Study on the application of aqueous two-phase extraction to purification of some proteins

Li Renqiang¹, Xu Siguang¹, Wu Zhongping², Fang Ling¹
(1. Department of Biotechnology, Jinan University, Guangzhou, 510632, China;  2. Department of Chemistry, Jinan University, Guangzhou, 510632, China)

Received on Sep. 25, 2005; Supported by the Grant-in-Aid for scientific research to Li R from Jinan University, China (640073).

Abstract Based on the advantage of aqueous two-phase extraction (ATPE) in bioseparation and preliminary experiments that all heme-containing proteins may be concentrated into the salt phase in aqueous two-phase system, ATPE was applied to practice for the preparation of these heme-containing protein Lumbricus terrestris hemoglobin (LtHb), cytochrome c (Cyt c) and peroxidase (POD) from biomaterials. Results demonstrated that to apply ATPE to extracting process for preparation of LtHb from earthworm, for preparation of Cyt c from pork heart and for preparation of POD enzymatic product from radish were successful with the extracting process being short, operating easily with lower cost. SDS-PAGE, purification effect analysis and absorption spectral scan explained that very pure LtHb may be obtained, the extracting process in initial purification of Cyt c was short and effective, and POD product obtained was almost as good in quality as that by conventional salting out technique when ATPE were used.
Key words Aqueous two-phase extraction; Purification; Lumbricus terrestris hemoglobin; Cytochrome c; Peroxidase

Bioseparation is an important technique in biochemistry, and is also a challenge due to that the bioorganic compounds existed in a cell lysate is a complicated mixture, including various biomolecules such as nucleic acid, protein, carbohydrate, lipid, vitamin. Chromatographic methods, electrophoresis, centrifugation, precipitation are often performed in bioseparation. However, in the case of unknowing the character of separated compound, it is usually needed at least several steps to obtain the aim molecules. Bioseparation seems to be complicated.
    Aqueous two-phase extraction (ATPE) is a system in that two immiscible phases are formed when polymers such as poly(ethylene glycol)(PEG) are mixed with dextran, other polymers, or salts in particular concentration. The compound in ATPE system has an equilibrium distribution depending on its own surface properties such as charge and hydrophobicity, and on the physicochemical properties of the two phase, which can be manipulated by adjusting the factors such as the polymer molecular mass and concentration, type of phase forming salt, salt concentration, ionic strength, and pH. Recently ATPE has been widely used for protein separation [1,2] for it has the potential to produce a concentrated and purified product in one step when compared to the number of steps involved in conventional bioseparation process. ATPE is a simple separation process, and it offers gentle nontoxic environments for biomolecules. It is cost effective and can be scaled up easily, so it is especially suitable for the practical production.
    Based on the advantage of ATPE, ATPE composed of PEG and ammonium sulfate that was often used in practice was applied to the purification of useful biochemical product or medical protein in this study. According to the preliminary experiments that the heme-containing proteins such as cytochrome C (Cyt c), hemoglobin (Hb), peroxidase (POD) could be distributed completely in the phase of ammonium sulfate salt in PEG―(NH₄)₂SO₄ system, ATPE was used in the process of preparing these biochemical products from some biomaterials.

