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Folate-Sepharose column as an affinity absorbent for protein fractionation
ANALYTICAL
98. 184- 189 (1979)
BIOCHEMISTRY
Folate-Sepharose
Column as an Affinity Protein Fractionation FUMIO
Division
of Chemistr.v.
Nationul
for
SAWADA
of
Rdiolo,yicul
Received
January
Institute
Absorbent
Sciences,
Anugu~~u.
Chiba
City,
260 Jupun
22, 1979
Folate-Sepharose gel was prepared by direct coupling of fohc acid to epoxy-activated Sepharose. Bovine pancreatic ribonuclease A was adsorbed on the column at pH 5.0 and was desorbed with ligands which had an affinity for the enzyme. The pH dependency of the affinity of the enzyme for folic acid in the gel matrix was similar to that in solution. Bovine liver glutamate dehydrogenase exhibited a very strong affinity for the gel. Cationexchange character was the dominant factor in the chromatography of proteins on a folate-Sepharose column.
Folic acid, whose derivatives are metabolic cofactors in the one-carbon transfer reactions, has a unique affinity for bovine pancreatic ribonuclease A (RNase A) (1.2) and for liver glutamate dehydrogenase (3). The pteridine moiety of folic acid, which is essential for metabolic reactions, is not essential in both cases of interaction with these proteins. To obtain further information on the nature of the interaction of folic acid with a series of proteins, an affinity column, in which folic acid was coupled to an agarose gel matrix through the pteridine moiety, was prepared. This product proved effective in separating protein mixtures including RNase A and its derivatives. MATERIALS
egg-white lysozyme from Sigma Chemical Company; swine stomach pepsin from P.-L. Biochemical Inc.; and Asprrgilfus ribonuclease T, (RNase T,) from Sankyo Company, Tokyo. Performic acid-oxidized RNase A was prepared by the method of Hirs (4), carboxymethylhistidine-1 I9-RNase A (1 I9-His(cm)-RNase A)’ after Crestfield et crl. (5), and 4-thiouridylate-photosensitized RNase A was the same preparation described in Fig. 9c of Ref. (6). Folic acid andp-aminobenzoylglutamic acid (NH,BzGlu) were products of Tokyo Kasei Kogyo Company, Tokyo. Adenosine 5’-phosphate (A5’P) and uridine 3’(2’)-phosphate (U3’(2’)P) were from Boehringer. The latter was almost exclusively the 3’-isomer as estimated by the absorption ratio, A2,,,/A26,, (7).
AND METHODS
Materials
Prrpuratim
Epoxy-activated Sepharose 6B and Sephadex G-25 were products of Pharmacia Fine Chemicals. Separax S cellulose acetate film was the product of Fuji Photo Film Company, Tokyo. RNase A, cu-chymotrypsin, trypsin, and liver glutamate dehydrogenase (all from beef) were obtained from Boehringer/Mannheim; bovine chymotrypsinogen A and ovalbumin from Mann Research Laboratories; 0003-26971791130
184-06$02.00/O
Copynghl GI 1979 by Academx Prers, Inc. All rights of reproductwn m any form reserved
oj’Folatt> -Sephrosr
Gel
Eight grams of epoxy-activated Sepharose 6B was hydrated and washed in distilled water, equilibrated with 0.1 M sodium bicarbonate (pH 10.0). and the supernatant of ’ Abbreviations used: 119.Histcm), carboxymethylhistidine-119: NH,BzGlu,p-aminobenzoylglutamic acid, A5’P. adenosine 5’-phosphate: U3’(2’)P, uridine 3’(2’)phosphate. 184
FOLATE-SEPHAROSE
COLUMN
IN PROTEIN
the settled gel decanted. To the gel was added 100 ml of 0.01 M folic acid dissolved in 0.1 M bicarbonate (pH adjusted to 10.0 with NaOH) and the mixture was shaken for 16 h at 30°C in the dark. The brownish yellow gel was collected on a sintered glass filter and washed with 400 ml of bicarbonate buffer, then successively with 100 ml each of distilled water, 0.5 M NaCl in 0.1 M sodium bicarbonate (pH 8), 0.5 M NaCl in 0.1 M sodium acetate (pH 4), and distilled water. The washed gel was treated with the folic acid solution four more times. The residual epoxy groups in the gel were blocked by shaking the gel with 80 ml of I M monoethanolamine for 8 h at 30°C in the dark. Finally, the gel was thoroughly washed as described above. The settled volume of the gel was 23.2 ml. The amount of folic acid coupled to Sepharose was 5.4 pmol,lml as estimated from the glutamic acid contents obtained by amino acid analysis. The product could be stored under 0.02% NaN, :at 4°C at least I year. The putative structure of the product is shown in Scheme I.
