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Protein adsorbents intended for use in aqueous two-phase systems
ANAEYTICAL
BIOCHEMISTRY
138,4 1l-4 15 (1984)
Protein Adsorbents Intended for Use in Aqueous Two-Phase Systems PER 0. HEDMAN’
AND JAN-GUNNAR
GUSTAFSSON
Pharmacia Fine Chemicals AB, Vppsala, Sweden Received October 25, 1983 Preparation of adsorbents with high partition coefficients in polyethylene glycol-dextran and polyethylene glycol-phosphate systems is described. These adsorbents may be used to carry proteins away from the insoluble cell fraeplents generated during cell disruption. By chromatographic elution, proteins may be selectively desorbed in a reduced volume. KEY WORDS: adsorbent; phase system; partitioning; hexokinase; alcohol dehydrogenase; Protein A.
When microorganisms are mechanically disrupted to release valuable products, the homogenate will contain solid fragments which are difficult to remove. On a large scale, filtration and centrifugation may become prohibitively expensive. To overcome these difficulties aqueous two-phase partitioning has been utilized (1,2). The feasibility of such extraction procedures depends on the obtainable partition coefficient of the desired product. An increased partition coefficient is sometimes obtained with inert salt additives (3) or by addition of affinity ligands (4-7). For the extraction of proteins from a cell fragment containing homogenate, affinity ligands would be useful. Alternatively, affinity adsorbent particles may be used. Such particles could be packed into chromatography columns before protein desorption, thus facilitating product recovery in a reduced volume. This work describes particles which partition to the polyethylene glycol (PEG)2-rich phase in systems where cell fragments stay in the more polar dextran- or phosphate-rich phase, and their use for protein extraction. ’ To whom correspondence should be addressed at Pharmacia Fine Chemicals, P.O. Box 175, S-75 104 Uppsala, Sweden. 2 Abbreviations used: PEG, polyethylene glycol; CNBr, cyanogen bromide; BSA, bovine serum albumin; ADH, alcohol dehydrogenase.
MATERIALS
AND METHODS
Albumin (for culture media) batch 3691 was obtained from Povite Producten, Amsterdam, Holland. Alcohol dehydrogenase (ADH), glucose-6-phosphate dehydrogenase, hexokinase (HK), ATP, NAD, NADP, and human gammaglobulin (Cohn Fraction II) were from the Sigma Chemical Company, St. Louis, Missouri. Baker’s yeast was purchased from Jistbolaget, Sollentuna, Sweden. Cibacron blue F3GA was purchased from CibaGeigy. Protein A, Sephadex G-50 medium, Sephadex LH-20, blue Sepharose CLdB, epoxy-activated Sepharose 6B, octyl-sepharose CL-4B, phenyl-Sepharose CL-4B, and Dextran T500 were from Pharmacia Fine Chemicals, Uppsala, Sweden. Polyethylene glycols 1540 and 4000 were purchased from Merck and methoxy-polyethylene glycol3000 was purchased from Hoe&t (Lot No. GVS 07-0251). Preparation of cell homogenates. Baker’s yeast, 100 g, was suspended in 50 mM sodium phosphate, pH 7, 80 ml and mixed with 80 ml dry glass beads (0.5-mm diameter). The mixture was agitated at full speed with an Ultra Turrax 45 drive unit in a flow-through chamber cooled by circulating ice water in the jacket. To minimize local overheating the agitator was run for 8 X 1 min with 1-min intervals. 411
000%2697/84 $3.00 Copyright 8 1984 by Academic F’res. Inc. All rights of reproduction in any form reserved.
412
HEDMAN
AND GUSTAFSSON
Protein determinations.Alcohol dehydrogenase was determined as described by Worthington Biochemical Corporation and by Vallee and Hoch (8). Hexokinase was determined by reaction of glucose with ATP and by the coupled NADP reduction catalyzed by glucose-6-phosphate dehydrogenase (9). Albumin was determined by its absorbance at 280 nm in a l-cm cuvette. The absorbance reading was divided by 0.66 to obtain the albumin concentration (in mg/ml). Protein A was similarly determined by division of the 275-nm absorbance reading by 0.16.
