[14] Partitioning of blood proteins using immobilized dyes

154

PARTITIONING OF MACROMOLECULES

[14]

[14] P a r t i t i o n i n g o f B l o o d P r o t e i n s U s i n g I m m o b i l i z e d D y e s

By GERD BIRKENMEIER Introduction The increasing demand for blood products for use as therapeutic agents necessitates the development of effective protein separation techniques. Purification of individual plasma proteins is often associated with problems because of similarities in their physicochemical properties and because many of them are present at low concentrations. Affinity chromatography techniques have greatly improved protein purification. ~Thus, a large number of proteins have been purified from human plasma by use of reactive dyes as affinity ligands. 2,~ Immobilized Cibacron Blue F3G-A was applied to the fractionation and purification of albumin, 4,5 blood clotting factors,6 a-fetoprotein, 7 a~,-proteinase inhibitor and al-acid glycoprotein,8 and other proteins. 2 Since their introduction by Albertsson, 9 aqueous two-phase systems have been applied for the extractive separation of biological materials, such as proteins, membranes, organelles, and cells.~° The partitioning of a macromolecule is influenced by many factors, including the molecular weight and charge of the biomolecule, concentration and molecular weight of the polymer, type and concentration of added salt, pH, and temperature. H Addition of affinity ligands covalently coupled to one of the phaseforming polymers can often be used to alter the partitioning of a protein 1 C. R. Lowe and P. D. G. Dean, "Affinity Chromatography." Wiley, New York, 1974. 2 G. Kopperschl~ger, H.-J. B6hme, and E. Hofmann, in "Advances in Biochemical Engineering" (A. Fiechter, ed.), p. 101. Springer-Verlag, Berlin, 1982. 3 G. Kopperschliiger, this volume [11]. 4 j. Travis and R. Pannell, Clin. Chim. Acta 49, 49 (1973). 5 G. M. Ghiggeri, G. Candiano, G. Delfino, and C. Queirolo, Clin. Chim. Acta 145, 205 (1985). 6 A. C. W. Swart, B. H. M. Kop-Klassen, and H. C. Hemker, Haemostasis 1, 237 (1972/1973). 7 K. Huse, M. Himmel, G. Birkenmeier, M. Bohla, and G. Kopperschl~iger, Clin. Chim. Acta 133, 335 (1983). s G. Birkenmeier and G. Kopperschl~iger, J. Chromatogr. 235, 237 (1982). 9 P.-A. Albertsson, "Partition of Cell Particles and Macromolecules." Wiley, New York, 1960. l0 H. Walter, D. E. Brooks, and D. Fisher (eds.), "Partitioning in Aqueous Two-Phase Systems." Academic Press, Orlando, Florida, 1985. ii G. Johansson, this volume [3].

METHODS IN ENZYMOLOGY,VOL. 228

Copyright© 1994by AcademicPress, Inc. All fightsof reproductionin any form reserved.

[14]

PARTITIONING OF SERUM PROTEINS

155

in a selective manner) 2 As in affinity chromatography, textile dyes are also useful ligands in affinity partitioning. These dyes can be obtained in a great variety of molecular structures, and some have a striking affinity for certain proteins. Affinity partitioning has also proved to be a sensitive tool in the analytical study of the mechanisms of dye-protein interactions. For example, the topography of dye-binding sites on the protein surface, 12 the dynamics of protein structure, 13and the influence of ligand immobilization on the binding mechanism have all been studied in detail by use of this method. 14 Here I focus on the applications of aqueous two-phase systems in the study of interactions of dyes with individual serum proteins and on serum protein fractionation by use of affinity ligands and with multistep extractions [i.e., countercurrent distribution (CCD)] or partitioning in three-phase systems. Materials and Methods Human serum albumin (HSA) and prealbumin are prepared from fresh human serum by dye affinity chromatography.15 Human serum is obtained from presumably healthy blood donors. Prior to use, the serum is dialyzed against the respective buffer used in the partitioning experiments. Small precipitates are removed by centrifugation. Antisera to human serum proteins are obtained from Behringwerke AG (Marburg, Germany). Poly(ethylene glycol) (PEG) is purchased from Serva (Heidelberg, Germany) (PEG 6000) or from Union Carbide (New York, NY) (PEG 8000). Dextran (Dx) T500, Dx T70, Dx T40, and Ficoll 400 are obtained from PharmaciaLKB (Uppsala, Sweden).

