- Email: [email protected]
High-performance liquid chromatography of proteins by gel permeation chromatography
ANALYTICAL
BIOCHEMISTRY
184-188 (1981)
111,
High-Performance Liquid Chromatography Gel Permeation Chromatography’
of Proteins
by
ROBERT A. JENIK~ANDJOHN W. PORTER Lipid
Metabolism Laboratory, of Physiological
William Chemistry,
S. Middteton Memorial University of Wisconsin,
Veterans Hospital, and the Department Madison, Wisconsin 53706
Received August 5, 1980 The ability of two high-performance liquid chromatography gel permeation columns to separate proteins was evaluated. These columns gave satisfactory molecular weight separations for some, but not all, proteins tested. These results indicate that there are limitations in confidence of molecular weight determinations made by this technique.
High-performance liquid chromatography (hplc)3 of peptides and proteins is a new and rapidly growing technique (1,2). Separation of proteins by this technique by ion exchange, affinity, normal phase, reverse phase and gel permeation modes have been reported. Both normal and reversephase chromatography give excellent resolution of proteins, but require organic solvents for protein fractionation which limits their application for enzymes. It appears, therefore, that gel permeation and ion-exchange chromatography are the methods of choice for the preservation of structure and activity of enzymes. Several columns exist for gel permeation chromatography. This paper reports the results obtained with two of these columns. Ideally, these columns should separate proteins on the basis of their molecular weights. However, not all proteins behave ideally on 1 This investigation was supported in part by a Grant, AM 01383, from the National Institute of Arthritis and Metabolic Diseases of the National Institutes of Health, United States Public Health Service, and the Medical Research Service of the Veterans Administration. * Present address: Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106. 3 Abbreviations used: hplc, high-performance liquid chromatography; SDS, sodium dodecyl sulfate. 0003-2697/81/030184-05$02.00/O Copyright AU rights
B 1981 by Academic Press, Inc. of reproduction in any form reserved.
these columns. This limits the reliability of the technique as a method for determining, the molecular weights of proteins. However, the solute-column interaction can be used to an advantage in some separations. MATERIALS
AND METHODS
The proteins used in this study were purchased from the following sources: ferritin from Boehringer-Mannheim; bovine serum albumin, ovalbumin, chymotrypsinogen A, and ribonuclease A from Pharmacia; soybean trypsin inhibitor from Worthington; glucagon and cytochrome C (type III) from Sigma; insulin from Eli Lilly, and bovine immunoglobulin G from Bio-Rad. Pigeon liver fatty acid synthetase was purified as described previously (3). Blue Dextran 2000 was obtained from Pharmacia, dipotassium ATP from P-L Biochemicals, and dithiothreitol from Calbiochem. The hplc water was purchased from Alltech Associates and J. T. Baker Chemical Company. All other chemicals were reagent grade. The gel permeation chromatography columns used in this study were Waters Associates I-125 protein columns (7.8 mm x 30 cm) and a TSK gel 4000SW column (7.5 mm x 30 cm) from Varian. The Waters I-125 protein column is reported by the
184
PROTEIN
HIGH-PERFORMANCE
LIQUID
CHROMATOGRAPHY
185
column. Samples of 5 to 40 pg in volumes of 5 to 20 ~1 were injected with a Waters Model U6K injector. The column effluent was monitored at 220 or 230 nm with a Perkin-Elmer LC-55 variable wavelength detector or at 254 and 280 nm with a Waters Model 440 absorbance detector.