KOH and kept at 80°C for 100 min. The product was washed with 25 mM sodium phosphate, pH 7.4, distilled water, ethanol, distilled water, and finally 25 mM sodium phosphate, pH 7.4. RESULTS
AdsorbentPartitioning
In PEG-KPQ phase systems cell fragments accumulate in the bottom phosphate-rich phase. It is thus desirable to find adsorbents which may be quantitatively recovered from Preparationof PEG- and methoxy-PEG- the top phase. A number of Sephadex and substitutedparticles.Methoxy-PEG 3000, 12 Sepharose derivatives were partitioned at room g, or PEG 4000, 16 g, was dissolved in 50 ml temperature in a system containing 8.8% distilled water at 40°C. NaOH, 1.0 M, was w/w potassium phosphate and I 5.2% w/w PEG 4000. The observed partitioning is shown used to adjust the pH to 12.5-12.8. Waterin Table 1. rinsed epoxy-activated Sepharose 6B, 20 g, was added and the mixture was agitated on a rotary In PEG-dextran phase systems the polarity shaker for 18 h at 40°C. To block possible difference between the phases is usually lower remaining reactive groups, I5 ml ethylene than the difference in PEG-phosphate sysglycol was added and agitation was continued tems. Nevertheless, quantitative partitioning for 4 h. Finally the gel was washed, first with to the PEG-rich phase was observed with Ci50 mM sodium phosphate, pH 7.0, containing bacron blue-PEG-Sepharose in a system con1 M NaCl and then with 50 mM sodium phos- taining 6% w/w PEG 4000 and 7.2% w/w phate, pH 7.0. Dextran T 500. CNBr activation.PEG-Sepharose or methoxy-PEG-Sepharose, 15 ml, washed with iceTABLE 1 cold water, was added to 30 ml distilled water containing 67 mg/ml CNBr. With cooling to PARTITIONING BEHAVIOR OF DIFFERENT GEL TYPES 5°C the reaction was initiated by pH adjustPhase containing ment, with NaOH, to 11.3. After 6 min the Particle the particles gel was washed with ice-cold water. Attachmentofprotein-bindingligands.Hu- Sephadex G-50 medium Bottom man gammaglobulin, 200 mg, was bound to Sephadex LH-20 TOP 12 g (wet wt) activated gel in 10 ml 0.1 M PEG-Sepharose 6B Top Methoxy-PEG-Sepharose 6B NaHC03 containing 0.5 M NaCI. The mixture TOP Phenyl-Sepharose CL4B Top was agitated for 3 h at room temperature and CL-4B Bottom then left standing 48 h at 4°C. The gel was Octyl-Sepharose Blue Sepharose CL-6B Top or bottom then washed in 0.1 M sodium acetate, 0.5 M NaCl, pH 4, followed by 0.1 M NaHCOs, pH Note. The phase system contained 8.8% w/w potassium 8.3, and 20 mM sodium phosphate, pH 7.0. phosphate and 15.2% w/w PEG 4000. The top phase is relatively richer in PEG. The lower phase contains more This sequence was repeated twice. Cibacron phosphate. Phase compositions were not determined. The blue was bound by triazine coupling to PEGPEG and phosphate concentrations of the phases may be Sepharose or methoxy-PEG-Sepharose. Sed- estimated from phase diagrams published by Albertsson imented gel, 25 ml, was mixed with 25 ml (3) assuming that the added get particles do not change distilled water, 1 g Cibacron blue, and 10 mg the composition of the phase system.