Single-Tube Partitioning Phase systems are prepared essentially as outlined by Brooks and Norris-Jones) 6 To prepare a 10-g two-phase system of the composition given in Fig. 1, and using 20% (w/w) Dx and 40% (w/w) PEG stock solutions, the following solutions are weighed out in a centrifuge tube: 5 g of 20% Dx T500, 1.875 g of 40% PEG 6000, I g buffer (10-fold concentrated), and 0-2.125 g of the protein sample. If less than 2.125 g of protein solution is added the difference is made up with water. The systems are carefully mixed by 20-40 inversions of the sealed tube, and the phases 12 G. 13 G. 14 G. 15 G. 16 D.

Johansson, G. Kopperschl~tger, and P.-/~. Albertsson, Fur. J. Biochem. 131, 589 (1983). Kopperschl~iger and G. Johansson, Biomed. Biochim. Acta 44, 1047 (1985). Johansson and M. Joelsson, J. Chromatogr. 537, 219 (1991). Birkenmeier, B. Tsehechonien, and G. Kopperschl~iger, FEBS Lett. 174, 162 (1984). E. Brooks and R. Norris-Jones, this volume [2].

156

PARTITIONING OF MACROMOLECULES 1

2

3

z,.

5

6

7

8

[14]

9

FIG. 1. Effect of different dye-PEG derivatives on partitioningofprealbumin and albumin. Prealbumin (1.6/~M, black bars) and albumin (13.1/zM, white bars) were partitioned together at 0° in 2-g systems composed of 10% (w/w) dextran T500, 7.5% (w/w) PEG 8000, and 10 mM sodium phosphate buffer, pH 7.0. The respective dye-PEG derivatives constituted 1.6% of total PEG in the system. Partitioning of prealbumin (---) and albumin (..... ) in the absence of dye-PEG is indicated. Dye-PEG derivatives: (1) Cibacron Blue F3G-A; (2) Procion Red H-3B; (3) Procion Orange MX-G; (4) Procion Yellow MX-R; (5) Procion Scarlet HR-N; (6) Procion Yellow MX-4G; (7) Procion Scarlet MX-G; (8) Procion Yellow MX-GR; (9) Procion Brown MX-5BR; (10) Remazol Yellow GGL. [From G. Birkenmeier and G. Kopperschl~ger, Mol. Cell. Biochem. 73, 99 (1987). Reprinted by permission of Kluwer Academic Publishers.]

are then allowed to separate. The settling time may be reduced to less than 1 min by brief centrifugation at low speed. In case o f affinity partitioning one o f the polymers is substituted by a desired percentage o f a liganded polymer. F o r example, if the system above was to contain 1.6% liganded P E G , 1.6% (w/w) of the total P E G would be replaced by liganded P E G , and thus the system would contain 1.845 g P E G and 0.03 g liganded PEG.

Single-Tube Partitioning in Three-Phase Systems Three-phase systems of 5 g are prepared by mixing aqueous stock solutions o f D x T40 (20%), Ficol1400 (40%), and P E G 6000 (40%) including liganded polymer solution and sample to yield the desired total composition o f 10% (w/w) Dx T40, 7% (w/w) Ficoll 400, and 3.5% (w/w) P E G 6000. The systems are equilibrated at 22 °. Centrifugation for 10 min at 1000 g is used to speed phase separation. Samples for analysis are withdrawn from the phases, appropriately diluted with barbital buffer, and subjected to electroimmunodiffusion.

