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Precipitation of human serum proteins by polyethyleneglycol
CLINICA
BRIEF
CHIMICA ACTA
527
NOTES
Precipitation
of human serum proteins by polyethyleneglycol
Selective precipitation of specific proteins from heterogenous biological fluids is an important and valuable technique in many biochemical purification procedures. At present the most commonly used types of precipitants are inorganic salts, such as (NH&SO, (ref. I), and organic solvents, such as ethano12. The former are somewhat lacking in selectivity, and the latter require strict temperature and pH control. Precipitation of proteins by water-soluble polymers, such as polyethylene glycol, polypropylene glycol, polyvinylpyrrolidone and dextran has been reported by several workers. Polson et al.3 concluded that polyethylene glycol (PEG) of molecular weight 6000 was the most suitable precipitant for general use since its solutions are easy to handle and cause no denaturation of proteins. Although precipitation of proteins with PEG is pH-dependent, control of temperature and ionic strength are unnecessary. Zeppezauer and Brishammert have pointed out in addition that precipitation with PEG is strictly reversible and that the polymers are easily removed by gel filtration. Iverius and Laurent5 have presented some quantitative information on PEG precipitation of several plasma proteins, and their data supported Laurent’s hypothesi+’ which predicted that larger protein molecules would be precipitated more easily than smaller ones. In the present investigation specific antisera to human serum proteins were employed to provide further quantitative information on the precipitation of proteins by PEG. Fresh human serum, pH 8.0, was pooled from several donors and was processed immediately without storage. PEG (mol. wt. 6000), Union Carbide Co., U.S.A., was added to separate aliquots of serum to yield final PEG concentrations ranging from z to 21 o/o (w/v). The serum-PEG mixtures were held at 4’ for z h and were then centrifuged at 4000 rev./min for IO min. The supernatants were saved, and the precipitates were redissolved in 0.1 M Verona1 buffer of pH 8.4. The supernatant and precipitate fractions were then tested for presence of various proteins by double diffusion in agar. LKB immunodiffusion equipment (LKB Produkter, Stockholm, Sweden) was employed, and specific rabbit antisera to eight human serum proteins were purchased from Behringwerke AG, Marburg/L., West Germany. In this system all the antisera used were capable of detecting a minimum specific protein concentration of 0.02 to 0.05 mg/ml. The results are summarized in Table I and Fig. I. The “/b PEG values at which the first detectable decreases in concentrations of the various proteins in the supernatants occurred (Table I, middle column) were in agreement with those values published previously3. All eight proteins tested were detected in the precipitate fractions at very low PEG concentrations, and analysis of these precipitates with rabbit anti-human serum (Fig. I I) showed that these precipitates were quite complex mixtures. This surprising result may not represent a true initial precipitation of all the different proteins at 2-4% PEG, but instead is most likely due to entanglement of minor amounts of the smaller proteins in the precipitates of a,- and p-lipoprotein and IgM. Although, in general, heavier proteins were precipitated first, the order of pre-
528 TABLE
BRIEF NOTES I
Protein
c+ and /?-Lipoproteins IgM xi-Macroglobulin IgG IgA Haptoglobin Transferrin x,-Lipoprotein
l/o PEG (w/v) In&al appearance in ihk precipitate
First detectable decrease in concentratiojz in supernatant
3 2 4 3 3 4 3 3
4 4 10
* Specific protein was detected
9 12 IL 15 15
in supernatants
Total absence fvom supernatant
* *
at all PEG concentrations
tested (z-z I %).
Fig. 1. Double diffusion in agar of various supernatant and precipitate fractions after PEG precipitation of normal human serum. The center wells contain the antisera: A, anti-u,- and B-lipoprotein; B, anti-&M; C, anti-g,M; D, anti-IgG; E, anti-IgA; F, anti-haptoglobin; G, anti-transferrin; H and I, anti-whole human serum. A to H, peripheral wells contain PEG supernatants. I, peripheral wells contain PEG precipitates.
