- Email: [email protected]
Affinophoresis of trypsins with an anionic affinophore
Biochimica etBiophysicaActa, 802 (1984) 135-140
135
Elsevier
BBA21881
AFFINOPHORESIS OF TRYPSINS WITH AN ANIONIC AFFINOPHORE KIYOHITO SHIMURA and KEN-ICHI KASAI
Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-01 (Japan) (Received May 18th, 1984)
Key words.. Trypsin; Affinity electrophoresis;Affinity ligand
Affinophoresis (Shimura, K. and Kasai, K. (1982) J. Biochem. 92, 1615-1622) is a newly devised electrophoretic separation technique for biomolecules, using an affinophore. The affinophore is a macromolecular polyelectrolyte bearing affinity iigands. It migrates rapidly in an electric field, and molecules which have affinity for the ligand are carried with it and separated from other molecules. An anionic affinophore for trypsin was synthesized, p-Aminobenzamidine, a competitive inhibitor of trypsin, was coupled to one-fifth of the carboxyi groups of polyacrylyl-fl-alanyl-fl-alanine by the use of water-soluble carbodiimide and the residual carboxyls were converted to suffonate groups by coupling with aminomethanesulfonic acid. Affinophoresis was carried out in 1% agarose gel plates, and the protein bands were detected with Coomassie brilliant blue R250. Enhanced migrations of bovine and Streptomyces griseus trypsins towards the anode were observed with the anionic affinophore. The migrations of inactive forms prepared by active site modifications were scarcely affected. However, the affinophore was not effective for Streptomyces erythreus trypsin, an anionic trypsin, probably because of ionic repulsion between the anionic molecules. S. griseus trypsin was separated from Pronase by affinophoresis.
Introduction Affinophoresis [1], a new approach for the separation of biomolecules, is based on the specific affinity of a biomolecule to be separated and on the electric interaction as a driving force of separation. This technique requires a carrier molecule, the affinophore, which is a macromolecular polyelectrolyte containing affinity ligands. In an electric field, the affinophore rapidly migrates due to its charges, and carries with it molecules which have specific affinity for the ligand. Two main distinctive features of affinophoresis are apparent from its principle. First, afflnophore-
Abbreviation: TLCK, heptanone.
L-l-chloro-3-tosylamido-7-amino-2-
0304-4165/84/$03.00 © 1984 Elsevier Science Publishers B.V.
sis is effective for neutral substances or substances which have poor effective charges for electrophoresis. Second, no insoluble support to immobilize the affinity ligands is necessary, whereas it is indispensable for so-called 'affinity electrophoresis' [2,3] or affinity chromatography [4]. We have reported the first application of this technique [1] in which we prepared a cationic affinophore for trypsins: a dextran derivative containing diethylaminoethyl groups as cationic groups and m-aminobenzamidine as the affinity ligand. In this paper, we describe the affinophoresis of trypsins with a newly synthesized anionic affinophore, which contains sulfonate groups as ionic groups and p-aminobenzamidine [5] as the affinity ligand on a matrix of polyacrylyl-fl-alanylfl-alanine.
136 Materials and Methods
The following materials were obtained from commercial sources: p-aminobenzamidine monohydrochloride, L-l-chloro-3-tosylamido-7-amino2-heptanone (TLCK) (Sigma, St. Louis, MO, U.S.A.); Sepharose CL-6B (Pharmacia Fine Chemicals, Uppsala, Sweden); agarose GP-36 (Nakarai Chemicals Co., Kyoto); Pronase P [6] (Kaken Chemical Co., Tokyo); 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide [7] (Dojin Laboratories, Kumamoto, Japan). fl-Alanine methyl ester hydrochloride was synthesized from fl-alanine in methanol using thionyl chloride. It was hygroscopic. Purified Streptomyces erythreus trypsin [8] and Streptomyces griseus trypsin [9] were gifts from Professor F. Sakiyama (Protein Research Institute, Osaka University) and Dr. M. Nakano (Faculty of Pharmaceutical Sciences, Hokkaido University), respectively. Preparation of TLCK-treated trypsins (TLCK-trypsins) [10] was described previously [1]. Other chemicals of the purest grade available were obtained from Wako Pure Chemical Industries (Osaka).