[14]

PARTITIONING OF SERUM PROTEINS

157

Countercurrent Distribution of Serum Proteins A two-phase system composed of 5% (w/w) PEG 8000, 7.5% (w/w) Dx T70, 20 mM sodium phosphate buffer, pH 7.0, and 20 mM NaC1 is used for countercurrent distribution. A stock system of 150 g is prepared and equilibrated at 3° overnight. A thin-layer CCD apparatus with 60 chambers is u s e d . 9 The volume of each cavity in the lower phase is 0.79 ml. The chambers are loaded with 0.95 ml of the upper phase and 0.7 ml of the lower phase of the separated phase system. A sample system of the same composition weighing 10 g is prepared by including 0.45 ml of serum per gram of the two-phase system. Protein precipitation visible at the interface after mixing the phase system is removed by centrifugation (5000 g for 2 min). The two phases of the sample system are separated, and volumes as indicated above are added to chambers 0 and 1 of the plates. The number of transfers is chosen to be 58. A shaking time of 30 sec and a separation time of 10 min per transfer cycle are applied. All steps are carried out at 3°. After the run, 1 ml of 50 mM sodium phosphate buffer, pH 7.0, is added to each chamber for two-phase dilution to a single phase. The fractions are analyzed for total protein content and for individual serum proteins (see below). The partitioning of serum proteins in CCD is expressed as the partition ratio, G, defined as the percentage of protein in the mobile part of the system divided by the percentage of protein in the stationary part. An approximate calculation of G can be made from the peak position in the CCD run according to the relationship G = t/(n - ~), where ~ is the distance in the number of the tubes that the protein (peak position) has moved and n is the number of transfers.

Calculation of Partitioning in Single-Tube Experiments The partition coefficient, K, is calculated as the ratio of the concentration (c) in the top (t) and the bottom (b) phases, Kt/b = Ct/Cb. The effect of a polymer-bound ligand on the partitioning of a protein is expressed in terms of A log K, given by A log K = log K~r - log K0, where K 0 and Kafr are the partition coefficients of the protein in the absence and presende of the polymer-bound ligand in the system, respectively, other conditions being identical. The maximum A log K (A log Km~x) is obtained by plotting 1/A log K versus 1/ligand concentration.

Preparation of Dye-Polymer Conjugates Dye-Poly(ethylene glycol) Conjugates. The coupling of reactive dyes to PEG proceeds at 90° in aqueous solution under alkaline conditions by

158

PARTITIONING OF MACROMOLECULES

[14]

an nucleophilic reaction between reactive groups of the dyes and the OH group of the polymer. 12 For the coupling, 20 g of PEG 6000 and 0.2 g LiOH are dissolved in 46 ml distilled water and heated to 90 °. With continuous stirring 4.5 g of dye and 0.56 g LiOH are added stepwise over a period of 1 hr. After continued incubation for 1 hr the solution is stirred for an additional 16 hr at room temperature. Then 200 ml of distilled water and 200 ml of 3 M KCI are added, and the polymer is extracted three times with chloroform (200 ml each time). Chloroform is evaporated, and the resulting polymer (18 g) is dissolved in 400 ml water, mixed with DEAEcellulose (Reanal, Budapest, Hungary) (100 g wet weight), and stirred for I hr at room temperature. Unsubstituted PEG is removed by washing the material with excess water on a sintered glass filter. Elution of dyesubstituted PEG is accomplished by adding 3 M KC1 solution. Again, the d y e - P E G is extracted with chloroform (3 times 200 ml each time). The organic solutions (600 ml) are combined, and 100 g anhydrous N a 2 S O 4 (Merck, Darmstadt, Germany) is added to remove excess water. The solution is filtered, and the chloroform is removed by evaporation. The yield is between 200 mg and 2 g of d y e - P E G depending on the reactivity of the particular dye used. The degree of substitution ranges between 0.8 and 1.1 mol dye/mol PEG, indicating monosubstitution. Dye Conjugates of Dextran and Ficoll. Dx T40 (Mr 40,000) and Ficoll 400 (Mr 400,000) can be liganded to Remazol Yellow GGL (Hoechst AG, Frankfurt, Germany) according to the method of Johansson and Anderss o n . 17 Ten grams Dx T40 or Ficoll 400 is dissolved in 50 ml distilled water containing 4 g Na3PO4 • 12H20, I g NaCI, and 0.3 g Remazol Yellow GGL. The temperature of the mixture is slowly raised to 70° in a water bath, and the mixture is stirred for 1 hr. After cooling to room temperature the solution is dialyzed against excess distilled water to remove free dye. The polymer content in the final solution is 5.9% Ficoll 400 of 6.5% Dx T40 (based on polarimetric determination using [od25 values of + 199° and + 54.9 ° ml g- ~ d m - ~ for Dx and Ficoll, respectively). The degree of dye substitution as analyzed photometrically using the extinction coefficient of 13.4 mM cm -1 (396 nm) for Remazol Yellow GGL is 37/zmol dye/g Ficoll and 11/zmol dye/g Dx. Coupling of Cibacron Blue F3G-A (Serva) to polymers is accomplished in an analogous manner. After the reaction is completed the polymers are precipitated by the slow addition of ethanol (500 ml). The precipitate is dissolved in water and reprecipitated. This cycle is repeated three times so as to remove the uncoupled dye. The last precipitate is dialyzed against excess distilled water. The resulting solutions contain 4.32% Ficoll 400 17 G. Johansson and M. Andersson, J. Chromatogr. 303, 39 (1984).

