[23]
P R E C I P I T A T I O N OF P R O T E I N S W I T H P E G
[23] P r e c i p i t a t i o n
of Proteins with Polyethylene
301
Glycol
B y KENNETH C. INGHAM
This chapter updates a chapter on protein precipitation using polyethylene glycol that appeared in a previous volume in the series. 1 The use of nonionic, water-soluble polymers, in particular polyethylene glycol (PEG), for fractional precipitation of proteins was introduced in 1964 by Poison et al. 2 Papers appearing over the next two decades provided an improved understanding of the molecular basis of the protein-precipitating action of P E G and additional documentation of the unique advantages of this polymer o v e r other reagents used for this purpose. Although m u c h of the literature on this subject deals with purification of proteins from blood plasma, 3 the approach is applicable to any complex mixture. The principles involved have been clarified by studies with purified proteins, and the purpose of this chapter is to summarize briefly these principles with emphasis on practical information enabling the reader to assess the potential applicability of this technique to specific separation problems. Advantages of Polyethylene Glycol The advantages of P E G as a fractional precipitating agent stem primarily from its well-known benign chemical properties. Unlike ethanol and other organic precipitating agents, P E G has little tendency to denature or otherwise interact with proteins even when present at high concentrations and elevated temperatures. Careful experiments designed to test this principle revealed that P E G 4004 at concentrations up to 30% (w/v) had no detectable effect on the circular dichroic spectrum or thermal denaturation temperature of ribonuclease. 5 Subsequent studies confirmed this result for ribonuclease but suggested that P E G has a destabilizing effect with some proteins at elevated temperature. 6 This should be of no coni K. C. Ingham, this series, Vol. 104, p. 351. : A. Polson, G. M. Potgieter, J. F. Largier, G. E. F. Mears, and F. J. Joubert, Biochim. Biophys. Acta 82, 463 (1964). 3 y. L. Hao, K. C. Ingham, and M. Wickerhauser, in "Methods of Protein Fractionation" (J. M. Curling, ed.), p. 57. Academic Press, New York, 1980. 4 PEG, Poly(ethylene glycol), poly(ethylene oxide), polyoxyethylene. Chemical formula: HOCH2CHz(CH2CH:O)nCH2CHEOH.PEG 400 and PEG 4000 signify heterogeneous mixtures having nominal average molecular weights of 400 and 4000, respectively. 5 D. H. Atha and K. C. Ingham, J. Biol. Chem. 256, 12108 (1981). 6 L. L. Lee and J. C. Lee, Biochemistry 26, 7813 (1987). METHODS IN ENZYMOLOGY, VOL. 182
Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.
302
PURIFICATION PROCEDURES: BULK METHODS
[23]
cern when using the polymer in protein purification where elevated temperatures are seldom employed. The low heat of solution and the relative insensitivity of PEG precipitation curves to minor variations in temperature eliminate the need for controlling temperature during reagent addition. Another advantage of PEG over ethanol or ammonium sulfate is the shorter time required for the precipitated proteins to equilibrate and achieve a physical state suitable for large-scale centrifugation. The advantages of PEG in facilitating the growth of protein crystals is well documented. 7 Mechanism of Action Careful measurements with a variety of purified proteins indicate that their solubilities decrease exponentially with increasing concentration of PEG according to Eq. (1), log S = log So - t i c
(1)
where S is the solubility in the presence of PEG at concentration C (%, w/v) and So is the apparent intrinsic solubility obtained by extrapolation to zero PEG. 5 Plots of log S vs [PEG] exhibit striking linearity over a wide range of protein concentration, the slope for a given protein being relatively insensitive to pH and ionic strength, but markedly dependent on the size of the PEG up to about 6000 Da. The slopes also tend to increase with increasing size of the protein, reinforcing the popular notion of a steric exclusion mechanism whereby proteins are concentrated in the extrapolymet space, eventually exceeding their solubility limit under the given solution conditions. Although a quantitative explanation of this behavior is yet to come, it is clear that, in the absence of specific interactions, the sequence of precipitation of several proteins in a mixture will depend primarily on the ratios of their initial concentrations relative to their respective solubilities in the absence of PEG. Thus, even though larger proteins have steeper slopes, a large protein initially present at high concentration could precipitate later than a small one present at low concentration if the intrinsic solubility of the latter is much less than that of the former. Manipulation of the solution conditions is expected to improve the separation of a given pair of proteins to the extent that their intrinsic solubilities diverge. Selection of PEG Most workers use material with a nominal average molecular weight in the 4000-6000 range. Polymers larger than this offer no advantage, since
[23]
PRECIPITATION OF PROTEINS WITH P E G
303