PROTEIN
ADSORBENTS
IN AQUEOUS
Protein-AdsorbentPartitioning To demonstrate the partitioning in the presence of protein, Cibacron blue-PEG-Sepharose and Cibacron blue-methoxy-PEGSepharose (2 ml sedimented gel) were mixed with 3 ml BSA (9.4 mg/ml) in 25 InM phosphate, pH 7.2, for 45 min. After albumin adsorption a supematant sample was used to determine the protein content. From this measurement the amount of bound albumin was calculated. The adsorbent was then recovered from the PEG-rich phase and poured into a column (K16/20 Pharmacia Fine Chemicals). Residual unbound solutes were eluted with 25 mM phosphate, pH 7.2, prior to albumin elution with 2 M KC1 in the same buffer. Table 2 shows the amount of albumin bound and the recovery of albumin upon elution. Because albumin is desorbed from Cibacron blue gels at high ionic strengths, this partitioning was carried out in a low-salts PEGdextran phase system. To verify that inexpensive PEG-phosphate systems may be used, the Protein A-IgG interaction was chosen as a salt-insensitive model. y-Globulin-PEGSepharose or y-globulin-methoxy-PEG-Sepharose, 0.6 ml, was mixed with 1.94 mg Protein A in 0.1 M phosphate, pH 7.2, and left overnight. Addition of potassium phosphate and PEG to final concentrations of 9.4% wf w phosphate, 13.1% w/w PEG 4000, and 4.9% w/w PEG 1540 resulted in the formation of two phases. The top, PEG-rich, phase containing the gel was transferred to a column TABLE 2 ALBUMIN-BINDING
CAPACITIES
OBSERVED
Gel Cibacron bluePEG-Sepharose Cibacron bluemethoxy-PEGSepharose
WITH
BLUE
AND RECOVERIES PEG
GELS
Albumin bound OWml gel)
Recovery OWmI se0
3.5
3.6
100
4.9
4.3
88
Recovery w
TWO-PHASE
SYSTEMS
413
(K16/20 Pharmacia Fine Chemicals) where Protein A was eluted with 0.1 M glycine, pH 3.0. The amount of recovery of Protein A is shown in Table 3. When PEG-Sepharose or methoxy-PEGSepharose was used, no protein was eluted with 0.1 M glycine, pH 3. If the y-globulinsubstituted gel is incubated with 0.1 M glycine, pH 3.0, for 16 h, no A2g0increase is observed, indicating that under these conditions the gel does not release any y-globulin.
EnzymeExtractionfrom Baker’s YeastHomogenate Protein binding by Cibacron blue is dependent on the ionic strength. To investigate if inexpensive PEG-salt systems are practically useful with Cibacron blue-methoxy-PEG-Sepharose, extraction of nucleotide cofactordependent enzymes was attempted. A homogenate of 100 g moist yeast was mixed with 20 ml Cibacron blue-methoxy-PEG-Sepharose and diluted to 150 ml with a buffer containing 20 mM Tris-HCl, pH 6.4, 5 mM MgCIz, 0.4 mM EDTA, and 2 PM dithiothreitol ( 10). This mixture was agitated on a reciprocating shaker at 4-6°C for 2 h. Addition of PEG and potassium phosphate to final concentrations of 10 and 12%, respectively, resulted in the formation of two phases. The bottom phase contained cell fragments, whereas the adsorbent was found in the top phase. Phase separation was slow but could be completed in 1-3 min at 1OOOgforces. The adsorbent was recovered from the top phase and transferred into a chromatography column (K 16/20 Pharmacia Fine Chemicals). ADH was eluted into 5 mM NAD, pH 6.4, and recovered in 12 ml (Table 4). Then at pH 8.6, hexokinase was eluted with 5 mM NAD (data not shown) and recovered in 12 ml from the same adsorbent. The uv adsorption of NAD prevented uv monitoring of the elution; therefore, 3-ml fractions were collected and assayed for enzymatic activity. The combined ADH-rich fractions contained activity corresponding to 8 1 AAsdO min-’ - ml-‘. The recovery was low, as discussed below. To find an explanation
414
HEDMAN
AND GUSTAFSSON
TABLE 3 PROTEIN A RECOVERY -f-GLOBULIN-SUBSTITUTED
Gel y-Globulin-PEGSepharose y-Globulin-methoxyPEG-Sepharose
