglobin solution to be purified2SO42.85g of filler Na is added into 100ml of hemoglobin solution to be purified2SO4Obtaining a hemoglobin sample to be purified; before loading, use equilibrium solution 0.2M Na2SO4And/25 mM Tris-HCl balance affinity chromatography medium, wherein the medium used in the affinity chromatography is CM Sephadex-Sulfanilamide, and then the prepared hemoglobin sample to be purified is loaded until the sample is flush with the medium layer, so as to obtain flow-through liquid, namely the purified hemoglobin.
2. Preparation of high purity carbonic anhydrase
First eluate of affinity chromatography 30mM Na2SO4Washing away non-specifically adsorbed impurity substances by 5mM Tris-HCl; using a second eluent of 0.6M NaClO4/0.2M CH3COONa is continuously eluted, and the adsorbed carbonic anhydrase is washed away, so that the purified carbonic anhydrase is obtained, and the medium is renatured; using equilibrium solution 0.2M Na2SO4After 25mM Tris-HCl equilibrium chromatography column, the next purification cycle can be carried out.
In the embodiment, each peak protein is monitored and collected by an ultraviolet detector, and the specific spectrum result is shown in figure 1; the concentration of hemoglobin is measured by a blood gas analyzer, and the yield of hemoglobin calculated by the concentration of hemoglobin is 95.2 percent; the protein content and the protein content of each collected peak were determined by reducing SDS-PAGE method, see FIG. 2, and the results show: the purity of the collected hemoglobin can reach 99.99 percent, a protein band with about 30kD in the hemoglobin solution before purification disappears after purification, a single protein band with about 30kD appears in the second eluent, the molecular weight of the band is consistent with that of carbonic anhydrase, the band is preliminarily judged to be carbonic anhydrase, the yield is 95.6 percent, and the purity is 99.91 percent; products of the approximately 30kD band were further qualitatively detected by Western blot method, see fig. 3, and the results show: the pre-purification band of about 30kD was carbonic anhydrase, and the band of about 30kD that appeared in the second eluate was also carbonic anhydrase. The detection result shows that: the carbonic anhydrase and the hemoglobin are thoroughly separated after affinity chromatography, so that the high-purity carbonic anhydrase is obtained while the high-purity hemoglobin is obtained, and the effect is very ideal.
Example 2
1. Preparation of high purity hemoglobin
Fresh anticoagulated bovine blood was taken, the blood was washed three times with 0.9% NaCl, and erythrocytes were dissolved with cold water. Centrifuging at 18000g for 30 minutes at 4 ℃ to remove cell debris and undissolved cells, wherein the supernatant is the hemoglobin solution to be purified; adding filler Na to the hemoglobin solution to be purified2SO4Adding 1.40g of filler Na into 100ml of hemoglobin solution to be purified2SO4Obtaining a hemoglobin sample to be purified; before loading, use equilibrium solution 0.1M Na2SO4And/25 mM Tris-HCl balance affinity chromatography medium, wherein the medium used in the affinity chromatography is CM Sephadex-Sulfanilamide, and then the prepared hemoglobin sample to be purified is loaded until the sample is flush with the medium layer, so as to obtain flow-through liquid, namely the purified hemoglobin.
2. Preparation of high purity carbonic anhydrase
Affinity chromatography first eluate 20mM Na2SO4Washing away non-specifically adsorbed impurity substances by 5mM Tris-HCl; using a second eluent of 0.4M NaClO4/0.2M CH3COONa is continuously eluted, and the adsorbed carbonic anhydrase is washed away, so that the purified carbonic anhydrase is obtained, and the medium is renatured; using equilibrium solution 0.1M Na2SO4After 25mM Tris-HCl equilibrium chromatography column, the next purification cycle can be carried out.
The detection is carried out by adopting the same method as the example 1, the yield of the hemoglobin is 92.3 percent, and the purity of the hemoglobin is 99.92 percent; the purity of carbonic anhydrase was 99.87%, and the yield was 94.3%.