cipitation did not proceed strictly according to molecular weight. For example, a,M (mol. wt. 820000) precipitated at the same PEG concentration (12%) as IgG (mol. wt. 16oooo), and serum IgA (mol. wt. 160000) precipitated somewhat later (140/o). It was possible to cause complete precipitation of several different proteins from serum. Thus one could obtain either a selective removal of undesired low density (a, and 1) lipoproteins at 5% PEG (Fig. I A), or a selective concentration of desired macroglobulins, e.g. ctzM at 12% PEG (Fig. I C). In addition it should be mentioned Clin. Chim. Arta, LO (1968) 527-529
BRIEF
529
NOTES
that PEG of molecular weight 6000 seems to be removed quite effectively from the precipitate fractions by dialysis for extended periods (3-5 days). Thus the viscosity is decreased enough to permit application of the proteins to gel filtration columns without further dilution. In our laboratory selective PEG precipitation of IgM at 7% PEG has proved a valuable aid in concentrating macroglobulins from large volumes of serum prior to further purification by gel filtration and electrophoresis*yg. No loss of IgM antibody activity has been noted in our samples at any time during the precipitation procedure. ACKNOWLEDGEMENTS
This research was supported in part by the Swedish Medical Research Council (Project No. K67-16X-2184-01) and by the foundations “Konung Gustaf V:s Soirsfond” and “Therese and Johan Anderssons Minne”. One of us (B.C.) received a research stipend from the U.S.P.H.S. Medical Student Research Training Program through Harvard Medical School. We thank Mrs. Barbro Andersson for skilled technical assistance. R. CHESEBR~ S.-E. SVEHAG
Department of Immunology, National BacteviologicaELaborntory, Sto~k~o~~~~ ~S~leden~
I F. KENDALL, J. Clin. Invest., 16 (1937) 921. 2 E. COHN cl al., J. Am. Chew SOL, 68 (1946) 459. 3 A. POLSON, G. M. POTGIETER, J. F. LARGIER, G. E. F. MEARS AND F. J. JOUBERT, Biochim. Biophys. Acta, 82 (1964) 463. 4 M. ZEPPEZAUER AND S. BRISHAMMER, Biochim. Biophys. Acta, 94 (1965) 581. 5 P. IVERIUS AND T. LAURENT, Biochim. Biophys. Acta, 133 (1967) 371. 6 T. LAURENT,B~OC~~~. J., 89(1963) 253. 7 T. LAURENT, Acta Chem. Stand., 17 (1963) 2664. 8 S. SVF,HAG ef al.,Science, 158 (1967) 933, 9 B. CHESEBRO et al., J. Exptl. Med., 127 (1908) 399.
Received January 3, 1968 C&z. Chim.
A&,
20 (1968)
527-529
BRIEF
CHIMICA ACTA
527
NOTES
Precipitation
of human serum proteins by polyethyleneglycol
Selective precipitation of specific proteins from heterogenous biological fluids is an important and valuable technique in many biochemical purification procedures. At present the most commonly used types of precipitants are inorganic salts, such as (NH&SO, (ref. I), and organic solvents, such as ethano12. The former are somewhat lacking in selectivity, and the latter require strict temperature and pH control. Precipitation of proteins by water-soluble polymers, such as polyethylene glycol, polypropylene glycol, polyvinylpyrrolidone and dextran has been reported by several workers. Polson et al.3 concluded that polyethylene glycol (PEG) of molecular weight 6000 was the most suitable precipitant for general use since its solutions are easy to handle and cause no denaturation of proteins. Although precipitation of proteins with PEG is pH-dependent, control of temperature and ionic strength are unnecessary. Zeppezauer and Brishammert have pointed out in addition that precipitation with PEG is strictly reversible and that the polymers are easily removed by gel filtration. Iverius and Laurent5 have presented some quantitative information on PEG precipitation of several plasma proteins, and their data supported Laurent’s hypothesi+’ which predicted that larger protein molecules would be precipitated more easily than smaller ones. In the present investigation specific antisera to human serum proteins were employed to provide further quantitative information on the precipitation of proteins by PEG. Fresh human serum, pH 8.0, was pooled from several donors and was processed immediately without storage. PEG (mol. wt. 6000), Union Carbide Co., U.S.A., was added to separate aliquots of serum to yield final PEG concentrations ranging from z to 21 o/o (w/v). The serum-PEG mixtures were held at 4’ for z h and were then centrifuged at 4000 rev./min for IO min. The supernatants were saved, and the precipitates were redissolved in 0.1 M Verona1 buffer of pH 8.4. The supernatant and precipitate fractions were then tested for presence of various proteins by double diffusion in agar. LKB immunodiffusion equipment (LKB Produkter, Stockholm, Sweden) was employed, and specific rabbit antisera to eight human serum proteins were purchased from Behringwerke AG, Marburg/L., West Germany. In this system all the antisera used were capable of detecting a minimum specific protein concentration of 0.02 to 0.05 mg/ml. The results are summarized in Table I and Fig. I. The “/b PEG values at which the first detectable decreases in concentrations of the various proteins in the supernatants occurred (Table I, middle column) were in agreement with those values published previously3. All eight proteins tested were detected in the precipitate fractions at very low PEG concentrations, and analysis of these precipitates with rabbit anti-human serum (Fig. I I) showed that these precipitates were quite complex mixtures. This surprising result may not represent a true initial precipitation of all the different proteins at 2-4% PEG, but instead is most likely due to entanglement of minor amounts of the smaller proteins in the precipitates of a,- and p-lipoprotein and IgM. Although, in general, heavier proteins were precipitated first, the order of pre-
528 TABLE
BRIEF NOTES I
Protein
c+ and /?-Lipoproteins IgM xi-Macroglobulin IgG IgA Haptoglobin Transferrin x,-Lipoprotein
l/o PEG (w/v) In&al appearance in ihk precipitate
First detectable decrease in concentratiojz in supernatant
3 2 4 3 3 4 3 3
4 4 10
* Specific protein was detected
9 12 IL 15 15
in supernatants
Total absence fvom supernatant
* *
at all PEG concentrations
tested (z-z I %).