FROM GELS
Protein A recovered (wfml gel)
Recovery (%I
2.1
64
2.33
72
we applied a solution of pure ADH to a column containing blue methoxy-PEG-Sepharose. The enzyme was eluted with a gradient of O-l M KC1 in 20 mM Tris-HCl, pH 6.4. Elution occurred at 0.75 M KCl. DISCUSSION
A number of ligands with moderate to low polarities are useful in designing particles partitioning to PEG-rich phases from dextran- or phosphate-rich phases. Successful partitioning was obtained with PEG-, methoxy-PEG-, phenyl-, hydroxypropyl-, and, in some systems, Cibacron blue-substituted particles. Surprisingly octyl groups did not result in particles partitioning to the PEG-rich phase. PEGand methoxy-PEGderivatized particles were used to prepare particles with suitable partitioning behavior and the ability to bind proteins with additional ligands. The methoxyPEGderivatized particles showed the higher protein-binding capacities. This indicates that the major fraction of the protein-binding ligands is bound to the gel matrix rather than to the free end of the polyethylene glycol. A rather low recovery of ADH was obtained from crude yeast homogenate. In 100 ~1 pooled eluate we measured 8.1 AAraO min-’ which converts to 972 AL& min-’ for the combined fractions obtained from 100 g moist yeast. Racker (11) measured 62,000 AA340 min-’ in a homogenate from 200 g dried yeast. Taken that Racker used 8 times more yeast, our recovery was 12.5% of the available ADH. A number of reasons may explain the low
recovery. First, the binding capacity of the gel should be considered. Assuming that the albumin and ADH capacities are similar, 98 mg ADH could bind to 20 ml of the used adsorbent. One milligram ADH would reduce 300400 gmol NAD per minute corresponding to 600-800 AAs40 min-‘. This means that the added gel had considerably higher capacity than was used for ADH binding. However, competitive binding of other substances from the crude homogenate may have reduced the ADH capacity. A more plausible cause of the low ADH recovery is the effect of high salt concentrations. In the phase system used (with a phosphate mixture of 306.9 g K,HPO, to 168.6 g KH2P04, average density 1.1 g/ml) the phosphate molarity corresponding to 12% w/w is 0.84. In a cell homogenate-free system containing 12% w/w phosphate and 10% PEG 4000 the top phase would contain 8% phosphates, which is lower than the elution molarity, which is 0.75 M with KCl. The ionic strength in 0.67 M phosphate, however, is considerably higher than it is in 0.75 M KCl. If the ADH binding is ionic-strengthdependent only (independent of the kind of anion), this explains the low yield. TABLE 4 ADH ACTIVITYIN
3-ml FRACTIONSFROMCIBACRON BLUE-METHOXY-PEG-SEPHAROSE
Fraction 1 2 3 4 5 6 7 8 9 10 11 12
A,&
min-’ . ml-’ 0.6 0.6 0.6 0.6 0.6 1.1 >20 >20 >20 11 0.5 0
Nate. ADH was eluted with a 5 mM NAD buffer. The enzyme activity determined in the collected fractions was determined according to Vallee and Hoch (8).
PROTEIN
ADSORBENT-S
IN AQUEOUS
CONCLUSION
A new class of solid adsorbents that partition to PEG-rich phases in PEG-dextran or PEGsalt two-phases systems may be synthesized. By attachment of protein-binding ligands to these adsorbents, purified enzymes may be recovered in clear solution although neither filtration nor centrifugation is used to clarify the crude cell homogenates used as starting materials. A limitation to the application of these adsorbents is the need to find ionic-strengthindependent l&and-protein combinations if the inexpensive PEG-salt phase systems are to be useful. REFERENCES 1. Kroner, K. H., Stach, W., and Kula, M.-R. ( 1982) J. Chem. Technol. Biotechnol. 35 130-l 31. 2. Veide, A., Smeds, A.-L., and Enfors, S.-O. (1983) Biotechnol. Bioeng. 25(7), 1789-1800.
