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An ultrafiltration membrane for the resolution and purification of bovine alpha-lactalbumin
ANALYTICAL
BIOCHEMISTRY
18,
An Ultrafiltration and Purification
81-87 (1967)
Membrane for the Resolution of Bovine Alpha-Lactalbuminl
W. F. BLATT Biochemistry-Pharmacology Environmental
S. M. ROBINSON
AWD
Division, Medicine,
U. S. Natick,
Army Research
Institute
of
Massachusetts
F. M. ROBBINS Pioneering
Research
Division, Natick.
U. S. Army Massachusetts
Natick
Laboratories.
C. A. SARAVIS l’he
Protein
Foundation, Received
Jamaica August
Plain,
Massachusetts
23, 1966
Preformed ultrafiltration membranes,2 which serve as barriers to the diffusive transport of macromolecules, have been reported as a practical system for the rapid concentration of dilute protein solutions (1). Michaels (2) has discussed the chemical constitution of these polymer membranes and their potential biomedical applications. The recent formulation of a membrane (XM-4A) with a more ‘(expanded structure” (approximate retention value, 3035,000) suggested its use as a partitioning device for the separation of larger sized molecular species (membrane-partition chromatography). Prior to evaluation of the protein retention characteristics of the polymer, flow rates (in a commercially available concentration cell) were determined for distilled water, phosphate-buffered saline, pH 7.4, and 0.2yo human serum albumin3 using a 1.75# diameter membrane under a pressure of 50 psi. These were 149.8, 132.1, and 19.1 X lO+ ml min-l cm-*, respectively. Crystalline bovine a-lactalbumin (molecular weight 15,500)) prepared as described by Robbins and Kronman (3), provided a material phys‘This study WB,B supported in p8,rt by Contract No. 43-66-476 Institute of Arthritis and Metabolic Diseases, National Institutes DHEW. * The Amicon Corp., Cambridge, Mass. “Obtained from Merck, Sharp and Dohme, West Point, Pa. 81
from the of Health,
National USPHS,
‘.
0.2%
ALPHA-LACTALBUMIN I
prerrvra
filftafion
XM-4A FILTER (35,000)
Concentrate
UM-2
Concentrate
FILTER (500)
23
A
69
B
Fraction IO I-4
FILTRATE
8
Fio. 1. Separation, chromatographic evaluation and yield of bovine ru-lactalbumin. Left, separation scheme (membrane partition chromatography). Center, gel diffusion 82
A NEW
ULTRAFILTRATION
MEMBRANE
a3
ically able to diffuse through the membrane, but reputed to contain a small quantity of albumin as a contaminant (3, 4). Aliquots of a 2% solution (5 ml) were diluted to 50 ml with tris-saline buffer, pH 8.6, and ultrafiltered under 50 psi at an average flow rate of 90.0 X lo-’ ml min-’ cm-?. After concentration to 5 ml, the filter-retained sample (concentrate A) was rediluted to 50 ml and the ultrafiltration repeated. The filtrates were pooled and then reduced in volume (concentrate B) using a retention membrane (EM-2) of lower molecular weight (500). The filterable material in the latter separation (<500) was concentrated by flash evaporation under reduced pressure (filtrate). The protein content of all fractions was determined by biuret analysis. A summary of the separation scheme and the nomenclature of the various fractions is shown at the left of Figure 1; per cent protein distribution, based on the total recovered, is shown at the right of the figure. It is apparent that a substantial portion of the ia-lactalbumin diffused through the XM-4A membrane with 23% remaining in the membrane retentate. We were able to isolate 69yo of the biuret-positive material, presumably with molecular limits of 500 through 35,000, in the second concentrate. No more than 8% of the total yield of biuret-positive material appeared in the final filtrate. Gross evaluation of the molecular size distribution of the starting material, as well as representative aliquots of each step during membrane fractionation, were provided by gel filtration on 1.5 X 50 cm columns of Sephadex G-100 at 5°C. The automated Spectrochrom’ apparatus was used for separation and evaluation in a manner previously described (5). Flow was maintained at 25 ml/hr using phosphate-buffered saline, pH 7.4, as the eluting solvent; the column eluate was monitored at 280 rnp, and 5 ml aliquots were collected. To facilitate visual comparison of the various fract.ions, approximately 6 111 g of protein was applied to the column for each separation. These graphic recordings are shown in the central portion of Figure 1. The various preparations can be identified from the flow scheme at the left. Components, separated on the basis of absorbance. are identified by the letters shown directly below the peaks. Four distinct fractions were observed following chromatographic evaluation of the st’arting material (Fig. l-1). Component A appears immediately after the void volume of the column has been expressed; component B appears in a volume fraction comparable to that observed for human * Beckman Instrument
Co., Spinco Div., Fullerton,
Calif.