50 40
RESULTS AND DISCUSSION
c I AW
06 05 04 03 02 I
01’
’
02
’
04
’
0.6
’
08
’
IO
’
12 k’
’
14
’
I6
’
I8
’
20
’
22
’
24
FIG. 1. A comparison of retention, k’, vs the log of the molecular weight for several protein standards on Waters I-125 protein columns. Two Waters I- 125 protein columns were connected in series and eluted at room temperature with 0.1 M potassium phosphate buffer, pH 7.0, at a flow rate of 2 ml/min. Elution of the compounds was monitored by light absorption at 220 nm. The capacity factor, k’, is a measure of retention of a solute on a column and is Riven by the equation: k’ = V, - V,,W,, where V, is the retention volume (i.e., flow rate x time) of the solute of interest and V, is the void volume of the column. V0 was determined with fenitin.
company to have exclusion limits of 2000 to 80,000 M,, and the TSK gel 4000SW column is reported to have exclusion limits of 5000 to l,OOO,OOO M,. A Waters Model 6000A solvent delivery system was used to pump solvents through the columns at flow rates of 1 to 2 ml/min. All buffers were made up in hplc water, passed through a C,, Sep-Pak (Waters), and then filtered and deaerated. All samples were filtered using a MF-1 microfilter (Bioanalytical Systems, Inc.) before injection onto the head of the
In gel permeation chromatography, solutes are separated by size with the larger molecules eluting ahead of the smaller ones. The retention time of a group of standards is plotted in Figs. 1 and 2. Not all of the protein standards exhibit a linear relationship between retention time and molecular weight. It is evident, therefore, that the columns do not function in the size exclusion mode for all compounds and that some type of solute-column interaction is occurring for several. Extraneous mechanisms such as ionic and hydrophobic effects and adsorption may affect the retention of a solute on gel permeation columns (2). Most gel permeation media contain certain amounts of negatively charged groups which give rise to an ion-exclusion effect (4). Ion exclusion is that phenomenon where the diffusion of anionic solutes into the interior of the gel is restricted because there is electrostatic repulsion between the solute and gel matrix (2,4). Hence, when low-ionic-strength buffers are used, the anionic solutes are eluted earlier than are nonionic solutes of similar hydrodynamic volume (2,4-6). On the Waters I-125 protein column there are negatively charged silanol groups on the silica support. Therefore, negatively charged molecules such as ATP or acyl carrier protein will be repelled by the silanol groups, and their molecular weights will be overestimated. Basic molecules such as cytochrome C and chymotrypsinogen A will be retarded or bound by electrostatic adsorption to the silanol groups, resulting in underestimations of their molecular
186
JENIK
AND
PORTER
I 0.4
L 06
I 0.8
lxlsoaoTO6050403020f 0 x E .P s IO-0 : EY 7g 65432-
I
I 0.2
I 1.0
1 1.2
I 1.4
k’
FIG. 2. A comparison of retention, k’, vs the log of the molecular weight for several protein standards on a TSK gel 4000SW column. Samples were eluted with 0.2 M potassium phosphate buffer, pH 7.0, at room temperature and a flow rate of 1 ml/mitt. Elution of the standards was detected by light absorption at 280 nm. k’ was defined in the legend to Fig. 1 and V,, was determined with Blue Dextran 2000.
