ANALYTICAL
BIOCHEMISTRY
72, 38-44 (1976)
Isoelectric Focusing in a Sephadex Column T. J. O’BRIEN~,
H. H. LIEBKE~,
Department Allan
of Biological Sciences, Hancock Foundation,
H. S. CHEUNG~,
AND L. K. JOHNSON
University of Southern Los Angeles, Cal(fornia
Received June 17, 1975; accepted November
California, 90007
11, 1975
An isoelectric focusing technique on a Sephadex column is described. This technique provides for the fractionation of amphoteric compounds in a pH gradient and allows for better than 95% recovery of the focused material. Focusing in this system over a pH range of 3.5- 10 reaches equilibrium in approximately 5 hr and thereby reduces the exposure of more labile proteins to long focusing times.
Isoelectric focusing (IEF) was developed for the purification and fractionation of amphoteric compounds. Systems utilizing this technique have employed density gradients (sucrose, glycerol, ficol) or acrylamide gels as support media (1,3,4). Flat beds of Sephadex and cylinders of acrylamide gels have also been used in conjunction with isoelectric focusing to achieve preparative separation of mg quantities of proteins (2,5). Although IEF in density gradients is most frequently employed, its use requires relatively large amounts of materials and long focusing times and is not supportive of proteins precipitated at their isoelectric point (6,7). IEF in acrylamide gels and flat beds of Sephadex alleviates these limitations but makes protein recovery and direct detection of enzyme activities difficult. For example, in acrylamide gel IEF, the focused zones may only be recovered subsequent to fractionation and solubilization from individual gel slices. The length of time necessary for this procedure could prove to be a limiting factor in the detection and recovery of more labile enzyme species. The detection of focused proteins in flat bed systems most frequently utilizes the paper print technique (8). Although this method of visualizing focused zones is probably the most sensitive and can be extended to enzymatic assays if one prepares a chromatographic paper impregnated with the enzyme substrate (9), it does not allow for the convenient re1 Present address: Department of CytoGenetics, City of Hope National Medical Center, Duarte, California 91010; to whom reprint requests should be addrssed. * Present address: Department of Human Genetics, Yale University, 333 Cedar Street, New Haven, Connecticut 06510. 3 Present address: University of Southern California, School of Medicine/Rheumatology Section, 2025 Zonal Avenue, OCD-130, Los Angeles, California 90033. 38 Copyright 0 I!976 by Academic Press. Inc. All rights of reproduction in any form reserved.
ISOELECTRIC
FOCUSING
IN SEPHADEX
39
covet-y of the individual protein species. This is especially necessary for the further purification or analysis of an individual band. The IEF method described herein has combined the high load capacities obtainable in granular gels (5,9) with the simple elution procedures of Sephadex chromatography. In so doing, most of the above mentioned limitations have been overcome while retaining the high resolving power of the technique. MATERIALS
AND METHODS
The apparatus consists of a 4 x 30 cm water-jacketed glass column containing a 2.5 x 20 cm G-15 Sephadex bed (Pharmacia) (Fig. 1). The Sephadex, preswollen in distilled water and deaerated under vacuum, is packed by continuous gravity flow through a half-bed volume of distilled water. The use of a low porosity gel such as G-15 overcomes for the most part bead collapse and air bubble formation sometimes encountered with high porosity gels. The Sephadex bed is supported by a 2.5 x 8 cm Teflon elution plug under a 25 mm Millipore filter (0.45), and after pack-
r
FIG. 1. Diagram of the isoelectric focusing apparatus.