Example 3
1. Preparation of high purity hemoglobin
Fresh anticoagulated bovine blood was taken, the blood was washed three times with 0.9% NaCl, and erythrocytes were dissolved with cold water. Centrifuging at 18000g for 30 minutes at 4 ℃,removing cell debris and undissolved cells, wherein the supernatant is the hemoglobin solution to be purified; adding filler Na to the hemoglobin solution to be purified2SO4Adding 4.30g of filler Na into 100ml of hemoglobin solution to be purified2SO4Obtaining a hemoglobin sample to be purified; before loading, use equilibrium solution 0.3M Na2SO4And (2) a 50mM Tris-HCl balance affinity chromatography medium, wherein the medium used in the affinity chromatography is CM Sephadex-Sulfanilamide, and then the prepared hemoglobin sample to be purified is loaded until the sample is flush with the medium layer, so as to obtain a flow-through liquid, namely the purified hemoglobin.
2. Preparation of high purity carbonic anhydrase
First eluate of affinity chromatography 40mM Na2SO4Washing away non-specifically adsorbed impurity substances by 8mM Tris-HCl; with a second eluent of 0.8M NaClO4/0.3M CH3COONa is continuously eluted, and the adsorbed carbonic anhydrase is washed away, so that the purified carbonic anhydrase is obtained, and the medium is renatured; using equilibrium solution 0.3M Na2SO4The column was equilibrated with 50mM Tris-HCl and then subjected to the next purification cycle.
The detection is carried out by adopting the same method as the example 1, the yield of the hemoglobin is 94.7 percent, and the purity of the hemoglobin is 99.89 percent; the purity of carbonic anhydrase was 99.85%, and the yield was 95.2%.
Example 4
The same procedure as in example 1 was repeated except that in the preparation of high purity carbonic anhydrase, oxidized ionic strength agent (NaClO) was used as the oxidized ionic strength agent in the second eluent, which was used in this example3The purity and yield of the produced carbonic anhydrase are shown in Table 1.
Example 5
The same procedure as in example 1 was conducted to produce high purity hemoglobin and high purity carbonic anhydrase, except that the oxidized ionic strength agent used in the second eluent was MgO in the production of high purity carbonic anhydrase2The purity and yield of the produced carbonic anhydrase are shown in Table 1.
TABLE 1
As can be seen from Table 1, the effect of different ionic strength agents on the purity of carbonic anhydrase is small, but from the viewpoint of the effect on the yield, NaClO was selected4As an ionic strength agent, the yield of carbonic anhydrase can be obviously improved.
Comparative example 1
The same procedure as in example 1 for preparing high purity hemoglobin and high purity carbonic anhydrase was conducted except that CaSO was used as the filler in the hemoglobin solution to be purified and the equilibrium solution4The yield and purity of hemoglobin are shown in Table 2.
Comparative example 2
The same procedure as in example 1 for preparing high purity hemoglobin and high purity carbonic anhydrase was conducted except that CaHPO was used as the filler in the hemoglobin solution to be purified and the equilibrium solution4The yield and purity of hemoglobin are shown in Table 2.
Comparative example 3
The same procedure as in example 1 for preparing high purity hemoglobin and high purity carbonic anhydrase, except that no bulking agent was added to the hemoglobin solution to be purified, the yield and purity of hemoglobin are shown in Table 2.
Comparative example 4
The same procedure as in example 1 for preparing high purity hemoglobin and high purity carbonic anhydrase, except that no filler Na was added to the hemoglobin solution to be purified2SO4And no filler Na is added into the equilibrium liquid2SO4The yield and purity of hemoglobin are shown in Table 2.
TABLE 2
As can be seen from Table 2, CaSO was used in comparative examples 1 and 24And CaHPO4As the filler, Na was used in example 12SO4As bulking agents, different bulking agents were used to obtain hemoglobin yields that were comparable, but Na was used2SO4As a filling agent, the purity of the hemoglobin is remarkably improved, and the hemoglobin with the purity of 99.99 percent can be obtained. In comparative example 3, in which no filler was added to the hemoglobin sample to be purified, the yield was not much different from that of example 1, but filler Na was added2SO4The purity of the hemoglobin is obviously improved. In comparative example 4, in which no filler was added to the hemoglobin sample to be purified and no filler was added to the equilibrium solution, the yield was slightly decreased as compared with example 1, but filler Na was added2SO4Then, the purity of the hemoglobin obtained in example 1 is significantly improved and can reach 99.99%. Comparative example 3 compared with comparative example 4, the addition of filler to the equilibration solution slightly improved the yield and purity of hemoglobin, while example 1 further added filler Na to the hemoglobin sample to be purified2SO4The purity of the hemoglobin is obviously improved. Therefore, through the above comparative experiments, Na is selected according to the invention2SO4The filler can effectively improve the purity of the hemoglobin as a filler for loading a hemoglobin sample to be purified and a balanced solution.
