Method for isolating oxygen-carrying hemerythrin from a sipunculid
By collecting coelomic fluid from Sipuncula foetida, centrifuging, lysing, and multi-stage tangential flow ultrafiltration, combined with freeze-drying, high-purity oxygen-carrying active hemopurin was successfully isolated from Sipuncula foetida and maintained. This solved the problem of separation difficulties in existing technologies and enabled the acquisition of key raw materials for red blood cell substitutes.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUANGXI MEDICAL UNIVERSITY
- Filing Date
- 2026-03-12
- Publication Date
- 2026-05-29
AI Technical Summary
Existing technologies cannot effectively isolate high-purity oxygen-carrying active hemopurin from Sipunculus nudus, which limits the application of Sipunculus nudus hemopurin in red blood cell substitutes.
The coelomic fluid of Sipuncula was collected, and crude hemopurin was obtained by centrifugation and lysis. The solution was then purified by multi-stage tangential flow ultrafiltration and subsequently freeze-dried to preserve the activity of the hemopurin.
It has achieved the separation and long-term activity maintenance of high-purity hemopurin, with a purity of over 95%, which is suitable for the research and development of red blood cell substitutes and promotes the intensive processing of Sipunculus nudus.
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Figure CN122103299A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of protein separation and purification technology, and in particular to a method for separating oxygen-carrying active hemoviolet protein from Sipuncula foetida. Background Technology
[0002] The research and development of red blood cell substitutes has been ongoing for more than 40 years, with hundreds of designs based on mammalian hemoglobin (Hb). However, due to the limitations of the basic structure of the Hb molecule, despite various modifications and alterations, the problems of molecular stability and vascular toxicity caused by decomposition products cannot be fundamentally solved, and the substitutes cannot effectively replace human red blood cells in carrying oxygen in the long term.
[0003] In recent years, some research centers have turned to finding more stable respiratory proteins from lower organisms, such as hemerythrin (Hr), which holds promise for fundamentally eliminating the limitations of hemoglobin-based erythrocyte substitutes. Sipuncula are marine invertebrates belonging to the phylum Sipuncula. Hemerythrin exists in coelomic fluid in a free state or is encapsulated by pigment cells. It consists of eight 13.5 kDa subunits, with a molecular weight of 108 kDa, approximately 1.6 times the size of hemoglobin. Its physicochemical properties are more stable than hemoglobin molecules, and it performs its physiological oxygen-carrying function without relying on the protection and support of erythrocytes, possessing unique advantages for development as a erythrocyte substitute. Recent studies have reported that hemerythrin-based multi-type erythrocyte substitutes (such as M101) have greater advantages than Hb-based oxygen carriers in clinical trials, animal experiments, and ex vivo organ protection, and related technologies are continuously developing.
[0004] However, the inability to obtain sipunculus hemopurin samples and the lack of reports on specific methods for isolating sipunculus hemopurin hinder the advancement of independent research and development in this field. There is an urgent need to establish a method for isolating hemopurin to obtain raw materials and promote research and development. Summary of the Invention
[0005] The technical problem to be solved by this invention is: how to obtain high-purity oxygen-carrying active hemopurin from Sipuncula worms.
[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: The present invention provides a method for separating oxygen-carrying active hemoviolet protein from Sipuncula foetida, which involves collecting pigment cells from the coelomic fluid of Sipuncula foetida, lysing them to prepare crude protein solution, obtaining high-purity hemoviolet protein solution through multi-stage tangential flow ultrafiltration, and then making it into lyophilized powder to maintain long-term activity.
[0007] The separation method of the present invention includes the following steps:
[0008] S1. Preparation of crude blood purple protein solution: Collect coelomic fluid of Sipunculus nudus, centrifuge to separate coelomic fluid cells, lyse and remove pigment cell fragments and other cells to obtain crude blood purple protein solution;
[0009] S2. Pour the crude hemopurin solution into the receiving bottle of the tangential flow ultrafiltration machine, dilute it with 5 to 10 times the volume of pure water, and perform tangential flow ultrafiltration sequentially using filter membranes with different pore sizes to retain 500 kDa-30 kDa molecules to obtain the purified hemopurin solution.
[0010] S3. Preparation of freeze-dried powder: The purified hemoviolet protein solution is frozen and then subjected to vacuum low-temperature freeze-drying to obtain hemoviolet protein freeze-dried powder that maintains oxygen-carrying activity for a long time.
[0011] The specific procedure for collecting the coelomic fluid of Sipunculus nudus is as follows: Select fresh Sipunculus nudus, wash them, and cut them open at the end along with the dorsal artery to collect the outflowing coelomic fluid.