40
O’BRIEN
Kr ,4L
ing is equilibrated with two-bed volumns of a solution containing 1.5% carrier ampholytes (LKB Produkter, Sweden) in 10% glycerol. The sample is also adjusted to 1.5% ampholytes, layered over the Sephadex, and allowed to enter the bed. Using about 10 ml of the ampholyteglycerol solution, the sample is then washed further into the column. A second Millipore filter is placed on the top of the Sephadex bed ensuring no air bubbles are entrapped. This filter acts as a partition between the Sephadex bed and a IO-ml polyacrylamide solution which is then poured over the filter. The solution consists of 14% acrylamide, 0.3% Bis, and 0.07% ammonium persulfate and is polymerized by the addition of 50 ~1 TEMED. Prior to polymerization of the acrylamide, a piece of nylon net is lowered into the solution to facilitate removal of the plug after termination of IEF. Upon completion of polymerization (approx 20 min) the column is inverted, the Teflon elution plug is removed, and a second acrylamide plug is layered over the bottom filter. After polymerization of the bottom plug the column is returned to its upright position and the lower end is immersed in the anode buffer containing 1% phosphoric acid (reagent grade). All entrapped air bubbles are removed with a syringe. The remaining upper portion of the column is filled with 1% ethanolamine (reagent grade) and serves as the cathode buffer reservoir. The cathode and anode electrodes are immersed in the upper and lower reservoirs, respectively, and connected to a 1000-V power unit (Buchler Scientific Instruments, Model 3-1014A). The column is then cooled by recirculating water at 1-4’C for 10 min. IEF is initiated at 800 constant volts (16 mA) and allowed to proceed to equilibrium as monitored by an eventual decline in the milliamperage to 2.5 mA (approx 4.5 hr) (Fig. 2). When focusing is completed the electrodes are disconnected, the buffer reservoir solutions are drained, and both ends of the column are thoroughly washed with distilled HZO. The apparatus
FIG. 2. Milliamperage increase and decline to equilibrium constant volts, 1.5% carrier ampholytes.
during a typical IEF run at 800
ISOELECTRIC
FOCUSING
IN SEPHADEX
41
is then inverted, and the anode plug is removed while rimming the acrylamide with a spatula to prevent vacuum formation. The filter disc is also removed and replaced by another ensuring no entrapped air between the Sephadex bed and the filter. The Teflon plug is then carefully inserted against the filter, and the elution tubing is clamped. The apparatus is next uprighted, and the cathode plug is removed in a similar fashion. The column may now be eluted with the ampholyte-glycerol solution or with distilled water. Following the passage of two-bed volumes, the apparatus is ready for reuse. RESULTS
AND DISCUSSION
In all isoelectric focusing procedures, samples are salt-free and the ambient temperature of the cooling system remains at 1-4’C. As demonstrated in Fig. 2, the current increases at the beginning of focusing (as the pH gradient becomes established), from approximately 16 mA after30 min, and thereafter declines and stabilizes at 2.5 mA. Although focusing equilibrium may readily be monitored by milliamperage stabilization, it is also useful to visualize the fractionation by including metachromatic marker proteins in the sample, such as ferritin, hemoglobin, myoglobin, or cytochrome C. It can be further noted that the rates of focusing of individual proteins varies with their respective molecular weights. A typical profile generated by the IEF of a solution of three different proteins is shown in Fig. 3. In this case, a sample volume of 5 ml was loaded onto the column. However, in practice the sample volume is not
FIG. 3. Isoelectric focusing of 1 mg each of ferritin (horse spleen, 2~ crystallized; Miles Laboratories), myoglobin (whale skeletal muscle, 2x crystallized; Calbiochem), and ribonuclease (bovine pancreas, 2~ crystallized; Calbiochem), 800 V, 4.5 hr. 1.5% carrier ampholytes (LKB), pH 3.5-10. Five-tenths milliliter fractions were collected and diluted to 1 ml of pH determinations which were measured at 4°C. Protein determinations are based on optical density measurements at 280 nm.
