Continuous Separation of Proteins by

Transcription

1 Continuous Separation of Proteins by Isoelectric Focusing Shintaro Furusaki and Tatsuji Kikuchi Department of Chemical Engineering, University of Tokyo, Tokyo, Japan 113 (Received Jan. 24, 1983) Continuous separation of proteins is studied by using electrophoresis. The apparatus is composed with electrophoresis cells which are separated by porous membranes. The value of ph is adjusted so as to be low near the anode and high near the cathode. Intermediate cells stand at intermediate values of ph. Some cells stand at ph's of the isoelectric points of desired proteins. By the driving forces originated by the electric potential field, proteins migrate toward the cells at their isoelectric points. This separation, continuous isoelectric focusing, is studied by measuring extent of migration of bovine serum albumin. Experimental results are discussed by use of a theory considering electrochemical mobility and flow pattern inside the cells. Key words : Isoelectric focusing, Separation, Proteins, Albumin separation called "Continuous Isoelectric Focusing" Introduction Liquid phase separation of proteins, such as enzymes and/or interferrons, is becoming a more important theme to study. Among a number of separation techniques, electrophoresis is one of the appropriate ways to separate proteins since it does not damage higher order structures of proteins which exist in the separation media. However, most of electrophoretic methods have been suitable to analytical applications and they are not adequate to be applied to large or moderate scale separations. Azuma et al1). studied continuous separation of proteins by use of electrophoresis and an apparatus with porous membrane. This process has been extended to be applied to separation of proteins having opposite electric charges2). According to these investigations the continuous separation process is useful for the protein systems possessing significant differences in electrophoretic mobilities. It is desired, however, that finer separation of mixture of several proteins is carried out and that more concentrated solutions of purified proteins are produced. To achieve these aims a novel continuous electrophsresis is proposed in this paper. This method is a modification of the previous studies1 `2), Buffer solutions of different ph are introduced to each electrophoresis cell which is separated by porous membranes. Proteins supplied to the electrophoresis cells migrate to the cell where ph of the solution is at the isoelectric point of each separated protein. It is the purpose of this paper to explain the separation technique and to study behaviors of proteins under the separation by use of a simple model system. Migration of bovine serum albumin (BSA) is studied as an example of the separation. Separation procedures Figure 1 shows a schematic picture of continuous isoelectric focusing. Buffer solutions flow upward through the cells which are bounded by porous membranes. The ph's of the solutions are different. It is higher in the cell which locates nearer to the cathode. Proteins are dissolved in the entering solutions. If the proteins A, B and C have isoelectric point (pi) of ph2, ph3 and

2 Furusaki, Kikuchi: Continuous Separation of Proteins by Isoelectric Focusing Fig. 1 Schematic picture of isoelectric focusing Fig. 2 Mobility of BSA in the solution whose ionic strength is zero, ueo Sign of ueo denotes the direction of migration, i. e. toward anode if ueo>0: toward cathode if ueo<0. Source numbers correspond to References. ph4, respectively, they will migrate to the cells having the ph values of the corresponding isoelectric points. Thus, proteins are forcused to the appropriate cells by difference of isoelectric points. So, more concentrated protein solution may be obtained by choosing proper experimental conditions. The process will work effectively when the supplied solutions contain mixtures of proteins. Each protein will be attracted to different exits. Experimental Measurment of the electric mobilities Measurement of the electric msbilities in buffer solutions was carried out by gel-electrophoresis1). This was recently modified3) so that migration rate of charged particles in a solution was measured by an electrophoresis cell with a porous membrane. The obtained mobility of BSA in infinitely dilute buffer solution, tieo, is given in Fig. 2. Actual mobilities ue can be calculated by the following equation1). Fig. 3 Experimental apparatus of continuous iso- where rp is radius of the protein particle and f(krp) is the Henry function of electrophoresis. The Debye- Huckel parameter is given by Eq. (2). From the value of ueo Fig. (2) 2, it can be concluded that the results by both methods agree well. The iso-

3 Fig. 4 Experimental condition to investigate migration of BSA (Dimensions are in mm.) electric point also agrees with the value, , i teratures4,5). Migration of BSA Experimental apparatus to study the continuous isoelectric focusing is shown in Fig. 3. The apparatus is composed of five cells. The boundaries between the cells are made of porous polytetrafluoroethylene in (PTFE) membrane. Its properties are given below: thickness 0.127mm, porosity 0.89, characteristic maximum pore diameter 3.7 m. The membrane was soaked in ca. 1% Triton X-100 ethanol solution in order to be moistened by water. The experimental condition is shown in Fig. 4. ctric potential field was supplied by use of a D.C. stabili zer. Ele The potential gradient was obtained from electric current and conductivity of the solution. BSA was introduced to the cell I only, where ph was the lowest. The inlet ph was adjusted by buffer solutions using 0.1M CH3COOH and 0.1M CH3COONa. The ph in the outlet solution was not the same as the inlet ph due to concentration polarization. It changed significantly in the cell near the electrodes. out at room temperature. Experiments were carried Velocities of the solutions in Fig. 5 Experimental results (c1 `c5) each cell were identical. Concentration of BSA was determined by UV absorption at 280 nm. Results of migration of BSA for three superficial velocities of liquid in the cells are given by the solid lines in Fig. 5. Here, the solution in the cells are considered to be mixed perfectly according to the consideration which will be described later. Therefore, the values of ph in the cells are considered same as those of the outlet solutions. BSA molecule has positive charge at ph lower than the isoelectric point, i.e It has negative charge at ph higher than the isoelectric point. It is obvious from the figure that BSA migrates toward the cell 3 where ph of the solution is at the isoelectric point of BSA. At the lowest velocity, 7.8 ~10-3 cm/s, most of BSA is found to be accumulated to the cell 3. BSA should not be transported to the cells 4 and 5, but experimentally there was a small amount in the cells. The reason for this phenomenon is not clear. It may be caused by some impurities in BSA.