weights and/or poor recovery from the column, The ion-exclusion effect can be suppressed by increasing the ionic strength of the mobile phase (4-6). This suppression of the ion-exclusion effect is attributed to screening of the charges of the solute and of the gel (4). Increasing the ionic strength also reduces the electrostatic adsorption of cationic proteins. However, the use of highionic-strength buffer does not totally alleviate these ionic effects (2,7). The choice of mobile phase affects the suppression of the ionic effects, Table 1. Acetate buffer is much poorer in suppressing ionic effects than the same concentration of phosphate buffer, and therefore ionic effects are more pronounced with the former. The deviation from a linear relation-
ship between the retention time and molecular weight of the standard proteins is decreased with the use of a phosphate mobile phase. The ability of the phosphate mobile phase to suppress the ionic effects more effectively than the acetate mobile phase is due to the higher ionic strength on equivalent concentration of phosphate buffer. The retention time of some proteins on gel permeation columns is affected by the choice and concentration of the mobile phase. It is apparent, therefore, that the molecular weights for unknown proteins obtained on these columns must be verified by other methods. The recovery of proteins from these columns varied from 60 to 95%. Since hplc is so rapid (~60 min) it appears that these columns would be ideal for chang-
PROTEIN TABLE COMPARISON WATERS
OF RETENTION
I-125
ACETATE
PROTEIN AND
HIGH-PERFORMANCE 1 TIMES
PHOSPHATE
Protein* Bovine serum albumin Ovalbumin Chymotrypsinogen A Soybean trypsin inhibitor Ribonuclease A Cytochrome C Insulin Glucagon
OF PROTEINS
ANALYSIS
COLUMN
ON IN
BUFFERS”
Ammonium acetatee (k’)”
Potassium phosphate’ (k’)”
0.11 0.17 0.70 0.34 0.63 1.15 1.70
0.13 0.20 0.44 0.37 0.46 0.59 1.67 1.20
” Determined on two Waters I-125 protein analysis columns (7.8 mm x 30 cm) in series at room temperature and a flow rate of 2 ml/mm. Protein elution was monitored by light absorption at 230 nm (acetate) or 220 nm (phosphate). * Listed in order of descending molecular weights. ’ 0.1 M, pH 7.0. d k’ is defined in the legend to Fig. 1.
LIQUID
187
CHROMATOGRAPHY
buffer concentration increases the ionic and adsorptive effects of the column, allowing two proteins with small differences in molecular weight to be separated (Fig. 3B). Changing the mobile phase, e.g., substituting ammonium acetate for potassium phosphate and increasing the column length, are also effective in achieving separations with these columns. The hplc on gel permeation columns can be a very useful analytical technique. Figure 4 shows the chromatographic elution pattern of ribonuclease A on a TSK gel 4000SW column. Gel filtration on Sephadex G-50 fine showed only one symmetrical lightabsorbing peak for this preparation of ribonuclease A. Furthermore, Tris/SDS/ urea polyacrylamide disc gel electro0.5-
A
04-
ing buffer systems or desalting samples. For example, due to the negatively charged nature of phosphate and acetate ions under certain experimental conditions and as with low-ionic-strength mobile phases, these salts will be excluded from the pores and comigrate with some proteins. Therefore, it is advisable to check the retention of the protein and buffer components separately before such a separation is attempted. It has been discussed above that the gel permeation columns available for hplc do not operate in the size exclusion model for all compounds. This can be used to an advantage in certain separations. In Fig. 3 the ionic and adsorptive effects of the I-125 protein column have been used to bring about the separation of ribonuclease A and cytochrome C. When 0.1 M potassium phosphate buffer, pH 7.0, is used, the ionic and adsorptive effects are minimized and the column functions mainly in a size exclusion mode (Fig. 3A). Decreasing the ionic strength of the mobile phase by lowering the
E
= 03R N t Y 3 B 02.
02.
z
or-
01 1
‘I(
o,-JL1L-J ,& 12345678
RETENTION
12345678 TIME Imtn)
FIG. 3. Separation of ribonuclease A and cytochrome C as a function of ionic strength of the mobile phase. Ribonuclease A (30&g) and cytochrome C (40 pg) were injected in a volume of 15 ~1 onto a single Waters I-125 protein column and eluted with (A) 0.1 M potassium phosphate buffer, pH 7.0, and (B) 0.04 M potassium phosphate buffer, pH 7.0, at a flow rate of 2 ml/mitt.