[0012] The lysis buffer consisted of Tris-HCl, NaCl, and H₂O, with Tris-HCl concentrations of 0–20 mM, NaCl concentrations of 0–50 mM, a pH of 7.4–8.5, and a lysis buffer-to-cell volume ratio of 1:1–10:1. The lysis conditions were: 4 °C, lysis time of 5–20 min.
[0013] The lysis buffer provided by this invention selectively lyses pigment cells, rather than physically disrupting all coelomic cells, to avoid the rupture of other contaminating cells and the release of various proteins, while maintaining the activity of hemoviolet proteins. The lysis buffer is weakly alkaline, has low osmotic pressure, and appropriate ionic strength, and is prepared from an aqueous solution of Tris-HCl and NaCl. After repeated testing, the optimal composition is: 10 mM Tris-HCl, 25 mM NaCl, pH 8.0, a cell lysis buffer to cell volume ratio of 10:1, and an optimal lysis time of 20 min at 4°C.
[0014] The tangential flow ultrafiltration step S2 is as follows: First, use an ultrafiltration membrane with a molecular weight cutoff of 500 kDa to remove macromolecular substances, and then use an ultrafiltration membrane with a molecular weight cutoff of 30 kDa to filter out small molecule mixed proteins and nucleic acids, resulting in purified hemopurin solution. Continue to use an ultrafiltration membrane with a molecular weight cutoff of 30 kDa for repeated filtration to obtain concentrated purified hemopurin solution.
[0015] Before performing step S2, a pre-filtration step is also provided. The pre-filtration process is as follows: ultrafiltration is performed sequentially using filter membranes with pore sizes of 0.65µm and 0.22µm, and the permeate is collected for later use.
[0016] The freezing conditions are: -20℃.
[0017] The vacuum cryogenic freeze-drying conditions are: vacuum degree 0.37 mbar, temperature -30℃, until dry powder is obtained.
[0018] This invention obtains pigment cells from the coelomic fluid of Sipuncula spp. Pigment cells are present in the coelomic fluid and blood vessels of Sipuncula spp., accounting for more than 70% of the total cells in the coelomic fluid. Viable pigment cells appear as round, transparent cells under a microscope, possessing a single nucleus and homogeneous cytoplasm. After HE staining, the nucleus is round or nearly round, deep purplish-red, and the cytoplasm is uniformly blue. The concentration of hemopurin is several tens of times higher than in the extracellular fluid. This invention employs a method of first filtering the coelomic fluid sequentially through 100μm and 50μm filters to remove tissue debris and foreign matter. Then, coelomic cells are obtained by centrifugation, followed by washing with buffer and centrifugation to obtain coelomic fluid cells, of which 70% are pigment cells.
[0019] This invention employs tangential flow ultrafiltration to separate hemopurin molecules from crude hemopurin solution. Based on the fact that the types of proteins contained in pigment cells are far fewer than those contained in the entirety of proteins in the crushed Sipuncula organism, this invention provides a simple and efficient tangential flow ultrafiltration technology. Through repeated testing, the concentration difference of hemopurin across the membrane after passing the filtrate through a 500 kDa pore size membrane and then through a 30 kDa pore size membrane resulted in a difference of over 1000 times, achieving optimal separation. To reduce membrane clogging in the tangential flow ultrafiltration unit, a supplementary technology using 0.65 µm and 0.22 µm pore size membranes for pre-filtration is also provided.
[0020] The step of obtaining the coelomic fluid of Sipunculus nudus in this invention requires cutting open the coelomic cavity of the Sipunculus nudus, preferably 1 cm from the tail end (cutting open along with the dorsal artery), which can ensure that the edible value of Sipunculus nudus as a high-end seafood food is not affected.
[0021] The present invention provides a method for separating oxygen-carrying active hemopurin from Sipuncula foetida, which employs vacuum freeze-drying to maintain the activity of hemopurin for a long period of time.
[0022] The beneficial effects obtained by this invention are as follows: This invention obtains a large amount of high-purity oxygen-carrying active hemopurin from Sipunculus nudus without affecting the edible value of Sipunculus nudus, using a simple method. After identification by SDS-PAGE electrophoresis, high-performance liquid chromatography, ultraviolet spectroscopy and other methods, the obtained hemopurin has good oxygen-carrying structure and function, with a purity of over 95%. It can be used as a key raw material for the research and development of hemopurin-based oxygen carriers (red blood cell substitutes), and will also help promote the upgrading of Sipunculus nudus farming to deep processing in the future. Attached Figure Description
[0023] Figure 1 The hemopurin SDS-PAGE electrophoresis identification image obtained by this invention clearly shows the characteristic bands (10-15 kDa subunits).