TWO-PHASE
SYSTEMS
415
3. Albertsson, P.-A. (197 1) Partitioning of Cell Particles and Macromolecules, 2nd. ed., pp. 88-9.5, AImquist & Wiksell, Stockholm/Wiley, New York. 4. Flanagan, S. D., and Barondes, S. H. (1975) J. Biol. Chem. 250, 1484-1489. 5. Mat&son, B., and Ling, T. G. I. (1980) J. Immunol. Methods 38,2 1l-223. 6. Kroner, K. H., Cordes, A., Schelper, A., Morr, M., Buckmann, A. F., and Kula, M.-R. (1982) in Affinity Chromatography and Related Techniques (Gribnau, T. C. J., Visser, J., and Nivard, R. J. F., eds.), pp. 49 l-501, Elsevier, Amsterdam. 7. Koppemchlager, G., and Johansson, G. ( 1982) Anal. Biochem. 124, 117- 124. 8. ValIee, B. L., and Hoch, F. L. (1955) Proc. Nat/. Acad. Sci. USA 41, 327. 9. Boehringer-Mannheim (1973) Biochimica Information, Part 1, p, 11, Mannheim. 10. Easterday, R. L., and Easterday, 1. M. (1974) in Immobilized Biochemicals and Affinity Chromatography (Dunlap, R. B., ed.), pp. 123-133, Plenum, New York. 11. Racker, E. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), p. 500, Academic Press, New York.
BIOCHEMISTRY
138,4 1l-4 15 (1984)
Protein Adsorbents Intended for Use in Aqueous Two-Phase Systems PER 0. HEDMAN’
AND JAN-GUNNAR
GUSTAFSSON
Pharmacia Fine Chemicals AB, Vppsala, Sweden Received October 25, 1983 Preparation of adsorbents with high partition coefficients in polyethylene glycol-dextran and polyethylene glycol-phosphate systems is described. These adsorbents may be used to carry proteins away from the insoluble cell fraeplents generated during cell disruption. By chromatographic elution, proteins may be selectively desorbed in a reduced volume. KEY WORDS: adsorbent; phase system; partitioning; hexokinase; alcohol dehydrogenase; Protein A.
When microorganisms are mechanically disrupted to release valuable products, the homogenate will contain solid fragments which are difficult to remove. On a large scale, filtration and centrifugation may become prohibitively expensive. To overcome these difficulties aqueous two-phase partitioning has been utilized (1,2). The feasibility of such extraction procedures depends on the obtainable partition coefficient of the desired product. An increased partition coefficient is sometimes obtained with inert salt additives (3) or by addition of affinity ligands (4-7). For the extraction of proteins from a cell fragment containing homogenate, affinity ligands would be useful. Alternatively, affinity adsorbent particles may be used. Such particles could be packed into chromatography columns before protein desorption, thus facilitating product recovery in a reduced volume. This work describes particles which partition to the polyethylene glycol (PEG)2-rich phase in systems where cell fragments stay in the more polar dextran- or phosphate-rich phase, and their use for protein extraction. ’ To whom correspondence should be addressed at Pharmacia Fine Chemicals, P.O. Box 175, S-75 104 Uppsala, Sweden. 2 Abbreviations used: PEG, polyethylene glycol; CNBr, cyanogen bromide; BSA, bovine serum albumin; ADH, alcohol dehydrogenase.
MATERIALS
AND METHODS
Albumin (for culture media) batch 3691 was obtained from Povite Producten, Amsterdam, Holland. Alcohol dehydrogenase (ADH), glucose-6-phosphate dehydrogenase, hexokinase (HK), ATP, NAD, NADP, and human gammaglobulin (Cohn Fraction II) were from the Sigma Chemical Company, St. Louis, Missouri. Baker’s yeast was purchased from Jistbolaget, Sollentuna, Sweden. Cibacron blue F3GA was purchased from CibaGeigy. Protein A, Sephadex G-50 medium, Sephadex LH-20, blue Sepharose CLdB, epoxy-activated Sepharose 6B, octyl-sepharose CL-4B, phenyl-Sepharose CL-4B, and Dextran T500 were from Pharmacia Fine Chemicals, Uppsala, Sweden. Polyethylene glycols 1540 and 4000 were purchased from Merck and methoxy-polyethylene glycol3000 was purchased from Hoe&t (Lot No. GVS 07-0251). Preparation of cell homogenates. Baker’s yeast, 100 g, was suspended in 50 mM sodium phosphate, pH 7, 80 ml and mixed with 80 ml dry glass beads (0.5-mm diameter). The mixture was agitated at full speed with an Ultra Turrax 45 drive unit in a flow-through chamber cooled by circulating ice water in the jacket. To minimize local overheating the agitator was run for 8 X 1 min with 1-min intervals. 411
000%2697/84 $3.00 Copyright 8 1984 by Academic F’res. Inc. All rights of reproduction in any form reserved.