1 MATERIALS AND METHODS
1.1 Materials
Earthworm (called Japan red earthworm) with 6-8 cm length, pork heart and radish (Rhaphanus sativus) were purchased from market. Horse heart Cyt c was from Sigma. All of the reagents used in the experiments were reagent grade and used as received. Solvent was distilled water. The instrument for absorption spectral scan was UV-VIS Spectrumlab 54 (Shanghai Lingguang Co., China) and for electrophoresis was applied by Beijing Liuyi Equipment Factory. Vacuum cold drying apparatus was Heto, High Technology of Scandinavia, Denmark.
1.2 Preparation of Lumbricus terrestris hemoglobin (LtHb) by ATPE
Washed earthworm body were bleeded in distill water using blender, the supernatant was obtained after centrifugation of the earthworm homagenate. In ATPE system with 12%(w/w) PEG6000 and 12%(w/w) ammonium sulfate, the hemoglobin in the supernatant was concentrated in salt phase. This process eliminated some proteins and enzymes dissolved in PEG phase, and concentrated the hemoglobin. After the centrifugation for removing the precipitate, the salt phase was dialyzed against 10mM/L phosphate buffer, pH5.1 overnight, of which the aim was to precipitate those hemoglobin due to their giant molecular size in near isoelectric point. This step would separate hemoglobin from other substances. The color substances were almost precipitated as predicted, and collected using centrifugation, dried by vacuum cold drying apparatus.
1.3 Application of ATPE to concentration and initial purification of Cyt c from pork heart
Pork heart with several hundreds grams were bleeded in weak acidic water for the extraction of Cyt c. The supernatant obtained after centrifugation was added with PEG6000 (12% w/w as final concentration) and (NH₄)₂SO₄ (12% w/w as final concentration) to make ATPE system, then collected the salt phase in which red substance were concentrated. After centrifugation, added 20% trichloroacetic acid (TCA) solution directly to the supernatant with 0.5% TCA as the final concentration to precipitate proteins, collected the color precipitate using centrifugation. Some water was added to the precipitate and the solution was dialyzed. The content of Cyt c was measured using O.D.₅₃₀ comparing to that of standard horse heart Cyt c.
1.4 Preparation of POD from radish by ATPE and by conventional salting out
Radish of about 300g was bleeded in distilled water and made the extraction for over 1 hr with agitation. Half of the supernatant obtained after centrifugation was used for ATPE and another half as the control using conventional salting out. PEG (12%, w/w) and (NH₄)₂SO₄ (12% w/w) was made in the supernatant to prepare ATPE system. The salt phase was dialyzed to remove (NH₄)₂SO₄ and the same volume of cold acetone was added to precipitate proteins. After centrifugation, cold acetone was again added to the supernatant according to 0.8∶1, collected the precipitate that would be POD sample after dialysis. As the control, the crude enzymatic supernatant was first salting out with 20% saturation of (NH₄)₂SO₄ to remove the precipitate, and again the second salt out with 80% saturation of (NH₄)₂SO₄ to collect the precipitate. After dialysis, the next steps to purify POD using cold acetone were as the same as that stated in ATPE system.
1.5 SDS-PAGE and visible absorption spectral scan
In SDS-PAGE that could determine the purity of protein, the purified protein or enzyme as the electrophoresis samples were first mixed with 1%SDS―1% mercaptoethanol solution in a boiled water-bath and then electrophoresis was performed in 3% concentrate―12% separation gel.
    The visible absorption spectra of LtHb and semifinished Cyt c obtained by ATPE were examined and compared to that of hemoglobin and Cyt c to convince whether the aim molecules were obtained.
1.6 Measurement of POD activity
Based on the mechanism that POD may catalyze 2-methoxyphenol to form color compound with the existence of hydrogen peroxide, POD activity could be examined and presented using O.D.₄₇₀. The concentration of POD (protein) could be calculated after reading O.D.₂₈₀ and O.D.₂₆₀ according to the equation:
Concentration of protein (mg/ml) = (1.45 O.D.₂₈₀-0.74 O.D.₂₆₀)×diluted fold
So POD activity could be presented as specific activity (O.D.₄₇₀/min_·protein weight).

2 RESULTS AND ANALYSIS
2.1 SDS-PAGE patterns and purification effect of ATPE               
LtHb is a giant molecule with the molecular mass 4-M [3-5], composed of more than 200 chains. Those subunits include four major O₂-binding chains, a, b, c, which form a disulfide-linked trimer, and monomer d. Additional structural chains, "linkers", are non-heme polypeptide L₁, L₂, and L₃. SDS-PAGE pattern of LtHb sample obtained by the process described above was shown in Figure 1. Molecular mass of three bands in the sample was near 22000, 17000 and 15000, respectively, after comparing to markers. Obviously based on the referent literature [4], the bottom band was d chain with 15960, and the band near it was the mixture of a, b and c subunit (they were 17525, 16254 and 17289, respectively). The upper band was L subunit that did not contain heme. At the position near 52000, some weak bands could be seen, which may be explained as partially disulfide bonds was remained in a, b and c trimer, so a little amount of trimer remained in SDS-PAGE sample. From Figure 1, LtHb may be convinced to be very pure. Due to all the color substance was precipitated in ATPE processing, so the recovery of LtHb was considered as 100%

Fig.1 SDS-PAGE pattern of LtHb product

Fig.2 SDS-PAGE pattern of semifinished Cyt c product

Table 1. Comparison of purification effect for Cyt c between ATPE and conventional techniques

    In the preparation of POD from radish by ATPE, SDS-PAGE pattern of its product was shown in Figure 3. They were almost the same in purity between conventional salting out and ATPE, which explained that the effect of ATPE was as good as by conventional salting out technique. They all enhanced greatly the purity comparing to the crude enzymatic solution as shown in Table 2. So many bands appeared in samples of ATPE or conventional salting out technique was due to some impurities remained or POD contained isoenzymes, but these did not affect the products as POD enzymatic product. Specific activity of crude enzyme, POD products made by conventional salting out, by ATPE was 0.077, 43.31 and 31.17 (O.D./mg·min), respectively, also demonstrated that ATPE was effective for the preparation of POD from radish due to that its advantage with quick and simple in extracting process and lower cost may remedy a defect in extracting purity. On the other hand, total enzymatic activity in ATPE was larger than that by conventional technique, which explained that the loss of enzyme protein in purification processing by ATPE was lower, and ATPE was suitable for the production on a large scales.