4 246 Furusaki, Kikuchi : Continuous Separation of Proteins by Isoelectric Focusing in each part is assumed homogeneous. When the two parts exist in the cell, recirculating flow will be generated due to buoyance. Momentum balance on laminar flow inside the cell leads to the following equation. Fig. 6 Mixing behavior of benzopurpurine 4B in the cell Fig. 7 A model of the flow field in the cell Pd is greater than pu Discussion Flow in the electrophoresis cells Mixing behavior in the electrophoresis cells affects the separation efficiency. Yokoyama7) studied the flow by feeding aqueous solution of benzopurpurine 4B (M. W. ca ). This is a red dye-staff with negative charge. So, the flow in the cells was visible. The dye-staff migrated toward the anode and flew upward along the porous membrane. Then, when it reached the liquid surface, it descended along the opposite membrane. Shortly after the inversion was observed, the solution began to become homogeneous due to the migration toward the anode. Schematic features of this event is given in Fig. 6. Approximate treatment of the flow field in the cell is attempted as follows by considering the high-density part and the low-density part in the cell. Figure 7 shows a schematic picture of the model. The cell is divided into the high-density part, i.e. the region 0 x (1-ƒÌ)l, and the low-density part, i.e. (1-ƒÌ)1<x<1. Density Fig. 8. From the figure it is understandable that the recirculating flow, or convection, is dominant in the cell

5 Therefore Fig. 8 Calculated velocity profile in the cell even by small unbalance of densities. In carrying out the electrophoresis separation by using membrane, there will be the density unbalance inside the cell due to concentration gradient of proteins. Then the velocity of the convective circulation is considerably larger than the superficial velocity. But the direction of recirculation will be opposite to the case of benzopurpurine 4B, i.e. downflow at high concentration zone of protein. When the recirculation prevails, the mixing behavior in the cell can be considered to be completely mixed as a first approximation. This consideration is a basic premise of the following analysis. Comparison of the separation data with Calculatedexpectation Assuming that flows in separating cells are in perfectly mixed scheme, separation in each cell can be calculated. Here, effect of diffusion can be considered negligible at the experimental conditions. If we know values of ue in Eq. (1), the exit concentration of BSA in the cell 1 is given by Eq. (15) Also for the cell 2, the following equation holds true. Since the cell 3 is at the isoelectric point of BSA, the protein will not migrate further toward the cells 4 and 5. the calculated outlet concentrations in each cell is given by the broken lines in Fig. 5. The coincidence of the experimental and calculated results does not seem satisfactory except the case of the lowest superficial velocity. However, the calculation seems to represent general behavior of the separation. The discreponcy at high superficial velocities may be diminished by considering the effect of longitudinal dispersion. This will affect the concentration distributions of not only the migrant but also the hydrogen ion. Due to the changes of the above concentration distributions the extent of migration of BSA may be affected significantly. Impurities and intermixing between cells may also influence the results. Further study on longitudinal dispersion and on its electrochemical effects is necessary to consider the above points. Conclusion Continuous isoelectric focusing to apply to separation of proteins is studied by use of an electrophoresis cell which is installed with porous polytetrafluoroethylene membrane. Proteins migrate toward the cells where ph's of the solutions are the isoelectric points of the corresponding proteins. Experimental results using BSA as a migrant are analyzed by a theory assuming completely mixed electrophoresis cells and utilizing mobility data of BSA. Calculated results show general behavior of the separation process but it is necessary to study further in order to fit the calculation better with the experimental data especially for high superficial velocity. Acknowledgement The anthors would like to express their appreciation to Japan membrane, and to Gore-tex Co., Ltd. for supplying the PTFE their experimental contributions. K. Nakata and K. Yokoyama for They are also indebted to K. Ueyama for discussions of the fluid dynamic study. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan (Grant No ). for

6 248 Furusaki, Kikuchi : Continuous Separation of Proteins by Isoelectric Focusing Nomenclature References 1) H. Azuma, S. Furusaki and T. Miyauchi: Kagaku Kogaku Ronbunshu, 5, 136 (1979) 2) K. Nakata, S. Furusaki and T. Miyauchi: Preprint 43rd Annual Meeting, Soc. Chem. Eng., Japan, p. 112, B301, Nagoya (1978) 3) S. Furusaki and N. Asai: Biotechnol. Bioeng., 25 in printing (1983). 4) Dictonary of Physics and Chemistry, 3rd. ed., p. 401 Iwanami Publ. Co. (1977) 5) Dictionary of Biology, 2nd. ed., p. 334, Iwanami Publ. Co. (1978) 6) J. R. Cann and J. G. Kirkwood: Cold Spring Symp. on Quant. Biol., 14, 9 (1950) 7) K. Yokoyama: B. S. Thesis, Univ. of Tokyo (1981)