[0024] Figure 2 The high-performance liquid chromatogram of hemopurin obtained by this invention has a peak area (i.e. purity) of 95.85%.
[0025] Figure 3 The ultraviolet spectrum of hemoviolet protein obtained by this invention shows the presence of diferric high-valence states (Fe) at 329 nm and 504 nm, respectively. 3+ –Fe 3+ Characteristic peaks and Fe(Ⅲ)–OOH⁻ oxygen species characteristic absorption peaks, indicating that the oxygen-carrying group structure is intact.
[0026] Figure 4 The curve of the ratio of deoxygenated hemoviolet protein content to deoxygenated hemoviolet protein aqueous solution deoxygenator obtained by the present invention shows that the sample can release (bind) oxygen molecules under different oxygen partial pressures. Detailed Implementation
[0027] Example 1
[0028] This embodiment describes a method for isolating hemopurin from Sipuncula foetida, the specific process of which is as follows:
[0029] 1. Preparation of crude hematuria protein solution:
[0030] (1) Wash the live star worms with clean water to remove dirt, mucus, etc.
[0031] (2) Soak the cleaned Sipunculus nudus in pure water at 20°C for 1 hour, and then wash it again after the waste in the body cavity is discharged.
[0032] (3) Cut the Sipuntia 1 cm from the tail end with scissors, collect the Sipuntia coelomic fluid in a 500 mL beaker, filter out foreign objects in the coelomic organs by passing the fluid through 100 μm and 50 μm filters in sequence, and collect the filtrate in a new beaker to obtain the Sipuntia coelomic fluid.
[0033] (4) Use a Pasteur pipette to dispense the liquid in the beaker into 50mL centrifuge tubes in 30mL portions, centrifuge for 5min at 3000rpm, discard the supernatant, and the precipitate is coelomic fluid cells.
[0034] (5) Add PBS buffer (pH 7.4), mix well by pipetting, centrifuge at 3000 rpm for 5 min at 4°C, and discard the supernatant. This step is to wash away cell adhesions, and can be repeated 1-2 times if necessary.
[0035] (6) Prepare the lysis buffer as follows: Tris-HCl concentration 10mM, NaCl concentration 25mM, pH value 8.0, and store at 4℃ for later use.
[0036] (7) Add the lysis buffer from step (6) to the coelomic cell fluid from step (5), with a cell lysis buffer to cell volume ratio of 5:1. Mix thoroughly by pipetting and place in a 4°C refrigerator for lysis for 20 min.
[0037] (8) Centrifuge for 5 minutes at 3000 rpm; take the supernatant, which is the crude hemopurine protein solution, seal it, and store it in a 4℃ refrigerator for later use.
[0038] 2. Tangential flow ultrafiltration (TFF)
[0039] (9) First rinse the tangential flow ultrafiltration system with 500mL of pure water, then pour the crude hemopurine protein solution into the bottle and dilute it with 5 times the volume of pure water;
[0040] (10) Select a 0.65µm pore size TFF filter membrane, pour the diluted crude hemopurin solution into a tangential flow ultrafiltration machine for ultrafiltration, adjust the pressure valve to 0.069-0.138 MPa, close the valve after one ultrafiltration, discard the retentate in the system, collect the outflow permeate for later use. The retentate is the large particulate impurities that need to be removed, and the permeate contains hemopurin.
[0041] (11) Select a 0.22µm pore size TFF filter membrane, pour the permeate into the tangential flow ultrafiltration machine for ultrafiltration again, adjust the pressure valve to 0.069-0.138 MPa, close the valve after one ultrafiltration, discard the retentate in the system, collect the outflowing permeate for later use. The retentate is the bacteria and other microorganisms that need to be removed, and the permeate contains hemopurin.
[0042] (12) Pour the filtrate obtained in step (11) into a tangential flow ultrafiltration machine, use a 500 kDa filter membrane, pressure 0.069-0.138 MPa, and after one ultrafiltration, close the valve, discard the retentate, and collect the outflowing permeate.
[0043] (13) Pour the permeate obtained in step (12) into a tangential flow ultrafiltration machine, replace it with a 30kDa filter membrane, and set the pressure to 0.069-0.138 MPa. Collect the retentate.
[0044] (14) Pour the filtrate obtained in step (13) into a tangential flow ultrafiltration machine, continue ultrafiltration for 3 to 5 times to concentrate, discard the outflowing permeate, and collect the retentate in the system as purified hemopurin concentrate.
[0045] 3. Preparation of lyophilized powder
[0046] (15) Freeze the high concentration and high purity hemopurine obtained in step (14) at -20°C.