chromatography of the membrane-prepared fractions on 1.5 x 50 cm columns of Sephadex G-100. Right, distribution in per cent of the total recovery of protein for the indicated fractions.
Concentrate
Alpha-lactalbumin
A
1.0
-
2-4
.
Fraction
Fraction
Albumin
1.0
I.0
Concentrate
B 2-5
\
1.0
Fraction
Fraction 10
Mixture
Filtrate
2-3
Fraction
Fraction
FIG. 2. Gel chromatography on 1.5 x 50 cm columns of Sephadex G-100. Identifica tion of the concentrates and filtrate can be made from Figure 1. 84
A NEW
ULTRAFILTRATION
85
MEMBRANE
serum albumin (see below), and, in all likelihood, represents the albumin impurity. The major peak, component C, comprises in excess of 92% of the total fraction. Component D appears in a volume fraction corresponding to the moieties of low molecular weight totally retarded by the column (K, > 0.9)) and, in part, consists of the UV-absorbing buffer salts used in the initial dilution and subsequent washes of the filter-retained sample prior to application to the column. The second pattern (Fig. 1-2) illustrative of the material comprising 23% of the total protein clearly indicates enrichment of the higher molecular weight moieties (A and B) with some retention of C. The C The Total and Distribution
TABLE 1 of the Contents of Fractions Derived from Membrane Partition Chromatography Distribution,*
Fraction
Albumin-a-lactalbumin (starting material) Concentrate A Concentrate B Filtrate
RecoryLn
74 23 3
%
A
n
C
D
0.3
22.2
64.6
12.9
2.0 0.2 1.6
36.9 0 0
61.1
90.9 12.5
0 8.9 85.9
* Recovery was determined by biuret analyses and based on the total yield. b Distributions were calculated from the total absorbance at 280 mp of the pooled subfractions obtained following column chromatography on Sephadex G-100.
peak dominates Figure l-3, which is devoid of component B, indicating separation of the albumin moiety from the lactalbumin. The filtrate (Fig. l-4) consists of fraction A, considerably reduced C, and the large salt-containing peak, D. The appearance of A in all membrane fractions and as the initial component in chromatographic separation is puzzling; it is possible that this material represents an aggregated form of one or more components. Further evidence for membrane filtration as an effective means of protein separation is given by the chromatographic data of Figure 2. In this series, equal amounts of bovine a-lactalbumin (Fig. 2-l) and human serum albumin (Fig. 2-2) were admixed to form the solution whose gel separation pattern is shown in 2-3. Membrane partition of this mixture was accomplished as described above. The fraction retained on the XM-4A filter is shown in Figure 2-4, that fraction passing through the membrane in 2-5, and the final filtrate in 2-6. The recovery of protein for each fraction given as per cent of the final yield, is shown in the second column of Table 1, whereas the distribution of the various chro-
86
BLATT,
ROBINSON,
ROBBINS,
AND
SARAVIS
FIG. 3. Titration immunodiffusion on cellulose acetate (initial concentrations are shown in Table 2) : (a) antigen well, (b) antisera trough, (c) immunoprecipitin lines indicating reaction.
matographic
components,
as determined
from the relative
absorbance of
the column eluates is indicated in the last four columns. The exclusion of albumin (component B) is clearly shown, as well as localization of the
lower molecular
weight moieties in the filtrate
fraction.
Further evidence substantiating the removal of albumin from either was obtained the crystalline or the admixed fraction of ,a-lactalbumin
by immunochemical evaluation of the various fractions using the titration-immunodiffusion procedure described by Saravis (6). In this technique, twofold serial dilutions of antigen were reacted with horse antihuman antisera,5 using preformed microporous cellulose acetate as the ‘Obtained from Netherlands Red Cross, Amsterdam, The Netherlands.