185
FRACTIONATION
with 100 ml of 1 M monoethanolamine for 24 hat 30°C to block the active epoxy groups and washed thoroughly as described. The product was colorless and had a tendency to stick on the surface of glassware. The putative structure of the product is shown in Scheme 1. Protein
Binding
to the F&ate - Sepharose
Gel A. Elation analysis. Protein was eluted from a folate-Sepharose column at 4°C with a concave NaCl gradient in sodium acetate at pH 5.0. Biospecific desorption of the bound protein from the column was achieved with 5 mM solutions of ligands at pH 5.0 and 4°C. B. Frontal analysis. A solution of protein was continuously applied onto a small column of folate-Sepharose gel and the concentration of protein in effluent fractions was measured. The dissociation constant ( Kd) of the protein-folate-gel complex was estimated using the equation K,[ = ~
L
v - vo
Preparation of Epoxy-Blocked Sepharose Gel
- [-%I,
where L is the total amount of folate attached to the gel column, V and V. are the elution volumes of the protein with and without interaction with the gel, respectively, and [E,] is the concentration of the protein solution (8).
Eight grams of epoxy-activated Sepharose 6B was treated with 0.1 M sodium bicarbonate at pH 10.0 in the same manner as in the preparation of folate-Sepharose except for the omission of folic acid. The gel was shaken
FOOH Sepharose-O-CH2-CH-CH20-CH2-CH2-CH2-CH*-CH*-C~~,-O-CH~-~H-CH~-NH‘. OH
OH
a)-CH,-NH-@O-&COOH OH
Folate-Sepharose
SepharOSe-O-CH2-CH-CHZO-CH2-CHZ-CH2-CH2-O-CH*-CH-CH*-NH-CH~-C~~-OH bH
Epoxy-Blocked
bH
Sepharose SCHEME
I
186
FUMIO
0
SAWADA
I
L
I
50
100
150
-NoCl
Gradlent
(O+O.lSM),pH
I
,
250 ml
200 5.0
-
;.5M
NaCl
FIG. 1. Elution of RNase A and its photoproducts from a folate-Sepharose column. Photosensitized RNase A was prepared by irradiation of the enzyme with near-ultraviolet light in the presence of 4-thiouridine 2’(3’)-phosphate as described in Fig. 9c of Ref (6). The column (I x 26 cm) was equilibrated with sodium acetate (pH 5.0 at 4°C. Na = 0.005 M). A solution of protein (0.5-0.6 mgi0.2-0.25 ml) was added to the column and was eluted at 4°C with a concave NaCl gradient consisting of 178 ml of the buffer in a mixer and 100 ml of 0.20 M NaCl in the buffer in a reservoir. The gradient curve is shown in Fig. 3. Enzyme activity was assayed with RNA as substrate (11). (0 0) Absorbance at 220 nm; (0 - - - 0) enzyme activity in arbitrary units. (Top) native RNase A; (bottom) photosensitized RNase A.