[0047] (16) Use a vacuum low-temperature freeze dryer to freeze dry the blood purple protein until a dry powder is obtained. The vacuum low-temperature freeze dryer is set to a vacuum degree of 0.37 mbar and a temperature of -30℃. Collect the powder and store it in a brown glass bottle at -80℃ in a dark and sealed container.
[0048] The hemoviolet lyophilized powder sample obtained in this embodiment showed characteristic protein subunit bands (10-15 kDa) when detected by SDS-PAGE electrophoresis. Figure 1 The visible peak area (i.e., purity) detected by high-performance liquid chromatography was 95.85%. Figure 2 Ultraviolet spectroscopy revealed the presence of a double iron high-valence state (Fe) at 329 nm. 3+ –Fe 3+ Characteristic absorption peaks of Fe(Ⅲ)–OOH⁻ oxides exist at 504 nm, such as... Figure 3 The chemical oxygen demand method was used to analyze the sample aqueous solution, and it was found that the content of deoxyhemoglobin increased with reaction time, as shown in the results. Figure 4 As shown.
[0049] Example 2
[0050] This embodiment describes an improved method for separating pigment cells from hemopurin in the process of isolating hemopurin from Sipunculus nudus.
[0051] Steps (1) to (5) are the same as in Example 1.
[0052] (6) Prepare pigment cell separation solution according to the volume ratio of Percoll cell separation solution to 1.5 M NaCl solution of 3:7, and take an appropriate amount into a centrifuge tube;
[0053] (7) Slowly add the resuspension of body cavity cells along the wall to the centrifuge tube containing pigment cell separation solution. The cell suspension will be visible above the cell separation solution with clear stratification in the middle. Place the tube in a centrifuge and centrifuge at 3000 rpm for 20 minutes to remove cell debris.
[0054] (8) The subsequent steps are the same as those in Example 1.
[0055] The purpose of adding this step in this embodiment is to separate the pigment cells from other cells, which theoretically can improve the purity of the crude hemoviolet protein solution. However, the subsequent method of this invention uses a selective lysis buffer, so other cells hardly rupture. The purity of the final hemoviolet protein sample is 96.21%, which is less than 1% different from the purity of the sample in Example 1. This indicates that this step is not a necessary step in this invention.
[0056] Example 3
[0057] This embodiment describes the method for preparing the lysis buffer and obtaining the optimal formulation in the method for isolating hemopurin from Sipunculus nudus.
[0058] The role of the lysis buffer is to selectively disrupt the clear cell membrane to release hemoviolet protein while protecting its functional activity. The actual effect depends on factors such as reaction temperature, pH, ionic strength (Tris-HCl and NaCl concentrations), lysis buffer / cell sample volume ratio, and reaction time. 4°C is a generally accepted optimal temperature for isolating active proteins in the biomedical field. The following describes the steps for determining the optimal lysis buffer formulation at this temperature.
[0059] 1. Selection of factors and levels for orthogonal experiments. Five factors were included: pH value of lysis buffer (A), Tris-HCl concentration (B), NaCl concentration (C), volume ratio of lysis buffer to cell sample (D), and lysis time (E). Preliminary experiments determined the approximate suitable ranges for each factor as follows: pH value (6–9), Tris-HCl concentration (0–30 mM), NaCl concentration (0–33 mM), and volume ratio of lysis buffer to cell sample (1:1–10:1). Subsequently, three levels were set for each factor to screen for the optimal scheme, as shown in Table 1.
[0060] Table 1. Factor Level Table for Orthogonal Experiment
[0061]
[0062] 2. Evaluation Criteria: a) Cell Lysis Score (0-10 points): Determined by visually observing the volume and number of remaining red blood cells at the bottom of the test tube. 10 points indicates complete lysis, and 0 points indicates no lysis; b) Protein Activity Score (0-10 points): The absorbance of the lysate supernatant at 504 nm (characteristic peak of oxygenated state) and 329 nm (characteristic absorption peak of diferroic center) was measured using a UV-Vis spectrophotometer. The A504nm / A329nm ratio was calculated. Protein activity score = (A504 / A329 ratio of this experimental group ÷ maximum A504 / A329 ratio among all groups) × 10. A higher ratio indicates a higher proportion of oxyhemoglobin (Oxy-Hr) in the sample and better activity; a lower ratio indicates that the protein has been oxidized (becomes Met-Hr) or deoxygenated (becomes Deoxy-Hr), resulting in decreased activity. c) The overall score = 30% cell lysis score + 70% protein activity score, reflecting the principle of prioritizing the protection of hemoglobin protein activity while also considering process efficiency.