42
O’BRIEN
ET ,4L
critical and in fact may approach the size of the void volume of the column (35 ml). As demonstrated in Fig. 3, a linear pH gradient, after elution, is obtained from 3.8 to 8.4. It may be noted that the effective pH range of the ampholytes used (3.5- 10) is restricted due to extension of the pH gradient into the acrylamide plugs. Migration of the acidic ampholyte species out of the Sephadex can be eliminated by adjusting the pH of the lower plug to 2.0 with phosphoric acid. Since the molecular weights of the focused proteins exceed the exclusion limits of the Sephadex G-15, gel-filtration properties do not alfect the elution profile of the proteins. The Sephadex acts merely as an anticonvectant and support medium, and thus the relative positions of the focused proteins are maintained upon elution. However, the smaller molecular weight ampholytes, less than the exclusion limits of G- 15 (l@), may be displaced somewhat relative to the proteins during elution, and therefore the pH of each eluted fraction might not represent the true p1 of the protein contained therein. The relative positions of individual proteins in the pH gradient, though, are always consistent on a run to run basis. For example, as in Fig. 3, ferritin with a reported p1 of 4.3-4.4 (6) reproducibly elutes at pH 4.2-4.4. The ~1’s of myoglobin and ribonuclease are 7.0-7.9 and 7.8-9.5, respectively (5,10,11), and they elute at pH 6.8-7.0 and 7.8-7.95, respectively. Focusing may also be carried out on a selective pH range basis to increase resolution of proteins with closely related ~1’s. Figure 4 shows a refocusing of the myoglobin from Fig. 3 over the pH range 6-8, and readily demonstrates its resolution into discrete peaks. The overall focusing profile was not found to change using this apparatus when increased amounts of sample are applied (up to 0.3 g). However, with increasing amounts of sample, the band width of the focused proteins increases accordingly. For example, I mg of ferritin is
-6.5
IO
FIG.
20 30 FRACTION
40 NUMBER
50
60
4. Refocusing of 1 mg of myoglobin over the pH range 6-8. Other conditions as in Fig. 3.
ISOELECTRIC
FOCUSING
43
IN SEPHADEX
I25
IO0
25
5.0
-I 20
30 FRACTIOU
40
50
60
70
NUMBER
FIG. 5. Isoelectric focusing of E. cok RNA polymerase (Miles Laboratories, K-12 strain). The equilibrated column was precooled to 2’C and 1.5 units were then applied in 4 ml of 1.5% amphohne and 10% glycerol. Other conditions were as in Fig. 3. Five-tenths milliliter fractions were collected and SO-PI ahquots of each fraction were assayed for enzyme activity (13).
distributed over six fractions (3 ml) while 300 mg is distributed in 16 fractions (8 ml). Therefore, using a pH 3.5-10 gradient with a slope similar to that in Fig. 3, proteins which have ~1’s of 1 pH unit or more apart can be fractionated completely when as much as 300 mg of each component is present. While isoelectric point precipitation may become a problem at higher protein concentrations and cause some tailing of the eluted peak, the user may find it advantageous to alleviate this problem by focusing in the presence of a nonionic detergent. In this regard, 0.5%, Brij-35 has already been demonstrated to prevent isoelectric point precipitation without altering the structure or isoelectric point of some proteins (12). Figure 5 demonstrates the fractionation of E. c& RNA polymerase by this method and represents 95.7% recovery of this relatively labile enzyme. This suggests that aggregation, molecular sieving, or other artifacts do not interfere with the fractionation and recovery of this enzyme. This particular method of IEF is therefore suitable for fractionation and quantitative retrieval of amphoteric species and offers distinct advantages over previously described IEF procedures for the following reasons: (I) Focusing periods are short (4.5 hr) compared to other semipreparative IEF systems; (2) quantitative recovery is possible in a concentrated volume; (3) volumes of up to 30 ml can be applied to the column which often avoids the necessity of concentrating the sample; and (4) eluted fraction volumes are suitable for enzymatic and other analysis. CONCLUSION
This method offers a means of utilizing IEF to fractionate enzymes and other amphoteric compounds which were heretofore difficult to re-
44
O’BRlEN
EZ- /tL.
trieve via conventional IEF methodology. The system can be constructed with inexpensive laboratory hardware and may be used on a semipreparative scale.