A NEW
ULTRAFILTRATION
ST
MEMBRANE
supporting medium. Figure 3 illustrates the results obtained with the starting albumin solution and the partition of the mixture, and Table 2 summarizes the total evaluation. The chromatographic data were confirmed; i.e., in those fractions passing through the XM-4A membrane, little or no albumin is present,, as shown by a procedure capable of determining 0.25 Fg of antigen. Quantitative
TABLE 2 Immunodiffusion with Anti-human
Fraction
Antisera
I+Oteiei,C~;cn.,
Human serum albumin Albumin-cone. A Albumin-cont. B Albumin-filtrate Bovine o-lactalbumin &act.-cont. $ a-lact.-cont. B or-lact.-filtrate Alb.-rr-lact.-cont. A Alb.-a-lack-cont. B Alb.-or-lact.-filtrate
Antigen
19.1 12.4 0.3 0.5 15.5 1.9 5.4 1.5 8.8 2.9 0.3
a Least discernible antigen tit.er, i.e., the final dilution antibody is still visible.
titera
>1/1024 > l/1024 l/2 (It) 0 l/16 l/16 112(*I 0 l/512 l/2 (i, 0 at’ which reaction with the
This new preparative method, while not affording complete resolution of components, shows considerable promise as a tool for protein fractionation. For lower molecular weight mixtures contaminated with albumin, the membrane described provides a rapid and inexpensive method for separating relatively large amounts of material without resorting to gel filtration. REFERENCES BLATT, W. F., FEIN~RQ, M. P., HOFFENBERQ, H. B., AND SARAVIS, c. A., &&?nce 3063,224 (1965). 2. MICHAELS, A. S., Ind. Eng. Ch.em. 57,32 (1965). 3. ROBBXNS, F. M., AND XRONMAN, M. J., Bbchim. Biophys. Acta 82, 186 (1964). 4. BENQTBSON, C!., HANSON, L. A., AND JOHANSSON, B. G., Acta Chem. Stand. 16, 127 1.
m32). 5. BLAT, W. F., AND PI-AN,
F. P., J. Chrmatog.,
6. SARAVKW, C. A., Am. J. Clin. Path. 54, 507 (1936).
in press
(1966).
BIOCHEMISTRY
18,
An Ultrafiltration and Purification
81-87 (1967)
Membrane for the Resolution of Bovine Alpha-Lactalbuminl
W. F. BLATT Biochemistry-Pharmacology Environmental
S. M. ROBINSON
AWD
Division, Medicine,
U. S. Natick,
Army Research
Institute
of
Massachusetts
F. M. ROBBINS Pioneering
Research
Division, Natick.
U. S. Army Massachusetts
Natick
Laboratories.
C. A. SARAVIS l’he
Protein
Foundation, Received
Jamaica August
Plain,
Massachusetts
23, 1966
Preformed ultrafiltration membranes,2 which serve as barriers to the diffusive transport of macromolecules, have been reported as a practical system for the rapid concentration of dilute protein solutions (1). Michaels (2) has discussed the chemical constitution of these polymer membranes and their potential biomedical applications. The recent formulation of a membrane (XM-4A) with a more ‘(expanded structure” (approximate retention value, 3035,000) suggested its use as a partitioning device for the separation of larger sized molecular species (membrane-partition chromatography). Prior to evaluation of the protein retention characteristics of the polymer, flow rates (in a commercially available concentration cell) were determined for distilled water, phosphate-buffered saline, pH 7.4, and 0.2yo human serum albumin3 using a 1.75# diameter membrane under a pressure of 50 psi. These were 149.8, 132.1, and 19.1 X lO+ ml min-l cm-*, respectively. Crystalline bovine a-lactalbumin (molecular weight 15,500)) prepared as described by Robbins and Kronman (3), provided a material phys‘This study WB,B supported in p8,rt by Contract No. 43-66-476 Institute of Arthritis and Metabolic Diseases, National Institutes DHEW. * The Amicon Corp., Cambridge, Mass. “Obtained from Merck, Sharp and Dohme, West Point, Pa. 81
from the of Health,
National USPHS,
‘.