[14]

PARTITIONING OF SERUM PROTEINS

159

and 5.6% Dx T40, respectively. The degree of substitution is 12/zmol dye/g Ficoll and 21 /xmol dye/g Dx, respectively, using the extinction coefficient of 13.6 mM cm -1 (610 nm) for Cibacron Blue F3G-A.

Analysis of Serum Proteins in Phase Systems Immunological Analysis. Protein concentration in the top and bottom phases can be obtained by rocket electroimmunoassay according to Laurell. TMGlass plates (8 × 9 cm) are coated with 15 ml of 1.5% agarose (Agarose Serva standard EEO) in sodium barbital buffer (0.1 M, pH 8.6). The concentration of the antiserum in the agarose ranges from 0.1 to 1% (v/v) depending on titer. Samples withdrawn from the phase system are diluted appropriately with barbital buffer and placed, in 5-/.d aliquots, into holes punched in the agarose. The appropriate dilutions of the standard serum and the samples are determined by the height of the precipitation peaks (1 to 5 cm) in the agarose. Electrophoresis can be performed in a Multiphor apparatus (Pharmacia-LKB) at 10 V/cm for 3 hr at 12°. After the run the plates are washed with saline solution overnight and finally air-dried. Staining of the precipitation lines is by Coomassie Blue R-250. Protein concentrations of the samples are calculated from a calibration curve using a double logarithmic plot of concentration versus peak height. Photometric Analysis. Quantitation of protein is based on the Bradford method. 19Aliquots from sample tubes obtained after CCD runs are mixed with 300/~1 of Bradford reagent in titer plates (96 wells) for 5 min. The absorbance is read at 610 nm in the ELISA-Reader Titertek Multiskan (Flow Laboratories, Meckenheim, Germany), and the protein concentration is calculated from a standard curve using bovine serum albumin (Boehringer, Mannheim, Germany) as standard. Results and Discussion The partitioning of proteins in aqueous two-phase systems depends on a number of factors, which include the composition of the two-phase system used and the surface properties of the protein. Resolution of serum proteins by partitioning in two-phase systems, which can be achieved by changing parameters such as pH, salt and ion composition, polymer concentration, and/or molecular weight of the polymers, is based on differences in surface properties of proteins such as charge or size. More selective partitioning of a particular protein can be effected by the use of specific ligands covalently bound to one of the phase-forming 18 C.-B. Laurell, Anal. Biochem. 15, 45 (1966). 19 M. M. Bradford, Anal. Biochem. 72, 248 (1976).

160

PARTITIONING OF MACROMOLECULES

[14]