Fig. 1. Double diffusion in agar of various supernatant and precipitate fractions after PEG precipitation of normal human serum. The center wells contain the antisera: A, anti-u,- and B-lipoprotein; B, anti-&M; C, anti-g,M; D, anti-IgG; E, anti-IgA; F, anti-haptoglobin; G, anti-transferrin; H and I, anti-whole human serum. A to H, peripheral wells contain PEG supernatants. I, peripheral wells contain PEG precipitates.
cipitation did not proceed strictly according to molecular weight. For example, a,M (mol. wt. 820000) precipitated at the same PEG concentration (12%) as IgG (mol. wt. 16oooo), and serum IgA (mol. wt. 160000) precipitated somewhat later (140/o). It was possible to cause complete precipitation of several different proteins from serum. Thus one could obtain either a selective removal of undesired low density (a, and 1) lipoproteins at 5% PEG (Fig. I A), or a selective concentration of desired macroglobulins, e.g. ctzM at 12% PEG (Fig. I C). In addition it should be mentioned Clin. Chim. Arta, LO (1968) 527-529
BRIEF
529
NOTES
that PEG of molecular weight 6000 seems to be removed quite effectively from the precipitate fractions by dialysis for extended periods (3-5 days). Thus the viscosity is decreased enough to permit application of the proteins to gel filtration columns without further dilution. In our laboratory selective PEG precipitation of IgM at 7% PEG has proved a valuable aid in concentrating macroglobulins from large volumes of serum prior to further purification by gel filtration and electrophoresis*yg. No loss of IgM antibody activity has been noted in our samples at any time during the precipitation procedure. ACKNOWLEDGEMENTS
This research was supported in part by the Swedish Medical Research Council (Project No. K67-16X-2184-01) and by the foundations “Konung Gustaf V:s Soirsfond” and “Therese and Johan Anderssons Minne”. One of us (B.C.) received a research stipend from the U.S.P.H.S. Medical Student Research Training Program through Harvard Medical School. We thank Mrs. Barbro Andersson for skilled technical assistance. R. CHESEBR~ S.-E. SVEHAG
Department of Immunology, National BacteviologicaELaborntory, Sto~k~o~~~~ ~S~leden~
I F. KENDALL, J. Clin. Invest., 16 (1937) 921. 2 E. COHN cl al., J. Am. Chew SOL, 68 (1946) 459. 3 A. POLSON, G. M. POTGIETER, J. F. LARGIER, G. E. F. MEARS AND F. J. JOUBERT, Biochim. Biophys. Acta, 82 (1964) 463. 4 M. ZEPPEZAUER AND S. BRISHAMMER, Biochim. Biophys. Acta, 94 (1965) 581. 5 P. IVERIUS AND T. LAURENT, Biochim. Biophys. Acta, 133 (1967) 371. 6 T. LAURENT,B~OC~~~. J., 89(1963) 253. 7 T. LAURENT, Acta Chem. Stand., 17 (1963) 2664. 8 S. SVF,HAG ef al.,Science, 158 (1967) 933, 9 B. CHESEBRO et al., J. Exptl. Med., 127 (1908) 399.
Received January 3, 1968 C&z. Chim.
A&,
20 (1968)
527-529