their solutions are more viscous and the precipitation curves are not much different from those obtained with PEG 6000. 2,5 Decreasing the molecular weight below 4000 spreads the precipitation of a mixture over a broader range of PEG concentrations. The improved resolution that might be thus anticipated is partially offset by the shallower slopes obtained for individual proteins. Nevertheless, Honig and Kula 8 found the degree of purification of 7-glucosidase from yeast extract to be about 2-fold greater with PEG 400 than with PEG 4000 or 6000. That PEG 400 is a liquid at room temperature whose solutions are substantially less viscous than those of the higher polymers, coupled with the potentially greater ease of removing it by molecular sieve methods, indicates a need for further comparisons. Analytical Precipitation Curve The following simple experiment is designed to quickly overcome ignorance about the amount of PEG required to precipitate a given protein(s) from a complex mixture. The scale of this experiment is dictated by the sensitivity of the assay employed; the availability of a radiolabeled tracer is a definite advantage. One dispenses a fixed amount (0.1-0.5 ml) of the mixture into a series of tubes (preferably in duplicate) to each of which is subsequently added an equal volume of buffer containing increasing amounts of PEG to produce a final concentration of 25-30% in the most concentrated tubes. It is important to buffer the PEG stock solutions to avoid PEG-induced changes in pH. 5,9 The increment in PEG concentration is arbitrary, but 3% (w/v) is adequate for initial screening. The vigor with which one mixes these solutions depends on the extent to which the desired protein(s) can withstand mechanical stress; gentle agitation on a vortex mixer is one approach. After 0.5-1.0 hr of incubation at room temperature or on ice, the samples are centrifuged and the percentage of the desired activity remaining in the supernatant liquid is determined. Inspection of the resulting "analytical precipitation curve" provides an estimate of the maximum concentration of PEG that can be added at one time without precipitating the protein of interest as well as the minimum concentration required to bring it out of solution, parameters that can then be more precisely defined with a second experiment that focuses on the relevant concentration range. With luck, the curve will fall either far to the left or far to the right on the PEG axis, defining a simple 7 A. McPherson, Jr., J. Biol. Chem. 251, 6300 (1976). 8 W. Honig and M.-R. Kula, Anal. Biochem. 72, 502 (1976). 9 G. Eichele, D. Karabelnik, R. Halonbrenner, J. N. Jansonius, and P. Christen, J. Biol. Chem. 253, 5239 (1978).
304
PURIFICATION PROCEDURES: BULK METHODS
[23]
one-step method for removing a large portion of unwanted macromolecules and/or concentrating the desired activity prior to further processing by other methods. Otherwise, it may be necessary to obtain a "PEG cut" via two precipitation steps utilizing in turn the maximum and minimum concentration of PEG referred to above. It is always possible to manipulate the precipitation curve horizontally along the PEG axis by varying solution conditions. For screening purposes, it is expedient to choose a fixed concentration of PEG that causes approximately 50% precipitation of the desired protein under a given set of solution conditions in order to determine rapidly the extent to which altering conditions such as pH and ionic strength might enhance or inhibit precipitation. The most gratifying result of this approach would be to identify substances or conditions that selectively influence the solubility of the desired protein. This concept is further developed in the following section. Influence of Protein-Protein and Protein-Ligand Interactions Studies with purified self-associating and heteroassociating proteins have shed some light on the role of protein-protein interactions on solubility in the presence of P E G ) ,1°-12 Based on the above-mentioned excluded volume considerations, one predicts that conditions that foster protein association should enhance precipitation because of the larger size of the complexes, wheres those that inhibit association would have the opposite effect. This is the case with almost all systems that have been examined. Of particular relevance in the present context was the observation 1° that bovine liver glutamate dehydrogenase at 2.8 mg/ml in 0.2 M potassium phosphate at pH 7.0, conditions known to promote extensive self-association, was quantitatively precipitated by PEG 4000 at concentrations above 15% (w/v). Such precipitation was completely inhibited, even at higher concentration of PEG, by the combined presence of l0 -3 M N A D H and GTP, cofactors known to reverse the self-association. Similar effects were observed with chymotrypsin, chymotrypsinogen, and fl-lactoglobulin A, in which cases self-association was manipulated by varying pH and ionic strength, parameters likely to be less selective. Nonspecific electrostatic interactions between oppositely charged proteins such as albumin and lysozyme can also have profound effects on solubility that are most pronounced at low ionic strength at a pH between the pI of each of the two proteins.ll While such interactions are frequently viewed as a i0 S. I. Miekka and K. C. Ingham, Arch. Biochem. Biophys. 191, 525 (1978). 11 S. I. Miekka and K. C. Ingham, Arch. Biochem. Biophys. 203, 630 (1980). 12 j. Wilf and A. P. Minton, Biochim. Biophys. Acta 671}, 316 (1981).