polymers. Thus, ligands with high specificity should extract a target protein from the bulk protein phase. Figure 1 depicts the result when 10 different dyes were screened for their ability to bind human serum albumin and prealbumin. Of the dyes tested only Remazol Yellow GGL-PEG affects the partitioning of prealbumin, thus indicating its specificity for binding to this protein. In contrast, the K of albumin undergoes significant changes when PEG derivatives with different dyes are used, with Cibacron Blue F3G-A-PEG found to have the greatest effect. The relatively high affinity of albumin for different dyes2° with great differences in chemical structure is not surprising since albumin is known to bind many different compounds including aromatics and fatty acids. 21 The selectivity of protein binding to Remazol Yellow GGL led us to study its quantitative aspects. This can be done by constructing partitioning curves as shown in Fig. 2. Proteins are partitioned in phases with increasing substitution of dye-PEG for bulk PEG. By use of an inverse plot of 1/A log K versus 1/dye concentration (Fig. 2B) the following binding data were obtained: A log Kmax of 0.6 and 0.54 and a relative affinity (0.5 x A log Km~x)of 110 and 45/xM for binding of albumin and prealbumin to Remazol Yellow GGL, respectively. When prealbumin is partitioned in the presence of excess albumin the binding of prealbumin to the dye is significantly increased, yielding a A log Kmax of 1.12, whereas the relative affinity remains constant. This increase has been explained by complex formation between albumin and prealbumin in the presence of the dye.15.22 Such analytical data obtained by partitioning are valuable parameters to compare and evaluate the affinity and extraction power of ligands for proteins. Competitive inhibition studies in two-phase systems were carried out so as to elucidate the Remazol Yellow GGL-protein interaction in more detail. As shown in Fig. 3, the binding of prealbumin to the dye can be reduced by the thyroid hormones T3 (L-3,3',5-triiodothyronine) and T4 (L-thyroxine), which are naturally bound ligand. These hormones were less effective in reducing the affinity partitioning of albumin. In agreement with data obtained by other methods, 22 the results indicate that Remazol Yellow GGL binds directly or close to the hormone-binding sites in prealbumin. Similarities in the structural and stereochemical features of thyroid hormones and dye may account for this assumption. By use of more than two polymers, systems consisting of several phases can be obtained. 9 Three-phase systems composed of Dx, Ficoll, 2o G. Birkenmeier, E. Usbeck, and G. Kopperschl~iger, Anal. Biochem. 136, 264 (1984). 2x F. W. Putnam, "The Plasma Proteins." Academic Press, New York, 1975. 22 G. Birkenmeier and G. Kopperschl~iger, Mol. Cell. Biochem. 73, 99 (1987).

[14]

PARTITIONING OF SERUM PROTEINS

161

A

1.0-

4

0.5

J 8

3o 1/d~ 6o

oTs REMAZOLYELLOW5GL-PEG(raM) FIG. 2. Effect on the partition coefficients of prealbumin and albumin of increasing concentrations of Remazol Yellow GGL-PEG. (A) Systems of 2 g were composed of 10% (w/w) dextran T500, 7.5% (w/w) PEG 6000 containing different quantities of Remazol Yellow GGL-PEG, 10 mM sodium phosphate buffer, pH 7.0, and protein. The log K of prealbumin and albumin in the absence of dye-PEG was - 0.39 and - 1.37, respectively. The concentration of 1 mM immobilized dye corresponds to a total replacement of 3.2% of the PEG by dye-PEG. Partitioning was carded out at 0°. Protein added: ([]) albumin (4.3 /zM); (Q) prealbumin (1.6/~M); (O) prealbumin (1.6/~M) plus albumin (13.1 ~M). (B) Reciprocal plot of the data in (A) (same symbols). [From G. Birkenmeier and G. Kopperschliiger, Mol. Cell. Biochem. 73, 99 (1987). Reprinted by permission of Kluwer Academic Publishers.]

and PEG allow proteins to be resolved among three phases. The possibility of binding specific ligands to each of the polymers could lead to increases in the selectivity of protein partitioning. In this context we have studied the distribution of albumin and prealbumin in a threephase system composed of 10% (w/w) Dx T40, 7% (w/w) Ficoll 400, and 3.5% (w/w) PEG 6000. 23 The volumes of the PEG-rich top phase, the Ficoll-rich middle phase, and the Dx-rich bottom phase were 24, 25, and 51%, respectively, of the total volume at 22°. Remazol Yellow GGL and Cibacron Blue F3G-A attached to different polymers were used as ligands because of their differential interaction with prealbumin and albumin. 23 p..,~. Albertsson and G. Birkenmeier, Anal. Biochem. 175, 154 (1988).