Synthesis of acrylyl-fl-alanine In a flask shielded from light, fl-alanine (17.8 g) and hydroquinone (20 mg) were dissolved in 50 ml of 2 M NaOH. Acrylyl chloride (25 g) was added in small portions over 30 min to the solution on an ice bath with vigorous stirring. The pH of the solution was maintained at 9-10 by the addition of 2 M NaOH. After another 5 min, the solution was acidified to pH 2 with 6 M HCI. The solution was frozen at - 8 0 ° C and dried over 3 days in the dark by evaporation, using a vacuum pump without heating. Acrylyl-fl-alanine was extracted from the solid three times, each time with 100 ml acetone, and the extract was applied to a column of silica gel (50 g, 10 x 3.5 cm i.d.). The column was eluted with acetone. The effluent containing acrylyl-flalanine (350 ml), detected by ultraviolet absorption measurement after thin-layer chromatography on silica gel (R F = 0.35, chloroform/acetic acid (4 : 1, v/v)), was concentrated in a rotary evaporator and then left to crystallize. The crystals were collected by filtration, washed twice with 20 ml of acetone/diethyl ether (1 : 1, v / v ) and dried in air.
The product was recrystallized from ethyl acetate by the addition of petroleum ether. Yield: 15.7 g (55%). M.p.: 94-98°C. Elemental analysis. Calc. for C6H9NO3: C, 50.34; H, 6.35; N, 9.80. Found: C, 49.97; H, 6.38; N, 9.71.
Synthesis of acrylyl-fl-alanyl-fl-alanine methyl ester fl-Alanine methyl ester hydrochloride (5.38 g), triethylamine (5.37 ml), hydroquinone (8 mg), acrylyl-fl-alanine (5.51 g) and dicyclohexylcarbodiimide (7.94 g) were added and dissolved consecutively in dichloromethane (38.5 ml), and the solution was left overnight at room temperature in the dark. Methanol (38.5 ml) was added, and the resultant slurry was stirred for 1 h at room temperature and then filtered. The filtrate was concentrated in a rotary evaporator, and the residue was dissolved in methanol (58 ml) containing 0.02% hydroquinone and filtered once more. The filtrate was passed through a column of Amberlite MB-1 (140 ml, 25 x 2.5 cm i.d., equilibrated with methanol) at a flow rate of 4 ml per min with methanol containing 0.02% hydroquinone as a solvent. The effluent containing the product (120 ml), detected by ultraviolet absorption measurement after thin-layer chromatography on silica gel (R v = 0.45, chloroform/methanol (8 : 1, v/v)), was evaporated and the residue was dissolved in chloroform (20 ml). The solution was applied to a column of silica gel (200 g, 36 x 3.4 cm, i.d.) equilibrated with chloroform/methanol (8 : 1, v / v ) containing 0.02% hydroquinone and eluted with the solvent at a flow rate of 2.5 ml per rain. The effluent containing the product (220 ml) was evaporated and the residue was crystallized from hot ethyl acetate (150 ml). Yield: 4.21 g (48%). M.p.: 129-131°C. Elemental analysis. Calc. for C10H16N204: C, 52.62; H, 7.07; N, 12.27. Found: C, 52.74; H, 7.11; N, 12.35.
Preparation of the affinophore (Fig. 1) Acrylyl-fl-alanyl-fl-alanine methyl ester (0.5 g, 2.2 mmol) was dissolved in water (4 ml) and mixed with 0.4 ml of 6 M NaOH. The solution was allowed to stand for 30 min on an ice bath to hydrolyze the ester. After neutralization with 6 M HC1, 50 ~tl of 10% (v/v) thioglycolic acid, which controls the degree of polymerization, and 100/~1 of 5% (w/v) ammonium persulfate were added.
137 The container was sealed after evacuation with a water-jet pump, and the monomer was polymerized at 70°C for 20 h. p-Aminobenzamidine hydrochloride [5] (75.5 mg, 0.44 mmol) was added to the solution of the polymer, and the pH was brought to 5 with 6 M HC1. 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride [7] (170 mg) was added in 3 portions over 5 min. After 1 h, almost all the p-aminobenzamidine was bound to the polymer (checked by ultraviolet absorption measurement after thin-layer chromatography on silica gel, n-butanol/acetic a c i d / w a t e r ( 4 : 1 : 2 , v/v)). A 1 ml portion of the reaction mixture was removed, diluted with water (4 ml), dialyzed against water and freeze-dried after neutralization with 1 M N a O H (product I, 0.1 g). To the residual solution, aminomethanesulfonic acid (220 mg) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (420 mg) were added in three portions over 5 rain. The pH was maintained between 4.5 and 5.0 with 6 M N a O H for 1 h. The solution was diluted with water (16 ml), dialyzed against water (5 1 x 3), neutralized with 1 M N a O H and freeze-dried (product II, 0.55 g).
a cold room (4°C). Proteins were stained according to the literature [11] except that a staining solution of 0.5% Coomassie brilliant blue R250 in methanol/acetic a c i d / w a t e r (4 : 1 : 5, v / v ) and a destaining solution of methanol/acetic a c i d / w a t e r (2 : 1 : 7, v / v ) were used.