[23]
PRECIPITATION OF PROTEINS WITH P E G
305
nuisance, to be minimized by maintaining near-physiological ionic strength, the possibility of using them to advantage in a purification scheme should be kept in mind. A more specific type of heteroassociation of the type that might be exploited in purification is the functional interaction between human plasma fibronectin and denatured collagen, i.e., gelatin. The precipitation curve for the plasma protein in phosphate-buffered saline shifted from 11% PEG to less than 3% PEG upon addition of gelatin, which by itself was not precipitated by PEG under these conditions. 13Since the complex between the two proteins is very stable, even at high ionic strength, it should be possible to precipitate fibronectin selectively from a complex mixture by this method. The contaminating gelatin could then be removed, e.g., by ion-exchange chromatography in the presence of urea. Although the advantage of this approach over affinity chromatography on immobilized gelatin is debatable, the example serves as an additional illustration of the application of bioaffinity principles to fractional precipitation. Any substance that interacts specifically with the desired protein has the potential to alter its solubility selectively and should thus be tested. Enzymes are ideal candidates for this approach, since they often interact with one or more effectors or cofactors, sometimes with large changes in the state of association. Methods of Removing PEG In many applications, PEG is used early in the purification scheme and is removed during subsequent chromatographic steps on ion-exchange or affinity columns to which PEG has no tendency to absorb. A word of caution is in order regarding the application of PEG-containing solutions to some exclusion columns, the performance of which can be significantly altered owing to osmotic effects of the polymer. 14Alternative approaches to removing PEG include ultrafiltration ~5,16and salt-induced phase separation ~7as reviewed) 8 The latter method is particularly useful for solutions containing relatively high concentrations of PEG and has the potential advantage that the protein may be concentrated in a low-volume, salt-rich phase. For many research purposes it is probably unnecessary to remove t3 K. C. Ingham, S. A. Brew, and S. I. Miekka, Mol. lmmunol. 20, 287 (1983). 14 K. Hellsing. J. Chromatogr. 36, 170 (1968). 15 T. F. Busby and K. C. Ingham, J. Biochem. Biophys. Methods 2, 191 (1980). 16 K. C. Ingham, T. F. Busby, Y. Sahlestrom, and F. Castino, in "Ultrafiltration Membranes and Applications" (A. R. Cooper, ed.), p. 141. Plenum, New York, 1980. 17 T. F. Busby and K. C. Ingham, Vox Sang. 39, 93 (1980). ~s K. C. Ingham and T. F. Busby, Chem. Eng. Commun. 7, 315 (1980).
306
PURIFICATION PROCEDURES: BULK METHODS
[23]
all traces of polymer from the final product, since it is optically transparent 19 and helps prevent loss of protein by absorption of glass. Summary Polyethylene glycol is a nondenaturing water-soluble polymer whose ability to precipitate protein from aqueous solution can be qualitatively understood in terms of an excluded volume mechanism. The increment in PEG concentration required to effect a given reduction in solubility is unique for a given protein-polymer pair, being insensitive to solution conditions and primarily dependent on the size of the protein and polymer. Selective manipulation of the solubility of specific proteins through control of their state of association or ligand environment can potentially remove some of the empiricism otherwise involved in fractional precipitation. Adequate methods for removing the polymer are available.
19 T h e low level o f U V a b s o r b a n c e frequently found in s o m e PEG preparations is not inherent to the p o l y m e r but is due to a small a m o u n t o f antioxidant s o m e t i m e s added by the m a n u f a c t u r e r .
