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High-performance size-exclusion chromatography: A buffer for the reliable determination of molecular weights of proteins
ANALYTICAL
BIOCHEMISTRY
121,
378-381 (1982)
High-Performance Size-Exclusion Chromatography: A Buffer for the Reliable Determination of Molecular Weights of Proteins FRANZ HEFTI Max-Planck-Institut
fiir
Psychiatric,
Abteilung
Neurochemie,
Martinvied,
Federal
Republic
of Germany
Received October 6, 1981 A high-performance size-exclusion chromatographic system is described that minimizes the ionic and hydrophobic interactions of proteins with the stationary phase. The system can be used to determine reliably the molecular weight of proteins between M, 10,000 and 70,000. A further application would be to separate proteins on a preparative (milligram) scale. Conditions critical for optimal resolution are discussed.
As compared to classical chromatography, high-performance liquid chromatography (HPLC)’ offers the advantages of a higher speed and of an improved quality of separation. This statement holds true for almost all compounds of low molecular weight, including proteins with molecular weights up to approximately 10,000. With larger proteins, however, successful applications of high-performance liquid chromatography are still rather scarce. The application of HPLC for such molecules seems most developed in the field of size-exclusion chromatography, for which several types of columns are commercially available (l-3). Unfortunately, these columns all exhibit a considerable degree of nonspecific retention of proteins, which limits their applicability for the determination of molecular weights (2). In the present report, I describe a mobile phase optimized to avoid such nonspecific interactions between proteins and the stationary phase. This high-performance size-exclusion chromatography system can determine reliably the molecular weights of proteins between 10,000 and 70,000 and is also useful for preparative separations. ’ Abbreviation used: HPLC, high-performance liquid chromatography. 0003-2697/82/060378-04$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
MATERIALS AND METHODS
The chromatographic equipment used consisted of a 6000A pump (Waters), a U6K loop injection valve (Waters), a 7.8 X 300mm I- 125 size-exclusion column (Waters), a variable-wavelength detector (LC-UV, Pye-Unicam) routinely set at 280 nm, and a chart recorder (Linseis Ltd., Selb, FRG). The mobile phase was a 0.08 M sodium phosphate buffer, pH 7.0, containing 0.32 M sodium chloride and 20% (v/v) ethanol (Merck, analytical grade). This buffer was filtered and degassed before use. Flow rates were between 0.1 and 2.0 ml/min and the pressure ranged accordingly from 50 to 1500 psi; the system was kept at room temperature. For injection, proteins were dissolved in the mobile phase. The effluent was collected manually for quantitative determination of recovery. Proteins were measured using the method of Lowry et al. with bovine serum albumin as standard (4). They were obtained from the following sources: aprotinin, avidin, isocitrate dehydrogenase, P-lactoglobulin, myoglobin, pepsin, horseradish peroxidase, trypsinogen (bovine pancreas), and soya bean trypsin inhibitor were from Sigma; catalase, bovine serum albumin (BSA), chymotrypsin, cytochrome c, chymotrypsinogen A, ferritin, hyaluronidase, and ovalbumin 378
HIGH-PERFORMANCE
SIZE-EXCLUSION
CHROMATOGRAPHY
379
OF PROTEINS
lo5 -
FIG. 1. Linear relationship between retention times and logarithm of molecular weights for standard proteins. An I-125 size-exclusion column (Waters) was used with a mobile phase of 0.08 M sodium phosphate buffer, pH 7.0, containing 0.32 M sodium chloride and 20% (v/v) ethanol (flow rate, 1 ml/ mitt; pressure, 400-600 psi; elution detected by monitoring at 280 nm). The void volume (V,) was determined with ferritin, the total volume (V,) with ‘Hz0 and with the tripeptide Tyr-Gly-Gly. The correlation curve was fitted using the least-squares method (correlation coefficient, r = 0.97).