This work was supported by a grant from the California Division of the American Cancer Society to the first author, Special Grant No. 643. The authors also wish to acknowledge Dr. B. C. Abbott, Dr. S. E. Allerton, and Dr. J. W. Beierle for their encouragement and suggestions; and Marilyn Cheung for preparation of the manuscript.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13,
Righetti, P., and Drysdale, J. W. (1971) Biochim. Biophys. Acfa 236, 17-28. Finlayson, G. R., and Chrambach, A. (197l)Amrl. Biochem. 40, 292-311. Catsimpoolas, N. (1970) Sepuration Science 5, 523-544. IIaglund, H. (1971) in Methods of Biochemical Analysis (Glick, D., ed,), Vol. 19, pp. l-104, Interscience, New York. Radola, B. J. (1%9) B&him. Bio~hys. Ac?a 194, 335-338. Rilbe, H. (19’70) in Frotides of the Biological Fluids, Vol. 17, pp. 369-382. Vesterberg, 0. (1971) in Methods in Enzymology (Jacoby, W. B., ed.), Vol. 22, pp. 389-412, Academic Press, New York. Radola, B. J. (I%@ J. Chromatography 38, 61-77. Radola, B. J. (1973) Annuls of New York Acad. Sci. 209, 127-143. Bobb, D. (1973) Ann& of New York Acad. Sci. 209,225-234. Jn The Encyclopedia of Bi~hemist~ (Williams, R. J., and Lansford, E. M., eds.), pp. 683, Reinhold Fublishing Corp., New York (1%7). Friesen, A. J., Jameson, J. C., and Ashton, F, EL (197l)Arzuf. Biochem. 41, 146-157. Blatti, S. P., Ingles, C. J., Lindell, T. J., Morris, P. W., Weaver, R. F., Weinberg, F., and Rutter, W. J. (1970) Cold Spring Harbor Symp. Quant. Biol. 35, 649-659.
BIOCHEMISTRY
72, 38-44 (1976)
Isoelectric Focusing in a Sephadex Column T. J. O’BRIEN~,
H. H. LIEBKE~,
Department Allan
of Biological Sciences, Hancock Foundation,
H. S. CHEUNG~,
AND L. K. JOHNSON
University of Southern Los Angeles, Cal(fornia
Received June 17, 1975; accepted November
California, 90007
11, 1975
An isoelectric focusing technique on a Sephadex column is described. This technique provides for the fractionation of amphoteric compounds in a pH gradient and allows for better than 95% recovery of the focused material. Focusing in this system over a pH range of 3.5- 10 reaches equilibrium in approximately 5 hr and thereby reduces the exposure of more labile proteins to long focusing times.
Isoelectric focusing (IEF) was developed for the purification and fractionation of amphoteric compounds. Systems utilizing this technique have employed density gradients (sucrose, glycerol, ficol) or acrylamide gels as support media (1,3,4). Flat beds of Sephadex and cylinders of acrylamide gels have also been used in conjunction with isoelectric focusing to achieve preparative separation of mg quantities of proteins (2,5). Although IEF in density gradients is most frequently employed, its use requires relatively large amounts of materials and long focusing times and is not supportive of proteins precipitated at their isoelectric point (6,7). IEF in acrylamide gels and flat beds of Sephadex alleviates these limitations but makes protein recovery and direct detection of enzyme activities difficult. For example, in acrylamide gel IEF, the focused zones may only be recovered subsequent to fractionation and solubilization from individual gel slices. The length of time necessary for this procedure could prove to be a limiting factor in the detection and recovery of more labile enzyme species. The detection of focused proteins in flat bed systems most frequently utilizes the paper print technique (8). Although this method of visualizing focused zones is probably the most sensitive and can be extended to enzymatic assays if one prepares a chromatographic paper impregnated with the enzyme substrate (9), it does not allow for the convenient re1 Present address: Department of CytoGenetics, City of Hope National Medical Center, Duarte, California 91010; to whom reprint requests should be addrssed. * Present address: Department of Human Genetics, Yale University, 333 Cedar Street, New Haven, Connecticut 06510. 3 Present address: University of Southern California, School of Medicine/Rheumatology Section, 2025 Zonal Avenue, OCD-130, Los Angeles, California 90033. 38 Copyright 0 I!976 by Academic Press. Inc. All rights of reproduction in any form reserved.