Analysis of the affinophore The affinophore solutions (1%, 0.1 ml) were evaporated, and the residues were hydrolyzed in 6 M HC1 at l l 0 ° C for 16 h in evacuated and sealed tubes. The content of p-aminobenzamidine was determined by spectrophotometry (X max = 292 nm, e = 1.54.10 4 M - ] • c m - 1 in 50 mM sodium phosphate buffer (pH 7.0)) with correction for the recovery during the hydrolysis (79%). The content of fl-alanine was determined with an amino acid analyzer. Results
Preparation of the anionic affinophore for trypsin Analysis of the affinophore (product II) showed 0
0
0
Affinophoresis Agarose powder (0.1 g), 1 ml of 1 M sodium phosphate buffer (pH 7.0) and water (8-9 ml) were mixed and heated on a boiling water bath. After dissolution, 1% ( w / v ) a f f i n o p h o r e solution was added to make a total volume of 10 ml, and the solution was spread on a Gel Bond film (12.5 × 8 cm; FMC, Marine Colloids Division, Rockland, ME, U.S.A.). The gel plate was placed on the cooling block (with circulating ice-cold water) of an electrophoretic apparatus (ATTO, SJ-1073, Tokyo) with its shorter edges facing the electrodes. A minimum amount of water was spread between the support film and the cooling block. Samples (2 /~1), which consisted of 4 ~g of trypsin (16 ~tg in the case of Pronase) or TLCK-treated trypsin and 8 ttg of the affinophore in 0.1 M sodium phosphate buffer (pH 7.2), were applied on the middle of the plate perpendicular to the longitudinal axis, using Sample Application Foil (LKB, 2117-206, Bromma, Sweden). The plate was bridged to the electrode solution, 0.1 M sodium phosphate buffer (pH 7.2). Electrophoresis was carried out at a constant current of 100 mA per plate for 40 min in
(5~q.)
I polyrnerizotion o
o
1) pABA(leq.)/WSC 2) H3NCH2SO~'/WSC ÷
NHz I'+
O
O /~
c~NH2 (leq.) (4eq.)
affinophore for trypsin Fig. 1. P r e p a r a t i o n of the a n i o n i c affinophore.
138
I
TGf
BD
20
30 40 tube number
50
Fig. 2. Gel chromatography of the affinophore. Product II (0.17 mg in 0.1 ml) was applied to a column of Sepharose CL-6B (50X 1 cm id.) equilibrated with 0.1 M sodium phosphate buffer (pH 7.0) and eluted with the buffer at a rate of 5.5 ml per h. Effluent was collected in fractions of 1 ml per tube, and the concentration of the affinophore was determined by ultraviolet absorption measurement. The arrows indicate the positions at which the following substances were eluted: BD, Blue dextran; TG, thyroglobulin (porcine, IU, (6.3-6.5).10’); IG, immunoglobuhn G (bovine, M, (1.551.55).10’); A, serum albumin (bovine, M, 6.6.104); G, glucose.
that 1% solution of the affinophore consists of 47 mM /3-alanine residue and 4.6 mM p-aminobenzamidine residue. This means that the ratio of the incorporated p-aminobenzamidine and the monomer in the final product was the same as that used in the reaction (Fig. 1). Titration of product I and product II showed that 95% of the carboxyl groups of product I was coupled with aminomethanesulfonic acid in product II (data not shown). Product II was applied to a column of Sepharose CLdB (Fig. 2). Almost all the affinophore was eluted behind thyroglobulin (M, 6.4. 105). Thus, we can expect that the affinophore would migrate practically freely in the 1% agarose gel used in the present experiments, because it is known that 1% agarose gel has a very low level of sieving effect for proteins with an M, lower than 5.105 [ll]. Zone electrophoresis of the affinophores was carried out under the same conditions as the affinophoresis described below. As shown in Fig. 3, product I (containing carboxyl groups) was heavily stained with the dye (lane 2). On the other hand, product II (containing sulfonate groups) was not stained (lane 3). In a separate experiment, in which the affinophores were detected by ultraviolet absorption measurements, product II migrated
Fig. 3. Agarose gel electrophoresis of the affinophores and trypsins. 2 pl each of S. griseus trypsin (4 pg, lane I), product 1 (20 pg. lane 2). product II (20 pg, lane 3) and S. erythreus trypsin (4 pg, lane 4) were applied to an agarose gel plate (12.5 x 8 cm) at the position marked as 0. Electrophoresis was carried out at 100 mA for 40 min. Dotted lines indicate the positions of the edges of the bridging sponges.