Binding of Folic Acid to Proteins in Solution The affinity of proteins for folic acid in solution was measured by the Hummel and Dreyer gel filtration method (9) on columns of Sephadex G-25 (fine). The molar ratios of the bound ligand to the proteins were calculated using the following values of molecular weight: RNase T, (11 ,OOO), RNase
A (13,700), lysozyme (13,900), trypsin (23,000), cY-chymotrypsin (25,300), chymotrypsinogen A (25,700), pepsin (34,200), ovalbumin (45,000), and glutamate dehydrogenase monomer (56,000). Cellulose
Acetate
Film Electrophoresis
Electrophoretic mobilities of proteins on a Separax S cellulose acetate film were
FOLATE-SEPHAROSE
COLUMN
IN PROTEIN
187
FRACTIONATION
determined at pH 5.0 and 4°C with correction for electroendoosmosis using dextran as a marker (10). Proteins on the film were detected by staining with 0.5% Coomassie brilliant blue R250 in acetic acid-methanolwater (1:4:5, v/v) or by fluorescence after the phthalaldehyde reaction.2
u 312?P
RESULTS
As shown in Figs. 1 and 3, RNase A bound firmly to folate-Sepharose gel under low salt concentration and was eluted from the column with 0.09 M NaCI.” The enzyme, photosensitized with 4-thiouridine 2’(3’)phosphate (6,11) was fractionated into several components with different degrees of enzyme activity (Fig. 1, bottom). The resolution of the peaks was much better than that obtained with a longer column of ionexchange resin Amberlite IRC-50 (6). The column also appears to be useful for a carboxymeth.ylated derivative of the enzyme, 119-His(cm)RNase A (cmR in Fig. 3). A small shoulder at the right-hand site is considered to be due to contamination by another inactive derivative of the enzyme, carboxymethylhistidineI2-RNase A. Biospecific desorption of RNase A from a folate- Sepharose column was acihieved with a 5 mM solution of three dilfferent ligands at pH 5.0 and 4°C. U3’(2’)P had the highest affinity and both A5’P and NH,BzGlu had a similar ability for desorption as :shown ? The cellulose acetate film was immersed for 1 min in 2% triethylamine-0.1% Brij 35 in 90% ethanol. It was immediately soaked, without drying, in 0.1% o-phthalaldehyde-0.4% mercaptoethanol-0. I’% Brij with 90% 35 in 90%’ ethanol for 2 min, and washed ethanol. After drying, protein was detected as a bluewhite fluorescent band under illumination with 365-nm light. :i RNase A was recovered quantitatively from the column if it was washed with 0.5 M NaCl after gradient elution with NaCI. The amounts of the fractions which emerged as a result of the treatment were somewhat variable in each experiment.
Buffer
i ... .. . e... ‘;oT
150 Effluent
0 200
ml
FIG. 2. Biospecific desorption of RNase A with ligands from a folate-Sepharose column. RNase A (0.5 mg) was eluted from a column of folate-Sepharose (I x 4 cm) with a 5 mM solution of a ligand in sodium acetate at pH 5.0 and 4°C. Ionic strength in the ligand solutions was 0.01 in all the cases. From top to bottom: elution with U3’(2’)P, AS’P, NH,BzGlu, and buffer.
in Fig. 2.” The data are compatible with the affinity of these ligands for the enzyme in solution at pH 5.0-5.2 and 23-25°C: the affinity constants are 1 x loj M for U2’(3’)P (12), 1 X 10” M for A5’P (l), and 5 x 10:’ M for NH,BzGlu (1). The affinity of RNase A for folate-sepharose gel, as estimated with frontal analysis, was maximum at about pH 5.6 (data not shown). This is entirely consistent with the affinity of folic acid for RNase A in solution (I). The K,t value for folate-RNase A complex in the gel matrix at pH 5.0 and 4°C was ’ The recovery of RNase A from commercial preparations eluted with these ligands from the column was about 90%.
188
FUMIO
SAWADA
estimated to be 9.8 x lo-” M. This value coincides well with 7.5 x lo-” M, the latter obtained in solution at pH 5.2 and 20°C (1).
Folute -Sephmose
Colurntz
A series of proteins were chromatographed on a folate-Sepharose column with NaCl gradient from 0 to 0.1 M pH 5.0 and 4°C (Fig. 3). Ovalbumin was not adsorbed to the column at all and lysozyme was eluted with a much higher concentration (0.45 M) of NaCl. Glutamate dehydrogenase bound firmly to folate-Sepharose; only one-fifth of the preparation was eluted despite the use of 2 M NaCl. The other proteins tested, including RNase A, chromatographed well
on the column although they showed different affinities for the gel. Except for two acidic proteins, ovalbumin and pepsin, most proteins were not adsorbed on epoxy-blocked Sepharose gel that did not contain folate. This is a clear indication that the folate moiety in folate-Sepharose is essential for the affinity of proteins. No clear relationship was observed between the affinity of proteins for folateSepharose gel and the affinity of these proteins for folic acid in solution: some proteins with similar affinities for folic acid in solution differed very much in their affinities for the gel. Electrophoretic mobilities of the proteins correlated roughly with their affinity for folate-Sepharose gel. Thus, basic protein
OV P RTI ctg
t
t
l0
I 50
I 100 Eff
lu
I 150 cnt
I 200
ml
GM
t 250
300
FIG. 3. Eluton patterns of proteins from a folate-Sepharose column. Proteins applied were ovalbumin (0~). pepsin (P). RNase T, (RT,), chymotrypsinogen A (Ctg), u-chymotrypsin (Ct), trypsin (T). RNase A (RA), I IY-(cm)-RNase (cmR). performic acid-oxidized RNase A (oxR). lysozyme (L), and glutamate dehydrogenase (GDH). The elution conditions were the same as described in Fig. 1. The NaCI gradient is indicated at the top of the figure. Arrows indicate the positions of addition of 0.5 M NaCI.