P R E C I P I T A T I O N OF P R O T E I N S W I T H P E G
[23] P r e c i p i t a t i o n
of Proteins with Polyethylene
301
Glycol
B y KENNETH C. INGHAM
This chapter updates a chapter on protein precipitation using polyethylene glycol that appeared in a previous volume in the series. 1 The use of nonionic, water-soluble polymers, in particular polyethylene glycol (PEG), for fractional precipitation of proteins was introduced in 1964 by Poison et al. 2 Papers appearing over the next two decades provided an improved understanding of the molecular basis of the protein-precipitating action of P E G and additional documentation of the unique advantages of this polymer o v e r other reagents used for this purpose. Although m u c h of the literature on this subject deals with purification of proteins from blood plasma, 3 the approach is applicable to any complex mixture. The principles involved have been clarified by studies with purified proteins, and the purpose of this chapter is to summarize briefly these principles with emphasis on practical information enabling the reader to assess the potential applicability of this technique to specific separation problems. Advantages of Polyethylene Glycol The advantages of P E G as a fractional precipitating agent stem primarily from its well-known benign chemical properties. Unlike ethanol and other organic precipitating agents, P E G has little tendency to denature or otherwise interact with proteins even when present at high concentrations and elevated temperatures. Careful experiments designed to test this principle revealed that P E G 4004 at concentrations up to 30% (w/v) had no detectable effect on the circular dichroic spectrum or thermal denaturation temperature of ribonuclease. 5 Subsequent studies confirmed this result for ribonuclease but suggested that P E G has a destabilizing effect with some proteins at elevated temperature. 6 This should be of no coni K. C. Ingham, this series, Vol. 104, p. 351. : A. Polson, G. M. Potgieter, J. F. Largier, G. E. F. Mears, and F. J. Joubert, Biochim. Biophys. Acta 82, 463 (1964). 3 y. L. Hao, K. C. Ingham, and M. Wickerhauser, in "Methods of Protein Fractionation" (J. M. Curling, ed.), p. 57. Academic Press, New York, 1980. 4 PEG, Poly(ethylene glycol), poly(ethylene oxide), polyoxyethylene. Chemical formula: HOCH2CHz(CH2CH:O)nCH2CHEOH.PEG 400 and PEG 4000 signify heterogeneous mixtures having nominal average molecular weights of 400 and 4000, respectively. 5 D. H. Atha and K. C. Ingham, J. Biol. Chem. 256, 12108 (1981). 6 L. L. Lee and J. C. Lee, Biochemistry 26, 7813 (1987). METHODS IN ENZYMOLOGY, VOL. 182
Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.
302
PURIFICATION PROCEDURES: BULK METHODS
[23]
cern when using the polymer in protein purification where elevated temperatures are seldom employed. The low heat of solution and the relative insensitivity of PEG precipitation curves to minor variations in temperature eliminate the need for controlling temperature during reagent addition. Another advantage of PEG over ethanol or ammonium sulfate is the shorter time required for the precipitated proteins to equilibrate and achieve a physical state suitable for large-scale centrifugation. The advantages of PEG in facilitating the growth of protein crystals is well documented. 7 Mechanism of Action Careful measurements with a variety of purified proteins indicate that their solubilities decrease exponentially with increasing concentration of PEG according to Eq. (1), log S = log So - t i c
(1)
where S is the solubility in the presence of PEG at concentration C (%, w/v) and So is the apparent intrinsic solubility obtained by extrapolation to zero PEG. 5 Plots of log S vs [PEG] exhibit striking linearity over a wide range of protein concentration, the slope for a given protein being relatively insensitive to pH and ionic strength, but markedly dependent on the size of the PEG up to about 6000 Da. The slopes also tend to increase with increasing size of the protein, reinforcing the popular notion of a steric exclusion mechanism whereby proteins are concentrated in the extrapolymet space, eventually exceeding their solubility limit under the given solution conditions. Although a quantitative explanation of this behavior is yet to come, it is clear that, in the absence of specific interactions, the sequence of precipitation of several proteins in a mixture will depend primarily on the ratios of their initial concentrations relative to their respective solubilities in the absence of PEG. Thus, even though larger proteins have steeper slopes, a large protein initially present at high concentration could precipitate later than a small one present at low concentration if the intrinsic solubility of the latter is much less than that of the former. Manipulation of the solution conditions is expected to improve the separation of a given pair of proteins to the extent that their intrinsic solubilities diverge. Selection of PEG Most workers use material with a nominal average molecular weight in the 4000-6000 range. Polymers larger than this offer no advantage, since
[23]
PRECIPITATION OF PROTEINS WITH P E G
303