I
188
JENIK AND PORTER
FC )
E
-Y
-c 1.005 4 N 5 -c I.010 y a 8 -c b.015 w z 1020
0.025 t
to analytical (micrograms) separations. One of the problems of gel permeation chromatography is the volume of the protein solution that can be injected onto the column. The best resolution is obtained on these columns when samples are injected in volumes smaller than 50 ~1. As the sample volume increases peak spreading occurs. After approximately 300 ~1 peak spreading interferes greatly with the resolution and separation of protein mixtures. It appears that hplc on ion-exchange or reverse-phase supports is better suited for semipreparative work on peptides and proteins (1,2,8). A disadvantage of reverse-phase hplc is the instability of most enzymes in the organic solvents used for elution. ACKNOWLEDGMENT
ki a0.005-
The authors would like to thank Dr. Kuni Takayama for making his hplc equipment available to us. o 2
4
1 1 I I ( 6 6 IO 12 14 16 I6 20 2; RETENTION TIME lmin)
4. Chromatography of ribonuclease A on a TSK gel 4000SW column. Ribonuclease A, 30 eg, was eluted from the column with 0.2 M potassium phosphate buffer, pH 7.0, at a flow rate of 1 ml/min. FIG.
phoresis showed only a single electrophoretic component with a molecular weight of approximately 14,000. From the hplc chromatogram (Fig. 4) it is noted that there is another component present in the ribonuclease A preparation. The amount of protein that can be applied to gel permeation columns is limited, and therefore this technique is usually confined
REFERENCES 1. Rubinstein, M. (1979) Anal. Biochem. 98, l-7. 2. Regnier, F. E., and Gooding, K. M. (1980) Anal. Biochem. 103, l-25. 3. Muesing, R. A., and Porter, J. W. (1975) in Methods in Enzymology (Lowenstein, J. M., ed.), Vol. 35, pp. 45-59, Academic Press, New York. 4. Stenlund, B. (1976) Advan. Chromatogr. 14, 37-74. 5. Crone, H. D., and Dawson, R. M. (1976) J. Chromntogr. 129, 91-96. 6. Crone, H. D. (1974) J. Chromatogr. 92, 127- 135. 7. Rokushika, S., Ohkawa, T., and Hatano, H. (1979) J. Chromatogr. 176, 456-461. 8. Lewis, R. V., Fallon, A., Stein, S., Gibson, K. D., and Undenfriend, S. (1980) Anal. Biochem. 104, 153-159.
BIOCHEMISTRY
184-188 (1981)
111,
High-Performance Liquid Chromatography Gel Permeation Chromatography’
of Proteins
by
ROBERT A. JENIK~ANDJOHN W. PORTER Lipid
Metabolism Laboratory, of Physiological
William Chemistry,
S. Middteton Memorial University of Wisconsin,
Veterans Hospital, and the Department Madison, Wisconsin 53706
Received August 5, 1980 The ability of two high-performance liquid chromatography gel permeation columns to separate proteins was evaluated. These columns gave satisfactory molecular weight separations for some, but not all, proteins tested. These results indicate that there are limitations in confidence of molecular weight determinations made by this technique.
High-performance liquid chromatography (hplc)3 of peptides and proteins is a new and rapidly growing technique (1,2). Separation of proteins by this technique by ion exchange, affinity, normal phase, reverse phase and gel permeation modes have been reported. Both normal and reversephase chromatography give excellent resolution of proteins, but require organic solvents for protein fractionation which limits their application for enzymes. It appears, therefore, that gel permeation and ion-exchange chromatography are the methods of choice for the preservation of structure and activity of enzymes. Several columns exist for gel permeation chromatography. This paper reports the results obtained with two of these columns. Ideally, these columns should separate proteins on the basis of their molecular weights. However, not all proteins behave ideally on 1 This investigation was supported in part by a Grant, AM 01383, from the National Institute of Arthritis and Metabolic Diseases of the National Institutes of Health, United States Public Health Service, and the Medical Research Service of the Veterans Administration. * Present address: Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106. 3 Abbreviations used: hplc, high-performance liquid chromatography; SDS, sodium dodecyl sulfate. 0003-2697/81/030184-05$02.00/O Copyright AU rights
B 1981 by Academic Press, Inc. of reproduction in any form reserved.