FOLATE-SEPHAROSE
COLUMN
which moved to the cathode had a higher affinity for the gel than did acidic protein with a positive charge. This observation suggests that the two carboxyl groups of the folate moiety play a dominant role in binding. DISCUSSION Sepharose derivatives coupled with folic acid or its analog, methotrexate, havle been utilized for purification of dihydrofolate reductase ( 13,14) and folate-binding protein (15). In these materials the pterins were covalently linked through carboxyl groups of glutamyl residues to the spacer arms attached to the agarose matrix. The structure of folate-Sepharose gel presented here is assumed to be reversed with respect to the site of coupling of the pterin to tihe gel matrix as illustrated in Scheme 1. The validity of the putative structure in which two carboxy1 groups are located at the terminus of the long chain is supported by the cationexchange nature of the gel. There is also supported by the fact that the interaction between RNase A and folic acid in the gel is virtually the same as that in solution where not the pteridine ring but the NH,BzGlu moiety of the ligand is essential (I). However, the exact site(s) for coupling between the pteridine ring and the fspacer arm is not clear. Several absorbents to which pyrimidine nucleotides have been coupled weire developed for affinity chromatography of RNase A (16,17). To these absorbents has been attributed a specific affinity for RNase A at the enzyme site for pyrimidine nucleotide, the site B,-/?,-I) (18). Folate-Sepharose gel differs in that it will interact with RNase A at a site for folic acid at or near the site B2-R2(1,2). thereby providing a different specificity. Folate- Sepharose gel resembles blue dextran-Sepharose, a group-specific affinity absorbent for nucleotide-binding proteins (19,20), in its polyaromatic and polyanionic characteristics. Although folate-Sepharose is not a group specific, it may find wide application in chromatography of basic proteins
IN PROTEIN
FRACTIONATION
as lysozyme which have a high affinity the gel in the alkaline region.
189 for
ACKNOWLEDGMENTS The author wishes to express his thanks to Professor T. Tobita, Chiba University, for amino acid analysis and to Dr. H. Ohyama and Dr. T. Yamada in the NIRS for the use of an electrophoresis apparatus.
REFERENCES 1. Sawada, F., Kanesaka. Y.. and Irie, M. (1977) Biochim. BiophyA. A~IU 479, 188- 197. 1. Torii. K.. Urata, Y., Iitaka. Y.. Sawada, F.. and Mitsui. Y. (1978) J. Biochrm. (Tokyo) 83, 1239- 1247. 3. White, W. E.. Jr., Yielding, K. L., and Krumdiek, C. L. (1976) Biochirn. Biophy,s. Actor 55, 361373. 4. Hirs,C. H. W.(1956)J. Bid. Chew. 219.611-622. 5. Crestfield, A. M.. Stein. W. H., and Moore. S. (1963) J. Bid. C‘hc~m. 234, 2413-2420. 6. Sawada. F.. and Kanbayashi. N. ( 1973)J. Bkhem. ( TdJYJJ 74, 459-47 1. 7. Cohn, W. E. (1955) ;,I The Nucleic Acids (Chargaff, E., and Davidson, J. N., eds.) Vol. 1. p. 513. Academic Press, London/New York. 8. Kasai, K., and lshii. S. (1975)J. Biochern. (Tokyo) 77, 261-264. 9. Hummel. J. P.. and Dreyer, W. (1962) Biochim. Bitrphy.\. Actu 63. 530-532. IO. Kunkek. H. G.. and Tiselius. A. (1959) .1. G‘c,lcr. Ph:~.\io/. 