412
HEDMAN
AND GUSTAFSSON
Protein determinations.Alcohol dehydrogenase was determined as described by Worthington Biochemical Corporation and by Vallee and Hoch (8). Hexokinase was determined by reaction of glucose with ATP and by the coupled NADP reduction catalyzed by glucose-6-phosphate dehydrogenase (9). Albumin was determined by its absorbance at 280 nm in a l-cm cuvette. The absorbance reading was divided by 0.66 to obtain the albumin concentration (in mg/ml). Protein A was similarly determined by division of the 275-nm absorbance reading by 0.16.
KOH and kept at 80°C for 100 min. The product was washed with 25 mM sodium phosphate, pH 7.4, distilled water, ethanol, distilled water, and finally 25 mM sodium phosphate, pH 7.4. RESULTS
AdsorbentPartitioning
In PEG-KPQ phase systems cell fragments accumulate in the bottom phosphate-rich phase. It is thus desirable to find adsorbents which may be quantitatively recovered from Preparationof PEG- and methoxy-PEG- the top phase. A number of Sephadex and substitutedparticles.Methoxy-PEG 3000, 12 Sepharose derivatives were partitioned at room g, or PEG 4000, 16 g, was dissolved in 50 ml temperature in a system containing 8.8% distilled water at 40°C. NaOH, 1.0 M, was w/w potassium phosphate and I 5.2% w/w PEG 4000. The observed partitioning is shown used to adjust the pH to 12.5-12.8. Waterin Table 1. rinsed epoxy-activated Sepharose 6B, 20 g, was added and the mixture was agitated on a rotary In PEG-dextran phase systems the polarity shaker for 18 h at 40°C. To block possible difference between the phases is usually lower remaining reactive groups, I5 ml ethylene than the difference in PEG-phosphate sysglycol was added and agitation was continued tems. Nevertheless, quantitative partitioning for 4 h. Finally the gel was washed, first with to the PEG-rich phase was observed with Ci50 mM sodium phosphate, pH 7.0, containing bacron blue-PEG-Sepharose in a system con1 M NaCl and then with 50 mM sodium phos- taining 6% w/w PEG 4000 and 7.2% w/w phate, pH 7.0. Dextran T 500. CNBr activation.PEG-Sepharose or methoxy-PEG-Sepharose, 15 ml, washed with iceTABLE 1 cold water, was added to 30 ml distilled water containing 67 mg/ml CNBr. With cooling to PARTITIONING BEHAVIOR OF DIFFERENT GEL TYPES 5°C the reaction was initiated by pH adjustPhase containing ment, with NaOH, to 11.3. After 6 min the Particle the particles gel was washed with ice-cold water. Attachmentofprotein-bindingligands.Hu- Sephadex G-50 medium Bottom man gammaglobulin, 200 mg, was bound to Sephadex LH-20 TOP 12 g (wet wt) activated gel in 10 ml 0.1 M PEG-Sepharose 6B Top Methoxy-PEG-Sepharose 6B NaHC03 containing 0.5 M NaCI. The mixture TOP Phenyl-Sepharose CL4B Top was agitated for 3 h at room temperature and CL-4B Bottom then left standing 48 h at 4°C. The gel was Octyl-Sepharose Blue Sepharose CL-6B Top or bottom then washed in 0.1 M sodium acetate, 0.5 M NaCl, pH 4, followed by 0.1 M NaHCOs, pH Note. The phase system contained 8.8% w/w potassium 8.3, and 20 mM sodium phosphate, pH 7.0. phosphate and 15.2% w/w PEG 4000. The top phase is relatively richer in PEG. The lower phase contains more This sequence was repeated twice. Cibacron phosphate. Phase compositions were not determined. The blue was bound by triazine coupling to PEGPEG and phosphate concentrations of the phases may be Sepharose or methoxy-PEG-Sepharose. Sed- estimated from phase diagrams published by Albertsson imented gel, 25 ml, was mixed with 25 ml (3) assuming that the added get particles do not change distilled water, 1 g Cibacron blue, and 10 mg the composition of the phase system.