with a velocity of 90% of that of product I under the same conditions as in the experiment in Fig. 3 (data not shown). Product II was preferred in the present work, because it does not interfere with the detection of proteins with Coomassie blue dye.
a3 5
-_ .__..(l..._._...___._.*____ . . . . .. .i
3 cm 0
-3
; Fig. 4. Affinophoresis of trypsins with the anionic affinophore (product II). Electrophoresis of trypsins and trypsins treated with TLCK (4 pg each) was carried out in the absence (A) or presence (B) of the affinophore (0.1%). Details of the procedure were as described in the text. Lane 1, S. griseus trypsin; lane 2, TLCK-S. grisew trypsin; lane 3, S. etythreus trypsin; lane 4, TLCK-S. etythreus trypsin; lane 5, bovine trypsin; lane 6, TLCK-bovine trypsin.
139
E~
!
!
i
I
!
I
A
B
4
i
I
IC
!
i
!
~3 ~2 o=
o
--~ 0 E -I
@
Ii
|
*
i
I
|11
0.05 0.1 0.05 0.1 0 0.05 0.1 0 concentration of affinophore ( w / v % )
Fig. 5. Dependency of the migration of trypsins on the concentration of the affinophore (product II). A, S. griseus trypsin; B, S. erythreus trypsin; C. bovine trypsin. O, native trypsins; O, TLCK-trypsins.
Affinophoresis of trypsins Trypsins and their inactive derivatives prepared by affinity labeling with T L C K [10] were subjected to affinophoresis (Fig. 4). Compared with the electrophoretogram without affinophore (plate A), the enhanced migration of S. griseus and bovine trypsin toward anode in the presence of the affinophore is evident (plate B). Such an effect was not observed for TLCK-treated S. griseus or bovine trypsin. This indicates that the active sites of the enzymes play a principal role in the formation of the trypsin-affiniphore complexes. For S. erythreus trypsin, an anionic trypsin (pI 4.0) [8], however,
Fig. 6. Separation of S. griseus trypsin from Pronase P by affinophoresis. Electrophoresis of S. griseus trypsin (4/Lg, lane 1), Pronase P (16 #g, lane 2) and TLCK-S. griseus trypsin (4 #g, lane 3) was carried out in the absence (A) or presence (B) of the affinophore (product II, 0.02%).
this anionic affinophore was not effective. The dependency of the migration of trypsins on the concentration of the affinophore in the agarose gel plate is summarized in Fig. 5. S. griseus trypsin reached the maximum migration at a lower concentration of the affinophore than bovine trypsin. This situation is consistent with the observation that benzamidine inhibits S. griseus trypsin 10times more strongly than bovine trypsin [12]. The slight effect of the affinophore on TLCK-treated S. griseus and bovine trypsin could be considered to be a result of ionic interactions.
Separation of S. griseus trypsin from Pronase by affinophoresis Pronase [6] is a mixture of proteinases, including S. griseus trypsin, produced by a strain of S. griseus. Specific separation of S. griseus trypsin from Pronase was achieved by affinophoresis (Fig.