35, 89- 118. I I. Sawada. F. (1969) J. Biochrnl. (7’okw) 65, 767776. I?. Sawada. F.. and Irie. M. ( 1%9)5. Bioch~m. (7’1k~~) 66, 415-418. 13. Kaufman, B. T. (1974) in Methods in Enzymology (Jakoby, W. B., and Wilchek. M.. eds.). Vol. 34, Part B. pp. 272-381. Academic Press. New York/San Francisco/London. 14. Whitely, J. B., Henderson, G. B.. Russel, A.. Singl, P.. and Zevely. E. M. (1977) Ad. Bkchern. 79, 42-51. 15. Waxman, S., and Schreiber, C. (1975) Bioc~hrntist~ 14, 5422-5428. 16. Wilchek, M.. and Gorecki, M. (1969) Eur. J. Biochern. II, 491-494. 17. Scofie1d.R. E.. Werner.R. P.,and Wo1d.F. (1977) Atrctl. Biochem. 77. 152- 157. 18. Richards, F.. Wyckoff. H. W.. Carlson. W. D.. Allewell. N. M.. Lee, B., and Mitsui, Y. (1971) Co/d Spring Hnrhor Symp. Quant. Bid. 36, 35.-43. 19. Thompson. S. T.. Cass. K. H., and Stellwagen. E. (1975) Proc. Not. Acud. Sci. USA 72, 669-672. 20. Procourt, J.-L., Thang. D.-C., and Thang, M.-N. ( 1978) Eur. J. Biochrm. 82, 355-362.
98. 184- 189 (1979)
BIOCHEMISTRY
Folate-Sepharose
Column as an Affinity Protein Fractionation FUMIO
Division
of Chemistr.v.
Nationul
for
SAWADA
of
Rdiolo,yicul
Received
January
Institute
Absorbent
Sciences,
Anugu~~u.
Chiba
City,
260 Jupun
22, 1979
Folate-Sepharose gel was prepared by direct coupling of fohc acid to epoxy-activated Sepharose. Bovine pancreatic ribonuclease A was adsorbed on the column at pH 5.0 and was desorbed with ligands which had an affinity for the enzyme. The pH dependency of the affinity of the enzyme for folic acid in the gel matrix was similar to that in solution. Bovine liver glutamate dehydrogenase exhibited a very strong affinity for the gel. Cationexchange character was the dominant factor in the chromatography of proteins on a folate-Sepharose column.
Folic acid, whose derivatives are metabolic cofactors in the one-carbon transfer reactions, has a unique affinity for bovine pancreatic ribonuclease A (RNase A) (1.2) and for liver glutamate dehydrogenase (3). The pteridine moiety of folic acid, which is essential for metabolic reactions, is not essential in both cases of interaction with these proteins. To obtain further information on the nature of the interaction of folic acid with a series of proteins, an affinity column, in which folic acid was coupled to an agarose gel matrix through the pteridine moiety, was prepared. This product proved effective in separating protein mixtures including RNase A and its derivatives. MATERIALS
egg-white lysozyme from Sigma Chemical Company; swine stomach pepsin from P.-L. Biochemical Inc.; and Asprrgilfus ribonuclease T, (RNase T,) from Sankyo Company, Tokyo. Performic acid-oxidized RNase A was prepared by the method of Hirs (4), carboxymethylhistidine-1 I9-RNase A (1 I9-His(cm)-RNase A)’ after Crestfield et crl. (5), and 4-thiouridylate-photosensitized RNase A was the same preparation described in Fig. 9c of Ref. (6). Folic acid andp-aminobenzoylglutamic acid (NH,BzGlu) were products of Tokyo Kasei Kogyo Company, Tokyo. Adenosine 5’-phosphate (A5’P) and uridine 3’(2’)-phosphate (U3’(2’)P) were from Boehringer. The latter was almost exclusively the 3’-isomer as estimated by the absorption ratio, A2,,,/A26,, (7).