these columns. This limits the reliability of the technique as a method for determining, the molecular weights of proteins. However, the solute-column interaction can be used to an advantage in some separations. MATERIALS
AND METHODS
The proteins used in this study were purchased from the following sources: ferritin from Boehringer-Mannheim; bovine serum albumin, ovalbumin, chymotrypsinogen A, and ribonuclease A from Pharmacia; soybean trypsin inhibitor from Worthington; glucagon and cytochrome C (type III) from Sigma; insulin from Eli Lilly, and bovine immunoglobulin G from Bio-Rad. Pigeon liver fatty acid synthetase was purified as described previously (3). Blue Dextran 2000 was obtained from Pharmacia, dipotassium ATP from P-L Biochemicals, and dithiothreitol from Calbiochem. The hplc water was purchased from Alltech Associates and J. T. Baker Chemical Company. All other chemicals were reagent grade. The gel permeation chromatography columns used in this study were Waters Associates I-125 protein columns (7.8 mm x 30 cm) and a TSK gel 4000SW column (7.5 mm x 30 cm) from Varian. The Waters I-125 protein column is reported by the
184
PROTEIN
HIGH-PERFORMANCE
LIQUID
CHROMATOGRAPHY
185
column. Samples of 5 to 40 pg in volumes of 5 to 20 ~1 were injected with a Waters Model U6K injector. The column effluent was monitored at 220 or 230 nm with a Perkin-Elmer LC-55 variable wavelength detector or at 254 and 280 nm with a Waters Model 440 absorbance detector.
50 40
RESULTS AND DISCUSSION
c I AW
06 05 04 03 02 I
01’
’
02
’
04
’
0.6
’
08
’
IO
’
12 k’
’
14
’
I6
’
I8
’
20
’
22
’
24
FIG. 1. A comparison of retention, k’, vs the log of the molecular weight for several protein standards on Waters I-125 protein columns. Two Waters I- 125 protein columns were connected in series and eluted at room temperature with 0.1 M potassium phosphate buffer, pH 7.0, at a flow rate of 2 ml/min. Elution of the compounds was monitored by light absorption at 220 nm. The capacity factor, k’, is a measure of retention of a solute on a column and is Riven by the equation: k’ = V, - V,,W,, where V, is the retention volume (i.e., flow rate x time) of the solute of interest and V, is the void volume of the column. V0 was determined with fenitin.
company to have exclusion limits of 2000 to 80,000 M,, and the TSK gel 4000SW column is reported to have exclusion limits of 5000 to l,OOO,OOO M,. A Waters Model 6000A solvent delivery system was used to pump solvents through the columns at flow rates of 1 to 2 ml/min. All buffers were made up in hplc water, passed through a C,, Sep-Pak (Waters), and then filtered and deaerated. All samples were filtered using a MF-1 microfilter (Bioanalytical Systems, Inc.) before injection onto the head of the
In gel permeation chromatography, solutes are separated by size with the larger molecules eluting ahead of the smaller ones. The retention time of a group of standards is plotted in Figs. 1 and 2. Not all of the protein standards exhibit a linear relationship between retention time and molecular weight. It is evident, therefore, that the columns do not function in the size exclusion mode for all compounds and that some type of solute-column interaction is occurring for several. Extraneous mechanisms such as ionic and hydrophobic effects and adsorption may affect the retention of a solute on gel permeation columns (2). Most gel permeation media contain certain amounts of negatively charged groups which give rise to an ion-exclusion effect (4). Ion exclusion is that phenomenon where the diffusion of anionic solutes into the interior of the gel is restricted because there is electrostatic repulsion between the solute and gel matrix (2,4). Hence, when low-ionic-strength buffers are used, the anionic solutes are eluted earlier than are nonionic solutes of similar hydrodynamic volume (2,4-6). On the Waters I-125 protein column there are negatively charged silanol groups on the silica support. Therefore, negatively charged molecules such as ATP or acyl carrier protein will be repelled by the silanol groups, and their molecular weights will be overestimated. Basic molecules such as cytochrome C and chymotrypsinogen A will be retarded or bound by electrostatic adsorption to the silanol groups, resulting in underestimations of their molecular
186
JENIK
AND
PORTER
I 0.4
L 06
I 0.8
lxlsoaoTO6050403020f 0 x E .P s IO-0 : EY 7g 65432-
I
I 0.2
I 1.0
1 1.2
I 1.4
k’
FIG. 2. A comparison of retention, k’, vs the log of the molecular weight for several protein standards on a TSK gel 4000SW column. Samples were eluted with 0.2 M potassium phosphate buffer, pH 7.0, at room temperature and a flow rate of 1 ml/mitt. Elution of the standards was detected by light absorption at 280 nm. k’ was defined in the legend to Fig. 1 and V,, was determined with Blue Dextran 2000.