ISOELECTRIC
FOCUSING
IN SEPHADEX
39
covet-y of the individual protein species. This is especially necessary for the further purification or analysis of an individual band. The IEF method described herein has combined the high load capacities obtainable in granular gels (5,9) with the simple elution procedures of Sephadex chromatography. In so doing, most of the above mentioned limitations have been overcome while retaining the high resolving power of the technique. MATERIALS
AND METHODS
The apparatus consists of a 4 x 30 cm water-jacketed glass column containing a 2.5 x 20 cm G-15 Sephadex bed (Pharmacia) (Fig. 1). The Sephadex, preswollen in distilled water and deaerated under vacuum, is packed by continuous gravity flow through a half-bed volume of distilled water. The use of a low porosity gel such as G-15 overcomes for the most part bead collapse and air bubble formation sometimes encountered with high porosity gels. The Sephadex bed is supported by a 2.5 x 8 cm Teflon elution plug under a 25 mm Millipore filter (0.45), and after pack-
r
FIG. 1. Diagram of the isoelectric focusing apparatus.
40
O’BRIEN
Kr ,4L
ing is equilibrated with two-bed volumns of a solution containing 1.5% carrier ampholytes (LKB Produkter, Sweden) in 10% glycerol. The sample is also adjusted to 1.5% ampholytes, layered over the Sephadex, and allowed to enter the bed. Using about 10 ml of the ampholyteglycerol solution, the sample is then washed further into the column. A second Millipore filter is placed on the top of the Sephadex bed ensuring no air bubbles are entrapped. This filter acts as a partition between the Sephadex bed and a IO-ml polyacrylamide solution which is then poured over the filter. The solution consists of 14% acrylamide, 0.3% Bis, and 0.07% ammonium persulfate and is polymerized by the addition of 50 ~1 TEMED. Prior to polymerization of the acrylamide, a piece of nylon net is lowered into the solution to facilitate removal of the plug after termination of IEF. Upon completion of polymerization (approx 20 min) the column is inverted, the Teflon elution plug is removed, and a second acrylamide plug is layered over the bottom filter. After polymerization of the bottom plug the column is returned to its upright position and the lower end is immersed in the anode buffer containing 1% phosphoric acid (reagent grade). All entrapped air bubbles are removed with a syringe. The remaining upper portion of the column is filled with 1% ethanolamine (reagent grade) and serves as the cathode buffer reservoir. The cathode and anode electrodes are immersed in the upper and lower reservoirs, respectively, and connected to a 1000-V power unit (Buchler Scientific Instruments, Model 3-1014A). The column is then cooled by recirculating water at 1-4’C for 10 min. IEF is initiated at 800 constant volts (16 mA) and allowed to proceed to equilibrium as monitored by an eventual decline in the milliamperage to 2.5 mA (approx 4.5 hr) (Fig. 2). When focusing is completed the electrodes are disconnected, the buffer reservoir solutions are drained, and both ends of the column are thoroughly washed with distilled HZO. The apparatus
FIG. 2. Milliamperage increase and decline to equilibrium constant volts, 1.5% carrier ampholytes.