AND METHODS
Materials
Prrpuratim
Epoxy-activated Sepharose 6B and Sephadex G-25 were products of Pharmacia Fine Chemicals. Separax S cellulose acetate film was the product of Fuji Photo Film Company, Tokyo. RNase A, cu-chymotrypsin, trypsin, and liver glutamate dehydrogenase (all from beef) were obtained from Boehringer/Mannheim; bovine chymotrypsinogen A and ovalbumin from Mann Research Laboratories; 0003-26971791130
184-06$02.00/O
Copynghl GI 1979 by Academx Prers, Inc. All rights of reproductwn m any form reserved
oj’Folatt> -Sephrosr
Gel
Eight grams of epoxy-activated Sepharose 6B was hydrated and washed in distilled water, equilibrated with 0.1 M sodium bicarbonate (pH 10.0). and the supernatant of ’ Abbreviations used: 119.Histcm), carboxymethylhistidine-119: NH,BzGlu,p-aminobenzoylglutamic acid, A5’P. adenosine 5’-phosphate: U3’(2’)P, uridine 3’(2’)phosphate. 184
FOLATE-SEPHAROSE
COLUMN
IN PROTEIN
the settled gel decanted. To the gel was added 100 ml of 0.01 M folic acid dissolved in 0.1 M bicarbonate (pH adjusted to 10.0 with NaOH) and the mixture was shaken for 16 h at 30°C in the dark. The brownish yellow gel was collected on a sintered glass filter and washed with 400 ml of bicarbonate buffer, then successively with 100 ml each of distilled water, 0.5 M NaCl in 0.1 M sodium bicarbonate (pH 8), 0.5 M NaCl in 0.1 M sodium acetate (pH 4), and distilled water. The washed gel was treated with the folic acid solution four more times. The residual epoxy groups in the gel were blocked by shaking the gel with 80 ml of I M monoethanolamine for 8 h at 30°C in the dark. Finally, the gel was thoroughly washed as described above. The settled volume of the gel was 23.2 ml. The amount of folic acid coupled to Sepharose was 5.4 pmol,lml as estimated from the glutamic acid contents obtained by amino acid analysis. The product could be stored under 0.02% NaN, :at 4°C at least I year. The putative structure of the product is shown in Scheme I.
185
FRACTIONATION
with 100 ml of 1 M monoethanolamine for 24 hat 30°C to block the active epoxy groups and washed thoroughly as described. The product was colorless and had a tendency to stick on the surface of glassware. The putative structure of the product is shown in Scheme 1. Protein
Binding
to the F&ate - Sepharose
Gel A. Elation analysis. Protein was eluted from a folate-Sepharose column at 4°C with a concave NaCl gradient in sodium acetate at pH 5.0. Biospecific desorption of the bound protein from the column was achieved with 5 mM solutions of ligands at pH 5.0 and 4°C. B. Frontal analysis. A solution of protein was continuously applied onto a small column of folate-Sepharose gel and the concentration of protein in effluent fractions was measured. The dissociation constant ( Kd) of the protein-folate-gel complex was estimated using the equation K,[ = ~
L
v - vo
Preparation of Epoxy-Blocked Sepharose Gel
- [-%I,
where L is the total amount of folate attached to the gel column, V and V. are the elution volumes of the protein with and without interaction with the gel, respectively, and [E,] is the concentration of the protein solution (8).
Eight grams of epoxy-activated Sepharose 6B was treated with 0.1 M sodium bicarbonate at pH 10.0 in the same manner as in the preparation of folate-Sepharose except for the omission of folic acid. The gel was shaken
FOOH Sepharose-O-CH2-CH-CH20-CH2-CH2-CH2-CH*-CH*-C~~,-O-CH~-~H-CH~-NH‘. OH
OH
a)-CH,-NH-@O-&COOH OH
Folate-Sepharose
SepharOSe-O-CH2-CH-CHZO-CH2-CHZ-CH2-CH2-O-CH*-CH-CH*-NH-CH~-C~~-OH bH
Epoxy-Blocked
bH
Sepharose SCHEME
I
186
FUMIO
0
SAWADA
I
L
I
50
100
150
-NoCl
Gradlent
(O+O.lSM),pH
I
,
250 ml
200 5.0
-
;.5M
NaCl
FIG. 1. Elution of RNase A and its photoproducts from a folate-Sepharose column. Photosensitized RNase A was prepared by irradiation of the enzyme with near-ultraviolet light in the presence of 4-thiouridine 2’(3’)-phosphate as described in Fig. 9c of Ref (6). The column (I x 26 cm) was equilibrated with sodium acetate (pH 5.0 at 4°C. Na = 0.005 M). A solution of protein (0.5-0.6 mgi0.2-0.25 ml) was added to the column and was eluted at 4°C with a concave NaCl gradient consisting of 178 ml of the buffer in a mixer and 100 ml of 0.20 M NaCl in the buffer in a reservoir. The gradient curve is shown in Fig. 3. Enzyme activity was assayed with RNA as substrate (11). (0 0) Absorbance at 220 nm; (0 - - - 0) enzyme activity in arbitrary units. (Top) native RNase A; (bottom) photosensitized RNase A.