0.2%
ALPHA-LACTALBUMIN I
prerrvra
filftafion
XM-4A FILTER (35,000)
Concentrate
UM-2
Concentrate
FILTER (500)
23
A
69
B
Fraction IO I-4
FILTRATE
8
Fio. 1. Separation, chromatographic evaluation and yield of bovine ru-lactalbumin. Left, separation scheme (membrane partition chromatography). Center, gel diffusion 82
A NEW
ULTRAFILTRATION
MEMBRANE
a3
ically able to diffuse through the membrane, but reputed to contain a small quantity of albumin as a contaminant (3, 4). Aliquots of a 2% solution (5 ml) were diluted to 50 ml with tris-saline buffer, pH 8.6, and ultrafiltered under 50 psi at an average flow rate of 90.0 X lo-’ ml min-’ cm-?. After concentration to 5 ml, the filter-retained sample (concentrate A) was rediluted to 50 ml and the ultrafiltration repeated. The filtrates were pooled and then reduced in volume (concentrate B) using a retention membrane (EM-2) of lower molecular weight (500). The filterable material in the latter separation (<500) was concentrated by flash evaporation under reduced pressure (filtrate). The protein content of all fractions was determined by biuret analysis. A summary of the separation scheme and the nomenclature of the various fractions is shown at the left of Figure 1; per cent protein distribution, based on the total recovered, is shown at the right of the figure. It is apparent that a substantial portion of the ia-lactalbumin diffused through the XM-4A membrane with 23% remaining in the membrane retentate. We were able to isolate 69yo of the biuret-positive material, presumably with molecular limits of 500 through 35,000, in the second concentrate. No more than 8% of the total yield of biuret-positive material appeared in the final filtrate. Gross evaluation of the molecular size distribution of the starting material, as well as representative aliquots of each step during membrane fractionation, were provided by gel filtration on 1.5 X 50 cm columns of Sephadex G-100 at 5°C. The automated Spectrochrom’ apparatus was used for separation and evaluation in a manner previously described (5). Flow was maintained at 25 ml/hr using phosphate-buffered saline, pH 7.4, as the eluting solvent; the column eluate was monitored at 280 rnp, and 5 ml aliquots were collected. To facilitate visual comparison of the various fract.ions, approximately 6 111 g of protein was applied to the column for each separation. These graphic recordings are shown in the central portion of Figure 1. The various preparations can be identified from the flow scheme at the left. Components, separated on the basis of absorbance. are identified by the letters shown directly below the peaks. Four distinct fractions were observed following chromatographic evaluation of the st’arting material (Fig. l-1). Component A appears immediately after the void volume of the column has been expressed; component B appears in a volume fraction comparable to that observed for human * Beckman Instrument
Co., Spinco Div., Fullerton,
Calif.
chromatography of the membrane-prepared fractions on 1.5 x 50 cm columns of Sephadex G-100. Right, distribution in per cent of the total recovery of protein for the indicated fractions.
Concentrate
Alpha-lactalbumin
A
1.0
-
2-4
.
Fraction
Fraction
Albumin
1.0
I.0
Concentrate
B 2-5
\
1.0
Fraction
Fraction 10
Mixture
Filtrate
2-3
Fraction
Fraction
FIG. 2. Gel chromatography on 1.5 x 50 cm columns of Sephadex G-100. Identifica tion of the concentrates and filtrate can be made from Figure 1. 84
A NEW
ULTRAFILTRATION
85
MEMBRANE
serum albumin (see below), and, in all likelihood, represents the albumin impurity. The major peak, component C, comprises in excess of 92% of the total fraction. Component D appears in a volume fraction corresponding to the moieties of low molecular weight totally retarded by the column (K, > 0.9)) and, in part, consists of the UV-absorbing buffer salts used in the initial dilution and subsequent washes of the filter-retained sample prior to application to the column. The second pattern (Fig. 1-2) illustrative of the material comprising 23% of the total protein clearly indicates enrichment of the higher molecular weight moieties (A and B) with some retention of C. The C The Total and Distribution
TABLE 1 of the Contents of Fractions Derived from Membrane Partition Chromatography Distribution,*
Fraction
Albumin-a-lactalbumin (starting material) Concentrate A Concentrate B Filtrate
RecoryLn
74 23 3
%
A
n
C
D
0.3
22.2
64.6
12.9
2.0 0.2 1.6
36.9 0 0
61.1
90.9 12.5
0 8.9 85.9
* Recovery was determined by biuret analyses and based on the total yield. b Distributions were calculated from the total absorbance at 280 mp of the pooled subfractions obtained following column chromatography on Sephadex G-100.