Fig.3 SDS-PAGE pattern of POD product
1：Conventional  2：ATPE  3：Crude enzyme

Table 2 Parameters of POD product made by different methods

a Total enzymatic activity = Specific activity × Total protein

2.2. Spectral scan analysis
In order to examine the character of LtHb, Cyt c product obtained by ATPE process, absorption spectra of these products were measured, which were shown in Figure 4 and Figure 5. These absorption spectra were as the same as those of pure LtHb or pure Cyt c (data not shown) in both oxide and reduced forms, which explained that the LtHb and Cyt c obtained by using ATPE remained normal characters.

Fig.4 Absorption spectra of Lumbyicus terrestris hemoglobin product in 450-650nm.
Solid line refers to oxide form and bold solid one to reduced form (adding small quantities of sodium dithionite to the samples to make reduced form)

Fig.5 Absorption spectra of semifinished Cyt c sample
Bold solid line refers to oxide form and solid one to reduced form (adding small quantities of sodium dithionite to the samples to make reduced form)

3 DISCUSSION
Based on the advantage of ATPE and preliminary experiments that all heme-containing proteins may be concentrated into the salt phase in aqueous two-phase system, ATPE was applied to practice for the preparation of these heme-containing proteins. Experimental results demonstrated that ATPE was very effective in extracting process for preparation of these valuable biochemical products, and the advantage of ATPE was very obvious in bioseparation. In fact, aqueous two-phase system may be composed of various compounds as we know, however, PEG and ammonium sulfate were used in this extracting process. This was at first that ammonium sulfate was good for protecting protein from molecular destruction at normal room temperature and secondly it was nontoxic and cheap, which made the process more economical. The composition of aqueous two-phase system should change as needed according to the character of purified aim substance due to that the aim substance has different equilibrium distribution in various aqueous two-phase systems. Many compounds, not only biomacromolecule but also inorganic compound or organic molecule with lower molecular mass may be separated and concentrated by ATPE. So ATPE is widely applied in compound separation. Composition of aqueous two-phase system deserved studied for different aim substance, which presents that ATPE is useful in chemical separation.
    Another factor to be considered in ATPE would be the concentration of composed substance used. The formation of aqueous two-phase system needed enough concentration of PEG or ammonium sulfate, phase diagram may be got through experiment, which has two-phase and one-phase regions. Concentrations of PEG and ammonium sulfate must be selected in two-phase region, and the formation of aqueous two-phase system is quicker as the concentration of PEG or ammonium sulfate increases, which would shorten the extracting process, but enhance the cost, especially the equilibrium distribution of some biomacromolecule are depending on the concentration of composed compounds. So the used concentration of composed substance should be determined based on experiment where whether the extracting effect is good. This problem was not existed in this study due to heme-containing proteins were all distributed in salt phase.

4. CONCLUSION
To apply ATPE to extracting process for the preparation of LtHb from earthworm, for the preparation of Cyt c from pork heart and for the preparation of POD enzymatic product from radish are successful. In general, the extracting process becomes short, operating easily with lower cost when ATPE are used. In the preparation of LtHb from earthworm using ATPE, very pure LtHb product may be obtained. In the application of ATPE to concentration and initial purification of Cyt c from pork heart, the extracting process is short and effective. For the preparation of POD product, ATPE is almost good in quality as conventional salting out technique.

REFERENCES
[1] Balasubramaniam D, Wilkinson C, Cott K V et al. Journal of Chromatography A, 2003. 989:119-129.
[2] Li Y, Beitle R R. Biotechnol. Prog. 2002,18:1054-1059.
[3] Ownby D W, Zhu H, Schneider K, Beavis R C, Chait B T et al.  J. Biol. Chem. 1993, June, 268: 13539-13547.
[4] Zhu H, Hargrove M, Xie Q, Nozaki Y et al. J. Biol. Chem. 1996 Nov, 271: 29999-30006.
[5] Zhu H, Ownby D W, Riggs C K, Nolasco N J et al.  J. Biol. Chem. 1996 Nov, 271: 30007-30021.

双水相萃取法应用于一些蛋白质的提取纯化的研究
李任强¹，徐思光¹，吴忠平²，方玲¹
（暨南大学生物工程学系，广州 510632；暨南大学化学系，广州 510632）
摘要 根据预试中发现所有含血红素蛋白质在双水相萃取中都浓缩于盐相和双水相萃取法在生化分离上的优点，把双水相萃取法应用于从生物材料中提取纯化蚯蚓血红蛋白、细胞色素C和过氧化物酶等含血红素的蛋白质。结果表明，在从蚯蚓中提取纯化蚯蚓血红蛋白，从猪心中提取纯化细胞色素C和从白萝卜中制备过氧化物酶制剂过程中应用双水相萃取法都相当成功，萃取时间短，成本低，容易操作。聚丙烯酰胺凝胶电泳、纯化效果比较及吸收光谱扫描等分析结果说明在这些提取纯化过程中应用双水相萃取可获得较纯的蚯蚓血红蛋白，细胞色素C的粗提时间短而有效，所得的过氧化物酶制剂在质量上与传统盐析法所得的几乎一样。
关键词 双水相萃取，纯化，蚯蚓血红蛋白，细胞色素C，过氧化物酶

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