Binding of Folic Acid to Proteins in Solution The affinity of proteins for folic acid in solution was measured by the Hummel and Dreyer gel filtration method (9) on columns of Sephadex G-25 (fine). The molar ratios of the bound ligand to the proteins were calculated using the following values of molecular weight: RNase T, (11 ,OOO), RNase
A (13,700), lysozyme (13,900), trypsin (23,000), cY-chymotrypsin (25,300), chymotrypsinogen A (25,700), pepsin (34,200), ovalbumin (45,000), and glutamate dehydrogenase monomer (56,000). Cellulose
Acetate
Film Electrophoresis
Electrophoretic mobilities of proteins on a Separax S cellulose acetate film were
FOLATE-SEPHAROSE
COLUMN
IN PROTEIN
187
FRACTIONATION
determined at pH 5.0 and 4°C with correction for electroendoosmosis using dextran as a marker (10). Proteins on the film were detected by staining with 0.5% Coomassie brilliant blue R250 in acetic acid-methanolwater (1:4:5, v/v) or by fluorescence after the phthalaldehyde reaction.2
u 312?P
RESULTS
As shown in Figs. 1 and 3, RNase A bound firmly to folate-Sepharose gel under low salt concentration and was eluted from the column with 0.09 M NaCI.” The enzyme, photosensitized with 4-thiouridine 2’(3’)phosphate (6,11) was fractionated into several components with different degrees of enzyme activity (Fig. 1, bottom). The resolution of the peaks was much better than that obtained with a longer column of ionexchange resin Amberlite IRC-50 (6). The column also appears to be useful for a carboxymeth.ylated derivative of the enzyme, 119-His(cm)RNase A (cmR in Fig. 3). A small shoulder at the right-hand site is considered to be due to contamination by another inactive derivative of the enzyme, carboxymethylhistidineI2-RNase A. Biospecific desorption of RNase A from a folate- Sepharose column was acihieved with a 5 mM solution of three dilfferent ligands at pH 5.0 and 4°C. U3’(2’)P had the highest affinity and both A5’P and NH,BzGlu had a similar ability for desorption as :shown ? The cellulose acetate film was immersed for 1 min in 2% triethylamine-0.1% Brij 35 in 90% ethanol. It was immediately soaked, without drying, in 0.1% o-phthalaldehyde-0.4% mercaptoethanol-0. I’% Brij with 90% 35 in 90%’ ethanol for 2 min, and washed ethanol. After drying, protein was detected as a bluewhite fluorescent band under illumination with 365-nm light. :i RNase A was recovered quantitatively from the column if it was washed with 0.5 M NaCl after gradient elution with NaCI. The amounts of the fractions which emerged as a result of the treatment were somewhat variable in each experiment.
Buffer
i ... .. . e... ‘;oT
150 Effluent
0 200
ml
FIG. 2. Biospecific desorption of RNase A with ligands from a folate-Sepharose column. RNase A (0.5 mg) was eluted from a column of folate-Sepharose (I x 4 cm) with a 5 mM solution of a ligand in sodium acetate at pH 5.0 and 4°C. Ionic strength in the ligand solutions was 0.01 in all the cases. From top to bottom: elution with U3’(2’)P, AS’P, NH,BzGlu, and buffer.
in Fig. 2.” The data are compatible with the affinity of these ligands for the enzyme in solution at pH 5.0-5.2 and 23-25°C: the affinity constants are 1 x loj M for U2’(3’)P (12), 1 X 10” M for A5’P (l), and 5 x 10:’ M for NH,BzGlu (1). The affinity of RNase A for folate-sepharose gel, as estimated with frontal analysis, was maximum at about pH 5.6 (data not shown). This is entirely consistent with the affinity of folic acid for RNase A in solution (I). The K,t value for folate-RNase A complex in the gel matrix at pH 5.0 and 4°C was ’ The recovery of RNase A from commercial preparations eluted with these ligands from the column was about 90%.