their solutions are more viscous and the precipitation curves are not much different from those obtained with PEG 6000. 2,5 Decreasing the molecular weight below 4000 spreads the precipitation of a mixture over a broader range of PEG concentrations. The improved resolution that might be thus anticipated is partially offset by the shallower slopes obtained for individual proteins. Nevertheless, Honig and Kula 8 found the degree of purification of 7-glucosidase from yeast extract to be about 2-fold greater with PEG 400 than with PEG 4000 or 6000. That PEG 400 is a liquid at room temperature whose solutions are substantially less viscous than those of the higher polymers, coupled with the potentially greater ease of removing it by molecular sieve methods, indicates a need for further comparisons. Analytical Precipitation Curve The following simple experiment is designed to quickly overcome ignorance about the amount of PEG required to precipitate a given protein(s) from a complex mixture. The scale of this experiment is dictated by the sensitivity of the assay employed; the availability of a radiolabeled tracer is a definite advantage. One dispenses a fixed amount (0.1-0.5 ml) of the mixture into a series of tubes (preferably in duplicate) to each of which is subsequently added an equal volume of buffer containing increasing amounts of PEG to produce a final concentration of 25-30% in the most concentrated tubes. It is important to buffer the PEG stock solutions to avoid PEG-induced changes in pH. 5,9 The increment in PEG concentration is arbitrary, but 3% (w/v) is adequate for initial screening. The vigor with which one mixes these solutions depends on the extent to which the desired protein(s) can withstand mechanical stress; gentle agitation on a vortex mixer is one approach. After 0.5-1.0 hr of incubation at room temperature or on ice, the samples are centrifuged and the percentage of the desired activity remaining in the supernatant liquid is determined. Inspection of the resulting "analytical precipitation curve" provides an estimate of the maximum concentration of PEG that can be added at one time without precipitating the protein of interest as well as the minimum concentration required to bring it out of solution, parameters that can then be more precisely defined with a second experiment that focuses on the relevant concentration range. With luck, the curve will fall either far to the left or far to the right on the PEG axis, defining a simple 7 A. McPherson, Jr., J. Biol. Chem. 251, 6300 (1976). 8 W. Honig and M.-R. Kula, Anal. Biochem. 72, 502 (1976). 9 G. Eichele, D. Karabelnik, R. Halonbrenner, J. N. Jansonius, and P. Christen, J. Biol. Chem. 253, 5239 (1978).
304
PURIFICATION PROCEDURES: BULK METHODS
[23]
one-step method for removing a large portion of unwanted macromolecules and/or concentrating the desired activity prior to further processing by other methods. Otherwise, it may be necessary to obtain a "PEG cut" via two precipitation steps utilizing in turn the maximum and minimum concentration of PEG referred to above. It is always possible to manipulate the precipitation curve horizontally along the PEG axis by varying solution conditions. For screening purposes, it is expedient to choose a fixed concentration of PEG that causes approximately 50% precipitation of the desired protein under a given set of solution conditions in order to determine rapidly the extent to which altering conditions such as pH and ionic strength might enhance or inhibit precipitation. The most gratifying result of this approach would be to identify substances or conditions that selectively influence the solubility of the desired protein. This concept is further developed in the following section. Influence of Protein-Protein and Protein-Ligand Interactions Studies with purified self-associating and heteroassociating proteins have shed some light on the role of protein-protein interactions on solubility in the presence of P E G ) ,1°-12 Based on the above-mentioned excluded volume considerations, one predicts that conditions that foster protein association should enhance precipitation because of the larger size of the complexes, wheres those that inhibit association would have the opposite effect. This is the case with almost all systems that have been examined. Of particular relevance in the present context was the observation 1° that bovine liver glutamate dehydrogenase at 2.8 mg/ml in 0.2 M potassium phosphate at pH 7.0, conditions known to promote extensive self-association, was quantitatively precipitated by PEG 4000 at concentrations above 15% (w/v). Such precipitation was completely inhibited, even at higher concentration of PEG, by the combined presence of l0 -3 M N A D H and GTP, cofactors known to reverse the self-association. Similar effects were observed with chymotrypsin, chymotrypsinogen, and fl-lactoglobulin A, in which cases self-association was manipulated by varying pH and ionic strength, parameters likely to be less selective. Nonspecific electrostatic interactions between oppositely charged proteins such as albumin and lysozyme can also have profound effects on solubility that are most pronounced at low ionic strength at a pH between the pI of each of the two proteins.ll While such interactions are frequently viewed as a i0 S. I. Miekka and K. C. Ingham, Arch. Biochem. Biophys. 191, 525 (1978). 11 S. I. Miekka and K. C. Ingham, Arch. Biochem. Biophys. 203, 630 (1980). 12 j. Wilf and A. P. Minton, Biochim. Biophys. Acta 671}, 316 (1981).