during a typical IEF run at 800
ISOELECTRIC
FOCUSING
IN SEPHADEX
41
is then inverted, and the anode plug is removed while rimming the acrylamide with a spatula to prevent vacuum formation. The filter disc is also removed and replaced by another ensuring no entrapped air between the Sephadex bed and the filter. The Teflon plug is then carefully inserted against the filter, and the elution tubing is clamped. The apparatus is next uprighted, and the cathode plug is removed in a similar fashion. The column may now be eluted with the ampholyte-glycerol solution or with distilled water. Following the passage of two-bed volumes, the apparatus is ready for reuse. RESULTS
AND DISCUSSION
In all isoelectric focusing procedures, samples are salt-free and the ambient temperature of the cooling system remains at 1-4’C. As demonstrated in Fig. 2, the current increases at the beginning of focusing (as the pH gradient becomes established), from approximately 16 mA after30 min, and thereafter declines and stabilizes at 2.5 mA. Although focusing equilibrium may readily be monitored by milliamperage stabilization, it is also useful to visualize the fractionation by including metachromatic marker proteins in the sample, such as ferritin, hemoglobin, myoglobin, or cytochrome C. It can be further noted that the rates of focusing of individual proteins varies with their respective molecular weights. A typical profile generated by the IEF of a solution of three different proteins is shown in Fig. 3. In this case, a sample volume of 5 ml was loaded onto the column. However, in practice the sample volume is not
FIG. 3. Isoelectric focusing of 1 mg each of ferritin (horse spleen, 2~ crystallized; Miles Laboratories), myoglobin (whale skeletal muscle, 2x crystallized; Calbiochem), and ribonuclease (bovine pancreas, 2~ crystallized; Calbiochem), 800 V, 4.5 hr. 1.5% carrier ampholytes (LKB), pH 3.5-10. Five-tenths milliliter fractions were collected and diluted to 1 ml of pH determinations which were measured at 4°C. Protein determinations are based on optical density measurements at 280 nm.
42
O’BRIEN
ET ,4L
critical and in fact may approach the size of the void volume of the column (35 ml). As demonstrated in Fig. 3, a linear pH gradient, after elution, is obtained from 3.8 to 8.4. It may be noted that the effective pH range of the ampholytes used (3.5- 10) is restricted due to extension of the pH gradient into the acrylamide plugs. Migration of the acidic ampholyte species out of the Sephadex can be eliminated by adjusting the pH of the lower plug to 2.0 with phosphoric acid. Since the molecular weights of the focused proteins exceed the exclusion limits of the Sephadex G-15, gel-filtration properties do not alfect the elution profile of the proteins. The Sephadex acts merely as an anticonvectant and support medium, and thus the relative positions of the focused proteins are maintained upon elution. However, the smaller molecular weight ampholytes, less than the exclusion limits of G- 15 (l@), may be displaced somewhat relative to the proteins during elution, and therefore the pH of each eluted fraction might not represent the true p1 of the protein contained therein. The relative positions of individual proteins in the pH gradient, though, are always consistent on a run to run basis. For example, as in Fig. 3, ferritin with a reported p1 of 4.3-4.4 (6) reproducibly elutes at pH 4.2-4.4. The ~1’s of myoglobin and ribonuclease are 7.0-7.9 and 7.8-9.5, respectively (5,10,11), and they elute at pH 6.8-7.0 and 7.8-7.95, respectively. Focusing may also be carried out on a selective pH range basis to increase resolution of proteins with closely related ~1’s. Figure 4 shows a refocusing of the myoglobin from Fig. 3 over the pH range 6-8, and readily demonstrates its resolution into discrete peaks. The overall focusing profile was not found to change using this apparatus when increased amounts of sample are applied (up to 0.3 g). However, with increasing amounts of sample, the band width of the focused proteins increases accordingly. For example, I mg of ferritin is
-6.5
IO
FIG.