188
FUMIO
SAWADA
estimated to be 9.8 x lo-” M. This value coincides well with 7.5 x lo-” M, the latter obtained in solution at pH 5.2 and 20°C (1).
Folute -Sephmose
Colurntz
A series of proteins were chromatographed on a folate-Sepharose column with NaCl gradient from 0 to 0.1 M pH 5.0 and 4°C (Fig. 3). Ovalbumin was not adsorbed to the column at all and lysozyme was eluted with a much higher concentration (0.45 M) of NaCl. Glutamate dehydrogenase bound firmly to folate-Sepharose; only one-fifth of the preparation was eluted despite the use of 2 M NaCl. The other proteins tested, including RNase A, chromatographed well
on the column although they showed different affinities for the gel. Except for two acidic proteins, ovalbumin and pepsin, most proteins were not adsorbed on epoxy-blocked Sepharose gel that did not contain folate. This is a clear indication that the folate moiety in folate-Sepharose is essential for the affinity of proteins. No clear relationship was observed between the affinity of proteins for folateSepharose gel and the affinity of these proteins for folic acid in solution: some proteins with similar affinities for folic acid in solution differed very much in their affinities for the gel. Electrophoretic mobilities of the proteins correlated roughly with their affinity for folate-Sepharose gel. Thus, basic protein
OV P RTI ctg
t
t
l0
I 50
I 100 Eff
lu
I 150 cnt
I 200
ml
GM
t 250
300
FIG. 3. Eluton patterns of proteins from a folate-Sepharose column. Proteins applied were ovalbumin (0~). pepsin (P). RNase T, (RT,), chymotrypsinogen A (Ctg), u-chymotrypsin (Ct), trypsin (T). RNase A (RA), I IY-(cm)-RNase (cmR). performic acid-oxidized RNase A (oxR). lysozyme (L), and glutamate dehydrogenase (GDH). The elution conditions were the same as described in Fig. 1. The NaCI gradient is indicated at the top of the figure. Arrows indicate the positions of addition of 0.5 M NaCI.
FOLATE-SEPHAROSE
COLUMN
which moved to the cathode had a higher affinity for the gel than did acidic protein with a positive charge. This observation suggests that the two carboxyl groups of the folate moiety play a dominant role in binding. DISCUSSION Sepharose derivatives coupled with folic acid or its analog, methotrexate, havle been utilized for purification of dihydrofolate reductase ( 13,14) and folate-binding protein (15). In these materials the pterins were covalently linked through carboxyl groups of glutamyl residues to the spacer arms attached to the agarose matrix. The structure of folate-Sepharose gel presented here is assumed to be reversed with respect to the site of coupling of the pterin to tihe gel matrix as illustrated in Scheme 1. The validity of the putative structure in which two carboxy1 groups are located at the terminus of the long chain is supported by the cationexchange nature of the gel. There is also supported by the fact that the interaction between RNase A and folic acid in the gel is virtually the same as that in solution where not the pteridine ring but the NH,BzGlu moiety of the ligand is essential (I). However, the exact site(s) for coupling between the pteridine ring and the fspacer arm is not clear. Several absorbents to which pyrimidine nucleotides have been coupled weire developed for affinity chromatography of RNase A (16,17). To these absorbents has been attributed a specific affinity for RNase A at the enzyme site for pyrimidine nucleotide, the site B,-/?,-I) (18). Folate-Sepharose gel differs in that it will interact with RNase A at a site for folic acid at or near the site B2-R2(1,2). thereby providing a different specificity. Folate- Sepharose gel resembles blue dextran-Sepharose, a group-specific affinity absorbent for nucleotide-binding proteins (19,20), in its polyaromatic and polyanionic characteristics. Although folate-Sepharose is not a group specific, it may find wide application in chromatography of basic proteins
IN PROTEIN
FRACTIONATION
as lysozyme which have a high affinity the gel in the alkaline region.
189 for
ACKNOWLEDGMENTS The author wishes to express his thanks to Professor T. Tobita, Chiba University, for amino acid analysis and to Dr. H. Ohyama and Dr. T. Yamada in the NIRS for the use of an electrophoresis apparatus.
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