[0063] 3. Orthogonal Experiment Design and Results. This orthogonal experiment included 5 factors and 3 levels, with a total of 18 experiments designed. The experimental design and results are shown in Table 2, and the range analysis is shown in Table 3.
[0064] Table 2: L 18 (3 7 Orthogonal experimental design and results
[0065]
[0066] Table 3: Range Analysis of Factor Levels
[0067]
[0068] Comprehensive analysis leads to the conclusion that the order of influence on the extraction efficiency of Sipunculus hemopurin is as follows: volume ratio of lysis buffer to cell sample (D) > NaCl concentration (C) > lysis time (E) > pH value of lysis buffer (A) > Tris-HCl concentration (B). The optimal lysis conditions are: pH value of 8.0, Tris-HCl concentration of 10 mM, NaCl concentration of 25 mM, volume ratio of lysis buffer to cell of 10:1, and lysis time of 20 min.
Claims
1. A method for isolating oxygen-carrying active hemoviolet protein from Sipuncula foetida, characterized in that, Includes the following steps: S1. Preparation of crude blood violet protein solution: Collect coelomic fluid of Sipunculus nudus, centrifuge to separate coelomic fluid cells, lyse pigment and remove other cells and pigment cell fragments to obtain crude blood violet protein solution; S2. Tangential flow ultrafiltration was performed sequentially using filter membranes with different pore sizes to retain molecules ranging from 500kDa to 30kDa, resulting in a purified hemopurin solution. S3. Preparation of freeze-dried powder: The purified hemoviolet protein solution is frozen and then subjected to vacuum low-temperature freeze-drying to obtain hemoviolet protein freeze-dried powder that maintains oxygen-carrying activity for a long time.
2. The method for isolating oxygen-carrying active hemoviolet protein from Sipuncula according to claim 1, characterized in that, The specific procedure for collecting the coelomic fluid of Sipunculus nudus is as follows: Select fresh Sipunculus nudus, wash them, and cut them open at the end along with the dorsal artery to collect the outflowing coelomic fluid.
3. The method for isolating oxygen-carrying active hemoviolet protein from Sipuncula according to claim 1, characterized in that, The lysis buffer consisted of Tris-HCl, NaCl, and H2O, with Tris-HCl concentration of 0–20 mM, NaCl concentration of 0–50 mM, and the remainder being water; the pH value was 7.4–8.5, and the volume ratio of lysis buffer to cells was 1:1–10:
1.
4. The method for isolating oxygen-carrying active hemoviolet protein from Sipuncula according to claim 1, characterized in that, The pyrolysis conditions were: 4℃ environment, 5-20 min.
5. The method for isolating oxygen-carrying active hemoviolet protein from Sipuncula according to claim 1, characterized in that, The lysis buffer composition was: Tris-HCl concentration 10mM, NaCl concentration 25mM, pH value 8.0, and cell lysis buffer to cell volume ratio 10:1; the lysis conditions were: 4℃ for 20 min.
6. The method for isolating oxygen-carrying active hemoviolet protein from Sipuncula according to claim 1, characterized in that, The tangential flow ultrafiltration step S2 is as follows: First, use an ultrafiltration membrane with a molecular weight cutoff of 500 kDa to remove macromolecular substances, and then use an ultrafiltration membrane with a molecular weight cutoff of 30 kDa to filter out small molecule mixed proteins and nucleic acids, resulting in purified hemopurin solution. Continue to use an ultrafiltration membrane with a molecular weight cutoff of 30 kDa for repeated filtration to obtain concentrated purified hemopurin solution.
7. The method for isolating oxygen-carrying active hemoviolet protein from Sipuncula according to claim 1, characterized in that, A coarse filtration step is also included. The pre-filtration step is set before step S2. The pre-filtration process is as follows: ultrafiltration is performed sequentially using filter membranes with pore sizes of 0.65µm and 0.22µm, and the permeate is collected for later use.
8. The method for isolating oxygen-carrying active hemoviolet protein from Sipuncula according to claim 1, characterized in that, The freezing conditions are: -20℃.
9. The method for isolating oxygen-carrying active hemoviolet protein from Sipuncula according to claim 1, characterized in that, The vacuum cryogenic freeze-drying conditions are: vacuum degree 0.37 mbar, temperature -30℃, until dry powder is obtained.
10. The method for isolating oxygen-carrying active hemoviolet protein from Sipuncula according to claim 1, characterized in that, Step S1, which involves removing pigment cell debris and other cells, specifically involves filtering the body cavity fluid sequentially using 100μm and 50μm filters.