weights and/or poor recovery from the column, The ion-exclusion effect can be suppressed by increasing the ionic strength of the mobile phase (4-6). This suppression of the ion-exclusion effect is attributed to screening of the charges of the solute and of the gel (4). Increasing the ionic strength also reduces the electrostatic adsorption of cationic proteins. However, the use of highionic-strength buffer does not totally alleviate these ionic effects (2,7). The choice of mobile phase affects the suppression of the ionic effects, Table 1. Acetate buffer is much poorer in suppressing ionic effects than the same concentration of phosphate buffer, and therefore ionic effects are more pronounced with the former. The deviation from a linear relation-
ship between the retention time and molecular weight of the standard proteins is decreased with the use of a phosphate mobile phase. The ability of the phosphate mobile phase to suppress the ionic effects more effectively than the acetate mobile phase is due to the higher ionic strength on equivalent concentration of phosphate buffer. The retention time of some proteins on gel permeation columns is affected by the choice and concentration of the mobile phase. It is apparent, therefore, that the molecular weights for unknown proteins obtained on these columns must be verified by other methods. The recovery of proteins from these columns varied from 60 to 95%. Since hplc is so rapid (~60 min) it appears that these columns would be ideal for chang-
PROTEIN TABLE COMPARISON WATERS
OF RETENTION
I-125
ACETATE
PROTEIN AND
HIGH-PERFORMANCE 1 TIMES
PHOSPHATE
Protein* Bovine serum albumin Ovalbumin Chymotrypsinogen A Soybean trypsin inhibitor Ribonuclease A Cytochrome C Insulin Glucagon
OF PROTEINS
ANALYSIS
COLUMN
ON IN
BUFFERS”
Ammonium acetatee (k’)”
Potassium phosphate’ (k’)”
0.11 0.17 0.70 0.34 0.63 1.15 1.70
0.13 0.20 0.44 0.37 0.46 0.59 1.67 1.20
” Determined on two Waters I-125 protein analysis columns (7.8 mm x 30 cm) in series at room temperature and a flow rate of 2 ml/mm. Protein elution was monitored by light absorption at 230 nm (acetate) or 220 nm (phosphate). * Listed in order of descending molecular weights. ’ 0.1 M, pH 7.0. d k’ is defined in the legend to Fig. 1.