were from Boehringer (Mannheim, FRG), and the tripeptide Tyr-Gly-Gly was from Serva (Heidelberg, FRG). RESULTS
AND DISCUSSION
On most commercially available high-performance size-exclusion columns run with simple phosphate buffers, many proteins are retained longer than would be expected on the basis of their molecular weights (2). Most probably, such increased retention occurs because of ionic and hydrophobic interactions with the stationary phase. In the mobile phase described in the present report, ionic interactions are prevented by a high concentration of ions; hydrophobic interactions are minimized by the addition of ethanol. The use of ethanol proved much superior to that of detergents (Triton X- 100, deoxycholate; data not shown). Using this mobile phase on the I-l 25 column, an excellent linear correlation was obtained between the retention time and the logarithm of the molecular weight of proteins between kf, 10,000 and 70,000 (Fig. 1). All proteins tested were eluted from the column with re-
coveries of around 80-90’S (Table 1). The addition of both a high salt concentration and ethanol was necessary to obtain high recoveries and reliable retention times. Many standard proteins eluted with longer retenTABLE RECOVERIES
Standard
I
OF STANDARD
protein
Cytochrome c Myoglobin Soya bean trypsin inhibitor Pepsin Ovalbumin Isocitrate dehydrogenase BSA
PROTEINS” Recovery (W) 94.3 94. I 93.0 81.2 80. I 86.2 78.7
’ Proteins were run on an I-l 25 column (Waters) with a 0.08 M sodium phosphate buffer, pH 7.0, containing 0.32 M sodium chloride and 20% (v/v) ethanol (flow rate, 0.5 ml/min). Five hundred micrograms of each protein was injected, elution of the proteins was monitored at 280 nm and the eluate corresponding to the absorbance peak was collected manually. Proteins were determined according to Lowry et al. using bovine serum albumin as standard (4). Standard proteins are listed according to their molecular weight.
FRANZ
E
, EC
D
HEFT1
I
1w
50
0
25
20
15
lo
5
0
6
L
2
0
MINUTES
FIG. 2. Relationship between resolution and flow rate. Chromatographic conditions were as described in Fig. 1. Flow rates were varied as indicated in the figure. A mixture of 50 rg catalase (A), 50 rg BSA (B), 50 pg chymotrypsin (C), 25 rg cytochrome c (D), and 50 pg aprotinin (E) was injected in 200 ~1 of buffer in each case.
tion times than were predicted on the basis of their molecular weights when ethanol or sodium chloride was omitted from the mobile phase. Some proteins tested contain significant amounts of carbohydrates (avidin, 11%; ovalbumin, 3%; peroxidase, 16%); they, nevertheless, eluted according to their molecular weights (Fig. 1). Carbohydrate chains therefore do not seem to alter the elution behavior of the proteins, even though they contribute to their molecular weights. The high-performance size-exclusion chro-
matography system described in this report may also be useful for small-scale preparative separations (Fig. 2). The quality of separation is affected by two major factors: (i) Decreasing the flow rate produces better resolution and sharper peaks (Fig. 2). This effect is readily explained as due to the slow mass transfer of proteins between the mobile and stationary phases (4). (ii) The resolution decreases when the volume in which the sample is injected is increased (Fig. 3). The injection volume appears to be more critical
A
D c
P I 150
lrn
M
0
L
k I 1M
ICC
0 I so
I 0
MINUTES
FIG. 3. Relationship between resolution and injection volume. Chromatographic conditions were as described in Fig. 1. Flow rate: 0.1 ml/min. A mixture of 50 fig BSA (A), 50 pg chymotrypsin (B), 25 pg cytochrome c (C), and 50 pg aprotinin (D) was injected in a volume of 20 pl of buffer (first injection) and in 1 ml of buffer (second injection).
HIGH-PERFORMANCE
SIZE-EXCLUSION
than the flow rate for good resolution; excellent separations were achieved only when injection volumes were lower than 500 ~1, which may necessitate concentration of the sample before injection. The quality of separation is also limited by the amount of protein applied. Using the same protein mixture as that shown in Fig. 2, a good separation was obtained when up to 2 mg of each protein was injected. A successful separation of proteins using high-performance size-exclusion chromatography has recently been reported; separation was achieved by deliberately using the nonspecific retention of one compound (2). In our experience, separations relying on nonspecific retention are difficult to reproduce. They vary especially with the amount of protein applied to the column; only minimal amounts can be separated this way, probably because the column has only a limited amount of nonspecific binding sites. Although separations based on nonspecific re-
CHROMATOGRAPHY
381
OF PROTEINS
tention can be achieved, it is also advantageous to work with a system purely based on size exclusion because the behavior of such a system can be predicted reliably. ACKNOWLEDGMENTS The author thanks Mrs. C. Cap for excellent technical assistance and Dr. Y. A. Barde for suggestions and criticisms. The study was carried out in the laboratory of Dr. H. Thoenen, whose interest and encouragement are gratefully acknowledged. The author was supported by the Alexander von Humboldt Stiftung, Bonn, FRG.
REFERENCES 1. Regnier,
F. E., and Gooding, 103, l-25. R. A., and Porter,
K. M.
(1980)
Anal.
Biochem.
2. Jenik,
Biochem.
3. Kimura,
111,
J. W.
(1981)
Anal.
184-188.
M., and Hotawa, Y. (198 I ) J. Chroma211, 290-294. 4. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265275. 5. Jones, B. N., Lewis, R. V., Paabo, S., Kojima, K., Kimura, S., and Stein, S. (1980) 1. Liq. Chromatogr. 3, 1373-l 383. togr.