[23]
PRECIPITATION OF PROTEINS WITH P E G
305
nuisance, to be minimized by maintaining near-physiological ionic strength, the possibility of using them to advantage in a purification scheme should be kept in mind. A more specific type of heteroassociation of the type that might be exploited in purification is the functional interaction between human plasma fibronectin and denatured collagen, i.e., gelatin. The precipitation curve for the plasma protein in phosphate-buffered saline shifted from 11% PEG to less than 3% PEG upon addition of gelatin, which by itself was not precipitated by PEG under these conditions. 13Since the complex between the two proteins is very stable, even at high ionic strength, it should be possible to precipitate fibronectin selectively from a complex mixture by this method. The contaminating gelatin could then be removed, e.g., by ion-exchange chromatography in the presence of urea. Although the advantage of this approach over affinity chromatography on immobilized gelatin is debatable, the example serves as an additional illustration of the application of bioaffinity principles to fractional precipitation. Any substance that interacts specifically with the desired protein has the potential to alter its solubility selectively and should thus be tested. Enzymes are ideal candidates for this approach, since they often interact with one or more effectors or cofactors, sometimes with large changes in the state of association. Methods of Removing PEG In many applications, PEG is used early in the purification scheme and is removed during subsequent chromatographic steps on ion-exchange or affinity columns to which PEG has no tendency to absorb. A word of caution is in order regarding the application of PEG-containing solutions to some exclusion columns, the performance of which can be significantly altered owing to osmotic effects of the polymer. 14Alternative approaches to removing PEG include ultrafiltration ~5,16and salt-induced phase separation ~7as reviewed) 8 The latter method is particularly useful for solutions containing relatively high concentrations of PEG and has the potential advantage that the protein may be concentrated in a low-volume, salt-rich phase. For many research purposes it is probably unnecessary to remove t3 K. C. Ingham, S. A. Brew, and S. I. Miekka, Mol. lmmunol. 20, 287 (1983). 14 K. Hellsing. J. Chromatogr. 36, 170 (1968). 15 T. F. Busby and K. C. Ingham, J. Biochem. Biophys. Methods 2, 191 (1980). 16 K. C. Ingham, T. F. Busby, Y. Sahlestrom, and F. Castino, in "Ultrafiltration Membranes and Applications" (A. R. Cooper, ed.), p. 141. Plenum, New York, 1980. 17 T. F. Busby and K. C. Ingham, Vox Sang. 39, 93 (1980). ~s K. C. Ingham and T. F. Busby, Chem. Eng. Commun. 7, 315 (1980).
306
PURIFICATION PROCEDURES: BULK METHODS
[23]
all traces of polymer from the final product, since it is optically transparent 19 and helps prevent loss of protein by absorption of glass. Summary Polyethylene glycol is a nondenaturing water-soluble polymer whose ability to precipitate protein from aqueous solution can be qualitatively understood in terms of an excluded volume mechanism. The increment in PEG concentration required to effect a given reduction in solubility is unique for a given protein-polymer pair, being insensitive to solution conditions and primarily dependent on the size of the protein and polymer. Selective manipulation of the solubility of specific proteins through control of their state of association or ligand environment can potentially remove some of the empiricism otherwise involved in fractional precipitation. Adequate methods for removing the polymer are available.
19 T h e low level o f U V a b s o r b a n c e frequently found in s o m e PEG preparations is not inherent to the p o l y m e r but is due to a small a m o u n t o f antioxidant s o m e t i m e s added by the m a n u f a c t u r e r .