LIQUID
187
CHROMATOGRAPHY
buffer concentration increases the ionic and adsorptive effects of the column, allowing two proteins with small differences in molecular weight to be separated (Fig. 3B). Changing the mobile phase, e.g., substituting ammonium acetate for potassium phosphate and increasing the column length, are also effective in achieving separations with these columns. The hplc on gel permeation columns can be a very useful analytical technique. Figure 4 shows the chromatographic elution pattern of ribonuclease A on a TSK gel 4000SW column. Gel filtration on Sephadex G-50 fine showed only one symmetrical lightabsorbing peak for this preparation of ribonuclease A. Furthermore, Tris/SDS/ urea polyacrylamide disc gel electro0.5-
A
04-
ing buffer systems or desalting samples. For example, due to the negatively charged nature of phosphate and acetate ions under certain experimental conditions and as with low-ionic-strength mobile phases, these salts will be excluded from the pores and comigrate with some proteins. Therefore, it is advisable to check the retention of the protein and buffer components separately before such a separation is attempted. It has been discussed above that the gel permeation columns available for hplc do not operate in the size exclusion model for all compounds. This can be used to an advantage in certain separations. In Fig. 3 the ionic and adsorptive effects of the I-125 protein column have been used to bring about the separation of ribonuclease A and cytochrome C. When 0.1 M potassium phosphate buffer, pH 7.0, is used, the ionic and adsorptive effects are minimized and the column functions mainly in a size exclusion mode (Fig. 3A). Decreasing the ionic strength of the mobile phase by lowering the
E
= 03R N t Y 3 B 02.
02.
z
or-
01 1
‘I(
o,-JL1L-J ,& 12345678
RETENTION
12345678 TIME Imtn)
FIG. 3. Separation of ribonuclease A and cytochrome C as a function of ionic strength of the mobile phase. Ribonuclease A (30&g) and cytochrome C (40 pg) were injected in a volume of 15 ~1 onto a single Waters I-125 protein column and eluted with (A) 0.1 M potassium phosphate buffer, pH 7.0, and (B) 0.04 M potassium phosphate buffer, pH 7.0, at a flow rate of 2 ml/mitt.
I
188
JENIK AND PORTER
FC )
E
-Y
-c 1.005 4 N 5 -c I.010 y a 8 -c b.015 w z 1020
0.025 t
to analytical (micrograms) separations. One of the problems of gel permeation chromatography is the volume of the protein solution that can be injected onto the column. The best resolution is obtained on these columns when samples are injected in volumes smaller than 50 ~1. As the sample volume increases peak spreading occurs. After approximately 300 ~1 peak spreading interferes greatly with the resolution and separation of protein mixtures. It appears that hplc on ion-exchange or reverse-phase supports is better suited for semipreparative work on peptides and proteins (1,2,8). A disadvantage of reverse-phase hplc is the instability of most enzymes in the organic solvents used for elution. ACKNOWLEDGMENT
ki a0.005-
The authors would like to thank Dr. Kuni Takayama for making his hplc equipment available to us. o 2
4
1 1 I I ( 6 6 IO 12 14 16 I6 20 2; RETENTION TIME lmin)
4. Chromatography of ribonuclease A on a TSK gel 4000SW column. Ribonuclease A, 30 eg, was eluted from the column with 0.2 M potassium phosphate buffer, pH 7.0, at a flow rate of 1 ml/min. FIG.
phoresis showed only a single electrophoretic component with a molecular weight of approximately 14,000. From the hplc chromatogram (Fig. 4) it is noted that there is another component present in the ribonuclease A preparation. The amount of protein that can be applied to gel permeation columns is limited, and therefore this technique is usually confined
REFERENCES 1. Rubinstein, M. (1979) Anal. Biochem. 98, l-7. 2. Regnier, F. E., and Gooding, K. M. (1980) Anal. Biochem. 103, l-25. 3. Muesing, R. A., and Porter, J. W. (1975) in Methods in Enzymology (Lowenstein, J. M., ed.), Vol. 35, pp. 45-59, Academic Press, New York. 4. Stenlund, B. (1976) Advan. Chromatogr. 14, 37-74. 5. Crone, H. D., and Dawson, R. M. (1976) J. Chromntogr. 129, 91-96. 6. Crone, H. D. (1974) J. Chromatogr. 92, 127- 135. 7. Rokushika, S., Ohkawa, T., and Hatano, H. (1979) J. Chromatogr. 176, 456-461. 8. Lewis, R. V., Fallon, A., Stein, S., Gibson, K. D., and Undenfriend, S. (1980) Anal. Biochem. 104, 153-159.