20 30 FRACTION
40 NUMBER
50
60
4. Refocusing of 1 mg of myoglobin over the pH range 6-8. Other conditions as in Fig. 3.
ISOELECTRIC
FOCUSING
43
IN SEPHADEX
I25
IO0
25
5.0
-I 20
30 FRACTIOU
40
50
60
70
NUMBER
FIG. 5. Isoelectric focusing of E. cok RNA polymerase (Miles Laboratories, K-12 strain). The equilibrated column was precooled to 2’C and 1.5 units were then applied in 4 ml of 1.5% amphohne and 10% glycerol. Other conditions were as in Fig. 3. Five-tenths milliliter fractions were collected and SO-PI ahquots of each fraction were assayed for enzyme activity (13).
distributed over six fractions (3 ml) while 300 mg is distributed in 16 fractions (8 ml). Therefore, using a pH 3.5-10 gradient with a slope similar to that in Fig. 3, proteins which have ~1’s of 1 pH unit or more apart can be fractionated completely when as much as 300 mg of each component is present. While isoelectric point precipitation may become a problem at higher protein concentrations and cause some tailing of the eluted peak, the user may find it advantageous to alleviate this problem by focusing in the presence of a nonionic detergent. In this regard, 0.5%, Brij-35 has already been demonstrated to prevent isoelectric point precipitation without altering the structure or isoelectric point of some proteins (12). Figure 5 demonstrates the fractionation of E. c& RNA polymerase by this method and represents 95.7% recovery of this relatively labile enzyme. This suggests that aggregation, molecular sieving, or other artifacts do not interfere with the fractionation and recovery of this enzyme. This particular method of IEF is therefore suitable for fractionation and quantitative retrieval of amphoteric species and offers distinct advantages over previously described IEF procedures for the following reasons: (I) Focusing periods are short (4.5 hr) compared to other semipreparative IEF systems; (2) quantitative recovery is possible in a concentrated volume; (3) volumes of up to 30 ml can be applied to the column which often avoids the necessity of concentrating the sample; and (4) eluted fraction volumes are suitable for enzymatic and other analysis. CONCLUSION
This method offers a means of utilizing IEF to fractionate enzymes and other amphoteric compounds which were heretofore difficult to re-
44
O’BRlEN
EZ- /tL.
trieve via conventional IEF methodology. The system can be constructed with inexpensive laboratory hardware and may be used on a semipreparative scale.
This work was supported by a grant from the California Division of the American Cancer Society to the first author, Special Grant No. 643. The authors also wish to acknowledge Dr. B. C. Abbott, Dr. S. E. Allerton, and Dr. J. W. Beierle for their encouragement and suggestions; and Marilyn Cheung for preparation of the manuscript.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13,
Righetti, P., and Drysdale, J. W. (1971) Biochim. Biophys. Acfa 236, 17-28. Finlayson, G. R., and Chrambach, A. (197l)Amrl. Biochem. 40, 292-311. Catsimpoolas, N. (1970) Sepuration Science 5, 523-544. IIaglund, H. (1971) in Methods of Biochemical Analysis (Glick, D., ed,), Vol. 19, pp. l-104, Interscience, New York. Radola, B. J. (1%9) B&him. Bio~hys. Ac?a 194, 335-338. Rilbe, H. (19’70) in Frotides of the Biological Fluids, Vol. 17, pp. 369-382. Vesterberg, 0. (1971) in Methods in Enzymology (Jacoby, W. B., ed.), Vol. 22, pp. 389-412, Academic Press, New York. Radola, B. J. (I%@ J. Chromatography 38, 61-77. Radola, B. J. (1973) Annuls of New York Acad. Sci. 209, 127-143. Bobb, D. (1973) Ann& of New York Acad. Sci. 209,225-234. Jn The Encyclopedia of Bi~hemist~ (Williams, R. J., and Lansford, E. M., eds.), pp. 683, Reinhold Fublishing Corp., New York (1%7). Friesen, A. J., Jameson, J. C., and Ashton, F, EL (197l)Arzuf. Biochem. 41, 146-157. Blatti, S. P., Ingles, C. J., Lindell, T. J., Morris, P. W., Weaver, R. F., Weinberg, F., and Rutter, W. J. (1970) Cold Spring Harbor Symp. Quant. Biol. 35, 649-659.