162

PARTITIONING OF MACROMOLECULES

[14]

11 ~0S~

O

~0

160" s60

THYROID HORHONECONCENTRATION{juH) FIG. 3. Effect of thyroid hormones on affinity partitioning of prealbumin and albumin. The proteins were partitioned at 0° in 2-g systems composed of 10% (w/w) dextran T500, 7.5% PEG (w/w) 6000, 10 mM sodium phosphate buffer, pH 7.25, and increasing hormone concentrations. The A log K refers to the difference in partitioning of proteins in the absence and presence of 0.5 mM Remazol Yellow GGL-PEG. T4 (L-thyroxine) (©, [2) and T3 fL-3,3',5-triiodothyronine) (Q, E) were dissolved in 10 mM NaOH, incubated with the proteins for 15 min at 22°, and added to the phase system. (O, 0) Prealbumin (1.6/~M) in the presence of 30/~M albumin; (n, II) albumin (30/~M). [From G. Birkenmeier and G. Kopperschl~iger, Mol. Cell. Biochem. 73, 99 (1987). Reprinted by permission of Kluwer Academic Publishers.]

Table I shows the relative distributions of the two proteins among the three phases in the absence and presence of the dyes. The rationale for testing two different ligands in one phase system was to achieve separation of the two proteins in a single extraction on the basis of specific dye interactions. This offers the advantage of steering the distribution of two proteins of a mixture into different phases simultaneously. As seen, in the absence of the ligands both proteins are found in the Dx-rich bottom phase. A very effective separation of albumin and prealbumin is achieved in the phase system containing the ligands Cibacron Blue F3G-A-Ficoll and Remazol Yellow GGL-Dx. More than 90% of the prealbumin is found in the bottom phase, whereas albumin partitions in favor of the Ficollrich middle phase (81%). However, the yield of a protein in each phase depends on the volume ratio. Thus, even in the experiment with Cibacron

[14]

163

PARTITIONING OF SERUM PROTEINS

TABLE I PARTITIONING OF ALBUMIN AND PREALBUMIN IN THREE-PHASE SYSTEMSa IN PRESENCE OF TWO DIFFERENT IMMOBILIZED DYESb

Relative amount of protein in the phases Albumin Dye-polymer combination ~ Without dye Cb-Fic/RY-Dx Cb-Dx/RY-Fic Cb-Dx/RY-PEG Cb-PEG/RY-Dx Cb-PEG/RY-Fic Cb-Fic.,'RY-PEG

Prealbumin

Top

Middle

Bottom

Top

Middle

Bottom

1 2 0 0 32 35 2

16 81 2 2 34 33 76

83 17 8 98 34 32 22

6 1 4 16 1 6 17

19 5 27 24 7 30 26

75 94 69 60 92 64 57

a Composition of the system: 10% (w/w) dextran T40, 7% (w/w) Ficoll 400, 3.5% (w/w) PEG 6000, 20 mM sodium phosphate buffer, pH 7.0, and 20 mM NaCI. The concentrations of albumin and prealbumin in the system were 5 and 1.1 /~M, respectively. Temperature, 22°. b From P.-/~. Albertsson and G. Birkenmeier, Anal. Biochem. 175, 154 (1988), by permission. c The concentrations of the dye-polymers in the phases were as follows: Cibacron Blue F3G-A-PEG (Cb-PEG) (1.6%), Cibacron Blue F3G-A-Ficoll (Cb-Fic) (6%), Cibacron Blue F3G-A-Dx (Cb-Dx) (1%), Remazol Yellow GGL-PEG (RY-PEG) (1.6%), Remazoi Yellow GGL-Ficoll (RY-Fic) (6%), and Remazol Yellow GGL-Dx (RY-Dx) (6%). The percentage of the respective dye-polymer derivatives represents the fraction of the total amount of the polymer in the system that is replaced by the dye-liganded polymer.