BIOCHEMISTRY
121,
378-381 (1982)
High-Performance Size-Exclusion Chromatography: A Buffer for the Reliable Determination of Molecular Weights of Proteins FRANZ HEFTI Max-Planck-Institut
fiir
Psychiatric,
Abteilung
Neurochemie,
Martinvied,
Federal
Republic
of Germany
Received October 6, 1981 A high-performance size-exclusion chromatographic system is described that minimizes the ionic and hydrophobic interactions of proteins with the stationary phase. The system can be used to determine reliably the molecular weight of proteins between M, 10,000 and 70,000. A further application would be to separate proteins on a preparative (milligram) scale. Conditions critical for optimal resolution are discussed.
As compared to classical chromatography, high-performance liquid chromatography (HPLC)’ offers the advantages of a higher speed and of an improved quality of separation. This statement holds true for almost all compounds of low molecular weight, including proteins with molecular weights up to approximately 10,000. With larger proteins, however, successful applications of high-performance liquid chromatography are still rather scarce. The application of HPLC for such molecules seems most developed in the field of size-exclusion chromatography, for which several types of columns are commercially available (l-3). Unfortunately, these columns all exhibit a considerable degree of nonspecific retention of proteins, which limits their applicability for the determination of molecular weights (2). In the present report, I describe a mobile phase optimized to avoid such nonspecific interactions between proteins and the stationary phase. This high-performance size-exclusion chromatography system can determine reliably the molecular weights of proteins between 10,000 and 70,000 and is also useful for preparative separations. ’ Abbreviation used: HPLC, high-performance liquid chromatography. 0003-2697/82/060378-04$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
MATERIALS AND METHODS
The chromatographic equipment used consisted of a 6000A pump (Waters), a U6K loop injection valve (Waters), a 7.8 X 300mm I- 125 size-exclusion column (Waters), a variable-wavelength detector (LC-UV, Pye-Unicam) routinely set at 280 nm, and a chart recorder (Linseis Ltd., Selb, FRG). The mobile phase was a 0.08 M sodium phosphate buffer, pH 7.0, containing 0.32 M sodium chloride and 20% (v/v) ethanol (Merck, analytical grade). This buffer was filtered and degassed before use. Flow rates were between 0.1 and 2.0 ml/min and the pressure ranged accordingly from 50 to 1500 psi; the system was kept at room temperature. For injection, proteins were dissolved in the mobile phase. The effluent was collected manually for quantitative determination of recovery. Proteins were measured using the method of Lowry et al. with bovine serum albumin as standard (4). They were obtained from the following sources: aprotinin, avidin, isocitrate dehydrogenase, P-lactoglobulin, myoglobin, pepsin, horseradish peroxidase, trypsinogen (bovine pancreas), and soya bean trypsin inhibitor were from Sigma; catalase, bovine serum albumin (BSA), chymotrypsin, cytochrome c, chymotrypsinogen A, ferritin, hyaluronidase, and ovalbumin 378
HIGH-PERFORMANCE
SIZE-EXCLUSION
CHROMATOGRAPHY
379
OF PROTEINS
lo5 -
FIG. 1. Linear relationship between retention times and logarithm of molecular weights for standard proteins. An I-125 size-exclusion column (Waters) was used with a mobile phase of 0.08 M sodium phosphate buffer, pH 7.0, containing 0.32 M sodium chloride and 20% (v/v) ethanol (flow rate, 1 ml/ mitt; pressure, 400-600 psi; elution detected by monitoring at 280 nm). The void volume (V,) was determined with ferritin, the total volume (V,) with ‘Hz0 and with the tripeptide Tyr-Gly-Gly. The correlation curve was fitted using the least-squares method (correlation coefficient, r = 0.97).