peak dominates Figure l-3, which is devoid of component B, indicating separation of the albumin moiety from the lactalbumin. The filtrate (Fig. l-4) consists of fraction A, considerably reduced C, and the large salt-containing peak, D. The appearance of A in all membrane fractions and as the initial component in chromatographic separation is puzzling; it is possible that this material represents an aggregated form of one or more components. Further evidence for membrane filtration as an effective means of protein separation is given by the chromatographic data of Figure 2. In this series, equal amounts of bovine a-lactalbumin (Fig. 2-l) and human serum albumin (Fig. 2-2) were admixed to form the solution whose gel separation pattern is shown in 2-3. Membrane partition of this mixture was accomplished as described above. The fraction retained on the XM-4A filter is shown in Figure 2-4, that fraction passing through the membrane in 2-5, and the final filtrate in 2-6. The recovery of protein for each fraction given as per cent of the final yield, is shown in the second column of Table 1, whereas the distribution of the various chro-
86
BLATT,
ROBINSON,
ROBBINS,
AND
SARAVIS
FIG. 3. Titration immunodiffusion on cellulose acetate (initial concentrations are shown in Table 2) : (a) antigen well, (b) antisera trough, (c) immunoprecipitin lines indicating reaction.
matographic
components,
as determined
from the relative
absorbance of
the column eluates is indicated in the last four columns. The exclusion of albumin (component B) is clearly shown, as well as localization of the
lower molecular
weight moieties in the filtrate
fraction.
Further evidence substantiating the removal of albumin from either was obtained the crystalline or the admixed fraction of ,a-lactalbumin
by immunochemical evaluation of the various fractions using the titration-immunodiffusion procedure described by Saravis (6). In this technique, twofold serial dilutions of antigen were reacted with horse antihuman antisera,5 using preformed microporous cellulose acetate as the ‘Obtained from Netherlands Red Cross, Amsterdam, The Netherlands.
A NEW
ULTRAFILTRATION
ST
MEMBRANE
supporting medium. Figure 3 illustrates the results obtained with the starting albumin solution and the partition of the mixture, and Table 2 summarizes the total evaluation. The chromatographic data were confirmed; i.e., in those fractions passing through the XM-4A membrane, little or no albumin is present,, as shown by a procedure capable of determining 0.25 Fg of antigen. Quantitative
TABLE 2 Immunodiffusion with Anti-human
Fraction
Antisera
I+Oteiei,C~;cn.,
Human serum albumin Albumin-cone. A Albumin-cont. B Albumin-filtrate Bovine o-lactalbumin &act.-cont. $ a-lact.-cont. B or-lact.-filtrate Alb.-rr-lact.-cont. A Alb.-a-lack-cont. B Alb.-or-lact.-filtrate
Antigen
19.1 12.4 0.3 0.5 15.5 1.9 5.4 1.5 8.8 2.9 0.3
a Least discernible antigen tit.er, i.e., the final dilution antibody is still visible.
titera
>1/1024 > l/1024 l/2 (It) 0 l/16 l/16 112(*I 0 l/512 l/2 (i, 0 at’ which reaction with the
This new preparative method, while not affording complete resolution of components, shows considerable promise as a tool for protein fractionation. For lower molecular weight mixtures contaminated with albumin, the membrane described provides a rapid and inexpensive method for separating relatively large amounts of material without resorting to gel filtration. REFERENCES BLATT, W. F., FEIN~RQ, M. P., HOFFENBERQ, H. B., AND SARAVIS, c. A., &&?nce 3063,224 (1965). 2. MICHAELS, A. S., Ind. Eng. Ch.em. 57,32 (1965). 3. ROBBXNS, F. M., AND XRONMAN, M. J., Bbchim. Biophys. Acta 82, 186 (1964). 4. BENQTBSON, C!., HANSON, L. A., AND JOHANSSON, B. G., Acta Chem. Stand. 16, 127 1.
m32). 5. BLAT, W. F., AND PI-AN,
F. P., J. Chrmatog.,
6. SARAVKW, C. A., Am. J. Clin. Path. 54, 507 (1936).
in press
(1966).