Blue F3G-A-PEG/Remazol Yellow GGL-Dx a 5-fold increase in the volume of the top phase would cause 71% albumin and 90% prealbumin to be extracted into the top and bottom phases, respectively. In this context the advantage of a three-phase system compared to a two-phase system is that at least one phase remains that can extract the impurities. Compared to single-step partitioning, further improvements in separation of serum protein mixtures can be achieved by use of a multistep extraction procedure such a s C C D . 9 Figure 4 demonstrates the separation of total serum after 58 transfers in a two-phase system composed of 5% (w/w) PEG 6000, 7.5% (w/w) Dx T70, 20 mM sodium phosphate buffer, pH 7.0. The protein peak with a maximum at tube 15 consists mainly of the albumin fraction. Immunological analysis of other proteins revealed significant differences in their partitioning behavior. In the two-phase system used, most of the proteins partition in favor of the lower phase (i.e., are to the left in the CCD train). However, almost complete separa-

164

PARTITIONING OF MACROMOLECULES

[14]

1412

-

- Protein

:

"- B- Lipoprotein

10

2

"6

12t

10

o

o

=

= Prealbumin

Atbumin

A

z~ Thyroxine-binding globulin

/ 10

20

~0

Tube numt~r

Fro. 4. Thin-layer countercurrent distribution of human serum in a two-phase system composed of 5% (w/w) PEG 6000, 7.5% (w/w) dextran T70, and 20 mM sodium phosphate buffer, pH 7.0. The ordinate shows the relative amounts of different proteins recovered in each tube. [From G. Birkenmeier, G. Kopperschliiger, P.-A. Albertsson, G. Johansson, F. Tjerneld, H.-E. ~kedund, S. Berner, and H. Wickstroem, J. Biotechnol. 5, 115 (1987), by permission.]

tion from albumin was achieved in the cases of al-acid glycoprotein, thyroxine-binding globulin, t~l-antichymotrypsin, and fl-lipoprotein. The extent of protein separation for individual serum components is given in Table II by the separation factor log flAlb, defined as the logarithm of the ratio between the G values of a protein and of albumin.

[14]

PARTITIONING OF SERUM PROTEINS

l

: .~2-MocroglobuIin = Tronsferr~ o

12.

165

-a ,zcProteinose inhibitor o :el-Acid glycopro~n

10 8 6-

{

2 i

"6

,~

12

10

- Go-group pro~in o Hoptog[obin Ceru[op[osmin o ~wAnfichymotrypsin

8-

64-

i

1o

,

20

v

30

io

;o

60

Tube number

FIG. 4. (Continued)

Proteins with similar partitioning behavior such as albumin, transferrin, and haptoglobin can be resolved successfully by introducing dye-PEG into a two-phase system. Replacement of 1/20 of the total PEG by Cibacron Blue F3G-A-PEG, for instance, changes the partitioning of albumin and fl-lipoprotein considerably, whereas relatively small partitioning changes result with transferrin, arproteinase inhibitor, prealbumin, and ceruloplasmin. Even a small retardation of movement of al-acid glycoprotein could be detected, which is probably due to both the result of repulsive ionic forces between the negatively charged dye and the protein (pI 2.7) and the lack of any affinity to the dye.

166

[14]

PARTITIONING OF MACROMOLECULES.

T A B L E II G VALUES OF SERUM PROTEINS OBTAINED BY COUNTERCURRENT DISTRIBUTION IN POLY(ETHYLENE GLYCOL)--DEXTRAN SYSTEMa IN ABSENCE AND PRESENCE OF POLYMER-BOUND DYES b

Without dye Protein

G value

log flAibc

Cibacron Blue F3G-A G value

Procion Yellow HE-3G

Procion Red HE-3B

log /3~ab~

G value

log ~Albc

G value

log ~Alb c

Albumin Prealbumin alAcid glycoprotein cq-Proteinase inhibitor Transferrin Haptoglobin Ceruloplasmin /3-Lipoprotein ~2-Macroglobulin