PROTEIN
ADSORBENTS
IN AQUEOUS
Protein-AdsorbentPartitioning To demonstrate the partitioning in the presence of protein, Cibacron blue-PEG-Sepharose and Cibacron blue-methoxy-PEGSepharose (2 ml sedimented gel) were mixed with 3 ml BSA (9.4 mg/ml) in 25 InM phosphate, pH 7.2, for 45 min. After albumin adsorption a supematant sample was used to determine the protein content. From this measurement the amount of bound albumin was calculated. The adsorbent was then recovered from the PEG-rich phase and poured into a column (K16/20 Pharmacia Fine Chemicals). Residual unbound solutes were eluted with 25 mM phosphate, pH 7.2, prior to albumin elution with 2 M KC1 in the same buffer. Table 2 shows the amount of albumin bound and the recovery of albumin upon elution. Because albumin is desorbed from Cibacron blue gels at high ionic strengths, this partitioning was carried out in a low-salts PEGdextran phase system. To verify that inexpensive PEG-phosphate systems may be used, the Protein A-IgG interaction was chosen as a salt-insensitive model. y-Globulin-PEGSepharose or y-globulin-methoxy-PEG-Sepharose, 0.6 ml, was mixed with 1.94 mg Protein A in 0.1 M phosphate, pH 7.2, and left overnight. Addition of potassium phosphate and PEG to final concentrations of 9.4% wf w phosphate, 13.1% w/w PEG 4000, and 4.9% w/w PEG 1540 resulted in the formation of two phases. The top, PEG-rich, phase containing the gel was transferred to a column TABLE 2 ALBUMIN-BINDING
CAPACITIES
OBSERVED
Gel Cibacron bluePEG-Sepharose Cibacron bluemethoxy-PEGSepharose
WITH
BLUE
AND RECOVERIES PEG
GELS
Albumin bound OWml gel)
Recovery OWmI se0
3.5
3.6
100
4.9
4.3
88
Recovery w
TWO-PHASE
SYSTEMS
413
(K16/20 Pharmacia Fine Chemicals) where Protein A was eluted with 0.1 M glycine, pH 3.0. The amount of recovery of Protein A is shown in Table 3. When PEG-Sepharose or methoxy-PEGSepharose was used, no protein was eluted with 0.1 M glycine, pH 3. If the y-globulinsubstituted gel is incubated with 0.1 M glycine, pH 3.0, for 16 h, no A2g0increase is observed, indicating that under these conditions the gel does not release any y-globulin.
EnzymeExtractionfrom Baker’s YeastHomogenate Protein binding by Cibacron blue is dependent on the ionic strength. To investigate if inexpensive PEG-salt systems are practically useful with Cibacron blue-methoxy-PEG-Sepharose, extraction of nucleotide cofactordependent enzymes was attempted. A homogenate of 100 g moist yeast was mixed with 20 ml Cibacron blue-methoxy-PEG-Sepharose and diluted to 150 ml with a buffer containing 20 mM Tris-HCl, pH 6.4, 5 mM MgCIz, 0.4 mM EDTA, and 2 PM dithiothreitol ( 10). This mixture was agitated on a reciprocating shaker at 4-6°C for 2 h. Addition of PEG and potassium phosphate to final concentrations of 10 and 12%, respectively, resulted in the formation of two phases. The bottom phase contained cell fragments, whereas the adsorbent was found in the top phase. Phase separation was slow but could be completed in 1-3 min at 1OOOgforces. The adsorbent was recovered from the top phase and transferred into a chromatography column (K 16/20 Pharmacia Fine Chemicals). ADH was eluted into 5 mM NAD, pH 6.4, and recovered in 12 ml (Table 4). Then at pH 8.6, hexokinase was eluted with 5 mM NAD (data not shown) and recovered in 12 ml from the same adsorbent. The uv adsorption of NAD prevented uv monitoring of the elution; therefore, 3-ml fractions were collected and assayed for enzymatic activity. The combined ADH-rich fractions contained activity corresponding to 8 1 AAsdO min-’ - ml-‘. The recovery was low, as discussed below. To find an explanation
414
HEDMAN
AND GUSTAFSSON
TABLE 3 PROTEIN A RECOVERY -f-GLOBULIN-SUBSTITUTED
Gel y-Globulin-PEGSepharose y-Globulin-methoxyPEG-Sepharose
FROM GELS