were from Boehringer (Mannheim, FRG), and the tripeptide Tyr-Gly-Gly was from Serva (Heidelberg, FRG). RESULTS
AND DISCUSSION
On most commercially available high-performance size-exclusion columns run with simple phosphate buffers, many proteins are retained longer than would be expected on the basis of their molecular weights (2). Most probably, such increased retention occurs because of ionic and hydrophobic interactions with the stationary phase. In the mobile phase described in the present report, ionic interactions are prevented by a high concentration of ions; hydrophobic interactions are minimized by the addition of ethanol. The use of ethanol proved much superior to that of detergents (Triton X- 100, deoxycholate; data not shown). Using this mobile phase on the I-l 25 column, an excellent linear correlation was obtained between the retention time and the logarithm of the molecular weight of proteins between kf, 10,000 and 70,000 (Fig. 1). All proteins tested were eluted from the column with re-
coveries of around 80-90’S (Table 1). The addition of both a high salt concentration and ethanol was necessary to obtain high recoveries and reliable retention times. Many standard proteins eluted with longer retenTABLE RECOVERIES
Standard
I
OF STANDARD
protein
Cytochrome c Myoglobin Soya bean trypsin inhibitor Pepsin Ovalbumin Isocitrate dehydrogenase BSA
PROTEINS” Recovery (W) 94.3 94. I 93.0 81.2 80. I 86.2 78.7
’ Proteins were run on an I-l 25 column (Waters) with a 0.08 M sodium phosphate buffer, pH 7.0, containing 0.32 M sodium chloride and 20% (v/v) ethanol (flow rate, 0.5 ml/min). Five hundred micrograms of each protein was injected, elution of the proteins was monitored at 280 nm and the eluate corresponding to the absorbance peak was collected manually. Proteins were determined according to Lowry et al. using bovine serum albumin as standard (4). Standard proteins are listed according to their molecular weight.
FRANZ
E
, EC
D
HEFT1
I
1w
50
0
25
20
15
lo
5
0
6
L
2
0
MINUTES
FIG. 2. Relationship between resolution and flow rate. Chromatographic conditions were as described in Fig. 1. Flow rates were varied as indicated in the figure. A mixture of 50 rg catalase (A), 50 rg BSA (B), 50 pg chymotrypsin (C), 25 rg cytochrome c (D), and 50 pg aprotinin (E) was injected in 200 ~1 of buffer in each case.
tion times than were predicted on the basis of their molecular weights when ethanol or sodium chloride was omitted from the mobile phase. Some proteins tested contain significant amounts of carbohydrates (avidin, 11%; ovalbumin, 3%; peroxidase, 16%); they, nevertheless, eluted according to their molecular weights (Fig. 1). Carbohydrate chains therefore do not seem to alter the elution behavior of the proteins, even though they contribute to their molecular weights. The high-performance size-exclusion chro-
matography system described in this report may also be useful for small-scale preparative separations (Fig. 2). The quality of separation is affected by two major factors: (i) Decreasing the flow rate produces better resolution and sharper peaks (Fig. 2). This effect is readily explained as due to the slow mass transfer of proteins between the mobile and stationary phases (4). (ii) The resolution decreases when the volume in which the sample is injected is increased (Fig. 3). The injection volume appears to be more critical
A
D c
P I 150
lrn
M
0
L
k I 1M
ICC
0 I so
I 0
MINUTES
FIG. 3. Relationship between resolution and injection volume. Chromatographic conditions were as described in Fig. 1. Flow rate: 0.1 ml/min. A mixture of 50 fig BSA (A), 50 pg chymotrypsin (B), 25 pg cytochrome c (C), and 50 pg aprotinin (D) was injected in a volume of 20 pl of buffer (first injection) and in 1 ml of buffer (second injection).
HIGH-PERFORMANCE
SIZE-EXCLUSION
than the flow rate for good resolution; excellent separations were achieved only when injection volumes were lower than 500 ~1, which may necessitate concentration of the sample before injection. The quality of separation is also limited by the amount of protein applied. Using the same protein mixture as that shown in Fig. 2, a good separation was obtained when up to 2 mg of each protein was injected. A successful separation of proteins using high-performance size-exclusion chromatography has recently been reported; separation was achieved by deliberately using the nonspecific retention of one compound (2). In our experience, separations relying on nonspecific retention are difficult to reproduce. They vary especially with the amount of protein applied to the column; only minimal amounts can be separated this way, probably because the column has only a limited amount of nonspecific binding sites. Although separations based on nonspecific re-
CHROMATOGRAPHY
381
OF PROTEINS
tention can be achieved, it is also advantageous to work with a system purely based on size exclusion because the behavior of such a system can be predicted reliably. ACKNOWLEDGMENTS The author thanks Mrs. C. Cap for excellent technical assistance and Dr. Y. A. Barde for suggestions and criticisms. The study was carried out in the laboratory of Dr. H. Thoenen, whose interest and encouragement are gratefully acknowledged. The author was supported by the Alexander von Humboldt Stiftung, Bonn, FRG.
REFERENCES 1. Regnier,
F. E., and Gooding, 103, l-25. R. A., and Porter,
K. M.
(1980)
Anal.
Biochem.
2. Jenik,
Biochem.
3. Kimura,
111,
J. W.
(1981)
Anal.
184-188.
M., and Hotawa, Y. (198 I ) J. Chroma211, 290-294. 4. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265275. 5. Jones, B. N., Lewis, R. V., Paabo, S., Kojima, K., Kimura, S., and Stein, S. (1980) 1. Liq. Chromatogr. 3, 1373-l 383. togr.