0.33 0.78 2.74 0.63

0 0.37 0.92 0.28

4.52 0.84 1.83 0.59

0 -0.73 -0.39 -0.88

3.29 1.03 2.74 0.59

0 -0.50 -0.08 -0.74

1.18 0.78 2.51 0.59

0 -1.18 0.33 -0.30

0.27 0.30 0.43 0.02 0.17

-0.09 -0.04 0.11 - 1.22 -0.29 0.37 -0.34

0.54 3 1.36 0.68 0.63 2.74 4.52 0.59

-0.78 -0.04 -0.38 - 0.68 -0.72 -0.08 0.14 -0.74

-0.59 -0.15 -0.30 d -0.59

0.78 0.15

- 1.09 -0.91 -0.91 0.39 - 1.13 -0.95 -0.22 -0.88

0.30 0.84 0.59 d 0.30

at-Antichymotrypsin Gc-Group protein

0.36 0.55 0.55 11.2 1 0.33 II 0.50 2.74 0.59

4.52 0.43

0.58 -0.43

° Composition of the two-phase systems: 5% (w/w) PEG 6000, 7.5% (w/w) dextran I"70, and 20 mM sodium phosphate buffer, pH 7.0. All other conditions are described in the materials and methods section. In the CCD runs containing dye-PEG, 5% of the total PEG was replaced by dye-liganded PEG. b From G. Birkenmeiei', G. KopperschBger, P.-A.. Albertsson, G. Johansson, F. Tjerneld, H.-E. Akerlund, S. Berner, and H. Wickstroem, J. Biotechnol. 5, 115 (1987), by permission. c The logarithmic separation factor, log flPab, relative to albumin, is defined by log/3~ab = log G p r o t © i n - log G n l b u m i n . d NO distinct peak position.

Table II also delineates the effect of a number of dyes on the resolution of serum proteins by CCD. These dyes show remarkable differences in fractionation properties. For example, Cibacron Blue F3G-A and, even more, Procion Yellow HE-3G separate serum a2-macroglobulin into two distinct fractions, each having a different affinity for the dyes, whereas Procion Red HE-3 is less effective in binding to this protein. Dyes in conjunction with CCD are, therefore, sensitive tools for the detection of protein heterogeneities. 24 The capacity of the phase systems for protein is high. Sample systems prepared for CCD contained serum in a concentration of 20-30 g/liter. Two to five times more material may be included in the phase system provided that it does not precipitate. 8 The precipitates that occurred were mainly immunoglobulins and high molecular weight plasma proteins. However, the solubility of these proteins increased in systems containing 24 G. Birkenmeier, G. Kopperschlfiger, P.-A. Albertsson, G. Johansson, F. Tjerneld, H.-E. ,~kerlund, S. Berner, and H. Wickstroem, J. Biotechnol. 5, 115 (1987).

[15]

METAL AFFINITY PARTITIONING

167

dye-PEG. This is in keeping with results showing that precipitation of proteins by PEG can be prevented by adding dye-PEG. 2s'26 Conclusion Aqueous phase systems without or with affinity ligands are highly useful tools for the purification and study of serum proteins. A large number of dyes are commercially available and can be coupled easily to different polymers without loss of protein-binding properties. The simplicity and rapidity of partitioning make this method especially attractive for prepurification of whole plasma as well as for the isolation of high value-low volume therapeutic products from human blood. 25 G. J o h a n s s o n , in " P r o t e i n - D y e Interaction: D e v e l o p m e n t s and Applications" (M. A. Vijayalakshmi and O. Bertrand. eds.), p. 165. Elsevier, L o n d o n , 1989. 26 G. Birkenmeier and G. Kopperschl~tger, J. Biotechnol. 12, 93 (1991).

[15] M e t a l Affinity P a r t i t i o n i n g

By BONG H. CHUNG, DARWIN BAILEY, and FRANCES H. ARNOLD Introduction The partitioning of biological materials in aqueous two-phase systems, for example, poly(ethylene glycol) (PEG)-dextran (Dx) systems, can be altered in a reproducible and predictable fashion using affinity ligands attached to one of the phase-forming polymers. Metal ion complexes such as copper(II) iminodiacetate [Cu(II)IDA] have several very attractive features as affinity ligands: they are stable under a wide range of solvent conditions and temperatures and are therefore easy to recycle; they can be incorporated into chromatographic supports with very high loading capacities; their interactions with target molecules are reasonably specific and are reversible under mild conditions; and they are inexpensive. 1 Metal ion complexes have been used as affinity ligands in aqueous I F. H. Arnold, Bio/Technology 9, 151 (1991).

METHODS IN ENZYMOLOGY,VOL. 228

Copyright © 1994by AcademicPress, Inc. All rights of reproduction in any form reserved.