Protein A recovered (wfml gel)
Recovery (%I
2.1
64
2.33
72
we applied a solution of pure ADH to a column containing blue methoxy-PEG-Sepharose. The enzyme was eluted with a gradient of O-l M KC1 in 20 mM Tris-HCl, pH 6.4. Elution occurred at 0.75 M KCl. DISCUSSION
A number of ligands with moderate to low polarities are useful in designing particles partitioning to PEG-rich phases from dextran- or phosphate-rich phases. Successful partitioning was obtained with PEG-, methoxy-PEG-, phenyl-, hydroxypropyl-, and, in some systems, Cibacron blue-substituted particles. Surprisingly octyl groups did not result in particles partitioning to the PEG-rich phase. PEGand methoxy-PEGderivatized particles were used to prepare particles with suitable partitioning behavior and the ability to bind proteins with additional ligands. The methoxyPEGderivatized particles showed the higher protein-binding capacities. This indicates that the major fraction of the protein-binding ligands is bound to the gel matrix rather than to the free end of the polyethylene glycol. A rather low recovery of ADH was obtained from crude yeast homogenate. In 100 ~1 pooled eluate we measured 8.1 AAraO min-’ which converts to 972 AL& min-’ for the combined fractions obtained from 100 g moist yeast. Racker (11) measured 62,000 AA340 min-’ in a homogenate from 200 g dried yeast. Taken that Racker used 8 times more yeast, our recovery was 12.5% of the available ADH. A number of reasons may explain the low
recovery. First, the binding capacity of the gel should be considered. Assuming that the albumin and ADH capacities are similar, 98 mg ADH could bind to 20 ml of the used adsorbent. One milligram ADH would reduce 300400 gmol NAD per minute corresponding to 600-800 AAs40 min-‘. This means that the added gel had considerably higher capacity than was used for ADH binding. However, competitive binding of other substances from the crude homogenate may have reduced the ADH capacity. A more plausible cause of the low ADH recovery is the effect of high salt concentrations. In the phase system used (with a phosphate mixture of 306.9 g K,HPO, to 168.6 g KH2P04, average density 1.1 g/ml) the phosphate molarity corresponding to 12% w/w is 0.84. In a cell homogenate-free system containing 12% w/w phosphate and 10% PEG 4000 the top phase would contain 8% phosphates, which is lower than the elution molarity, which is 0.75 M with KCl. The ionic strength in 0.67 M phosphate, however, is considerably higher than it is in 0.75 M KCl. If the ADH binding is ionic-strengthdependent only (independent of the kind of anion), this explains the low yield. TABLE 4 ADH ACTIVITYIN
3-ml FRACTIONSFROMCIBACRON BLUE-METHOXY-PEG-SEPHAROSE
Fraction 1 2 3 4 5 6 7 8 9 10 11 12
A,&
min-’ . ml-’ 0.6 0.6 0.6 0.6 0.6 1.1 >20 >20 >20 11 0.5 0
Nate. ADH was eluted with a 5 mM NAD buffer. The enzyme activity determined in the collected fractions was determined according to Vallee and Hoch (8).
PROTEIN
ADSORBENT-S
IN AQUEOUS
CONCLUSION
A new class of solid adsorbents that partition to PEG-rich phases in PEG-dextran or PEGsalt two-phases systems may be synthesized. By attachment of protein-binding ligands to these adsorbents, purified enzymes may be recovered in clear solution although neither filtration nor centrifugation is used to clarify the crude cell homogenates used as starting materials. A limitation to the application of these adsorbents is the need to find ionic-strengthindependent l&and-protein combinations if the inexpensive PEG-salt phase systems are to be useful. REFERENCES 1. Kroner, K. H., Stach, W., and Kula, M.-R. ( 1982) J. Chem. Technol. Biotechnol. 35 130-l 31. 2. Veide, A., Smeds, A.-L., and Enfors, S.-O. (1983) Biotechnol. Bioeng. 25(7), 1789-1800.
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