Claims (8)
1. A preparation method of carbonic anhydrase-free hemoglobin comprises the following steps:
a. fresh anticoagulated bovine blood, washing the blood with 0.9% NaCl, dissolving red blood cells with water, centrifuging to remove cell debris and undissolved cells, and obtaining a supernatant to-be-purified hemoglobin solution;
b. adding a filling agent Na into the hemoglobin solution to be purified obtained in the step a2SO4Obtaining a hemoglobin solution sample to be purified, and filling agent Na2SO4In an amount of 2.85g Na per 100ml of the hemoglobin solution to be purified2SO4；
c. Before loading, use equilibrium solution 0.2M Na2SO4And c, 25-50mM Tris-HCl equilibrium affinity chromatography medium, then loading the hemoglobin solution sample obtained in the step b to obtain flow-through liquid, namely purified hemoglobin.
2. The method of claim 1, wherein the affinity chromatography medium is CM Sephadex-Sulfanilamid.
3. The method for producing carbonic anhydrase-free hemoglobin of claim 1, wherein in step c, the equilibrium solution of 0.2M Na is used before loading2SO425mM Tris-HCl equilibration affinity chromatography medium.
4. A method for preparing carbonic anhydrase, comprising the steps of:
a. fresh anticoagulated bovine blood, washing the blood with 0.9% NaCl, dissolving red blood cells with water, centrifuging to remove cell debris and undissolved cells, and obtaining a supernatant to-be-purified hemoglobin solution;
b. adding a filling agent Na into the hemoglobin solution to be purified obtained in the step a2SO4Obtaining a hemoglobin solution sample to be purified, and filling agent Na2SO4In an amount of 2.85g Na per 100ml of the hemoglobin solution to be purified2SO4；
c. Before loading, use equilibrium solution 0.2M Na2SO425-50mM Tris-HCl equilibrium affinity chromatography medium, then loading the hemoglobin solution sample obtained in the step b to obtain a flow-through liquid, namely the purified hemoglobinWhite;
d. subjecting the first eluate to affinity chromatography to obtain 20-40mM Na2SO4Eluting non-specifically adsorbed impurities by using 5-8mM Tris-HCl;
e. eluting with affinity chromatography second eluent comprising oxidized ionic strength agent and CH3COONa, the concentration of ionic strength agent in the second eluent is 0.4-0.8M, CH3The concentration of COONa is 0.2-0.3M, and the carbonic anhydrase specifically adsorbed is eluted to obtain purified carbonic anhydrase.
5. The method for producing carbonic anhydrase according to claim 4, wherein, in the step e, the oxidative ionic strength agent is NaClO3And NaClO4。
6. The method for producing carbonic anhydrase according to claim 5, wherein, in the step e, the oxidative ionic strength agent is NaClO4。
7. The carbonic anhydrase production method according to claim 6, wherein, in the step e, the second eluent is 0.6M NaClO4/0.2M CH3COONa。
8. The process for producing carbonic anhydrase according to claim 4, wherein in step d, the first eluent is 30mM Na2SO4/5mM Tris-HCl。
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996029346A1 (en) * | 1995-03-23 | 1996-09-26 | Biopure Corporation | Stable polymerized hemoglobin blood-substitute |
| CN1308636A (en) * | 1998-07-10 | 2001-08-15 | 柏尔纯公司 | Method for purifying hemoglobin |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996029346A1 (en) * | 1995-03-23 | 1996-09-26 | Biopure Corporation | Stable polymerized hemoglobin blood-substitute |
| CN1308636A (en) * | 1998-07-10 | 2001-08-15 | 柏尔纯公司 | Method for purifying hemoglobin |
Non-Patent Citations (2)
| Title |
|---|
| A DIFFERENT STRUCTURAL FEATURE FOR CARBONIC ANHYDRASES IN HUMAN ERYTHROCYTES;Nazan DEMIR等;《PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY》;19971231;第27卷(第4期);第280页倒数第1、2段，第281页第1段 * |
| In Vitro and in Vivo Effects of Dantrolene on Carbonic Anhydrase Enzyme Activities;Ilhami Gulcin等;《Biol. Pharm. Bull.》;20040531;第27卷(第5期);613-616 * |
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