6). Discussion
In the previous paper, we described the principle of affinophoresis and its application to trypsins using a cationic affinophore [1]. In this report affinophoresis of trypsins using an anionic affinophore is presented. A new approach to preparation of affinophores proved to be successful. A hydrophilic polymer, polyacrylyl-fl-alanyl-fl-alanine, was chosen as the matrix of the affinophore. Its carboxyl group was readily coupled with amino ligands by the use of water-soluble carbodiimide. The density of the affinity ligand on the affinophore could be controlled by varying the amount of the affinity ligand used in the reaction as a limiting reactant. The hydrophilic dipeptide spacer, fl-alanyl-/3-alanine, which protrudes from the main chain of the polymer, should reduce not only steric hindrance but also ionic repulsion between the matrix of the affinophore and proteins. A considerable improvement was brought about by the conversion of the anionic groups of the affinophore from carboxyl to sulfonate groups, because product II was then no longer stainable by protein dyes. This greatly facilitated detection of the protein bands after affinophoresis. The anionic affinophore was ineffective for the
140
anionic trypsin, S. erythreus trypsin, in contrast to the cationic affinophore, which was most effective for S. erythreus trypsin as described previously [1]. A major cause of the ineffectiveness is presumably ionic repulsion between the affinophore and the enzyme. Therefore, use of an affinophore which has opposite charges to a protein to be separated by affinophoresis is recommended. One of the distinctive features of affinophoresis is that it can be carried out without any insoluble support. This feature is favorable for the electrophoretic separation of particles such as cells or organelles. Since each sub-set of lymphocytes has distinct surface markers, they should be separable with affinophores carrying specific binding proteins, for example, lectins or antibodies, etc. The scope for further development and application of affinophoresis seems considerable.
Acknowledgements We wish to acknowledge the generous gifts of purified S. erythreus trypsin from Prof. Fumio Sakiyama (Protein Research Institute, Osaka University) and purified S. griseus trypsin from Dr.
Michiharu Nakano (Faculty of Pharmaceutical Sciences, Hokkaido University). We are indebted to Ms. Mari Kubodera and Mr. Keishi Uemura for their technical assistance.
References 1 Shimura, K. and Kasai, K. (1982) J. Biochem. 92, 1615-1622 2 Takeo, K. and Nakamura, S. (1972) Arch. Biochem. Biophys. 153, 1-7 3 Ho~ej~i, V. and Kocourek, J. (1974) Biochim. Biophys. Acta 336, 338-343 4 Cuatrecasas, P., Wilchek, M. and Anfinsen, C.B. (1968) Proc. Natl. Acad. Sci. USA 61,636-643 5 Mares-Guia, M. and Show, E. (1965) J. Biol. Chem. 240, 1579-1585 6 Narahashi, Y. (1970) Methods Enzymol. 19, 651-664 7 Hoare, D.G. and Koshland, D.E., Jr. (1967) J. Biol. Chem. 242, 2447-2453 8 Yoshida, N., Sasaki, A. and Inoue, H. (1971) FEBS Lett. 15, 129-132 90lafson, R.W. and Smillie, L.B. (1975) Biochemistry 14, 1161-1167 10 Show, E. (1967) Methods Enzymol. 11,677-686 11 Miyake, J., Fehrnstr6m, H. and Wallenborg, B. (1977) LKB Application Note 310, LKB-Produkter, Bromma, Sweden 12 Kasai, K. and lshii, S. (1978) J. Biochem. 84, 1051-1060
135
Elsevier
BBA21881
AFFINOPHORESIS OF TRYPSINS WITH AN ANIONIC AFFINOPHORE KIYOHITO SHIMURA and KEN-ICHI KASAI
Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-01 (Japan) (Received May 18th, 1984)
Key words.. Trypsin; Affinity electrophoresis;Affinity ligand
Affinophoresis (Shimura, K. and Kasai, K. (1982) J. Biochem. 92, 1615-1622) is a newly devised electrophoretic separation technique for biomolecules, using an affinophore. The affinophore is a macromolecular polyelectrolyte bearing affinity iigands. It migrates rapidly in an electric field, and molecules which have affinity for the ligand are carried with it and separated from other molecules. An anionic affinophore for trypsin was synthesized, p-Aminobenzamidine, a competitive inhibitor of trypsin, was coupled to one-fifth of the carboxyi groups of polyacrylyl-fl-alanyl-fl-alanine by the use of water-soluble carbodiimide and the residual carboxyls were converted to suffonate groups by coupling with aminomethanesulfonic acid. Affinophoresis was carried out in 1% agarose gel plates, and the protein bands were detected with Coomassie brilliant blue R250. Enhanced migrations of bovine and Streptomyces griseus trypsins towards the anode were observed with the anionic affinophore. The migrations of inactive forms prepared by active site modifications were scarcely affected. However, the affinophore was not effective for Streptomyces erythreus trypsin, an anionic trypsin, probably because of ionic repulsion between the anionic molecules. S. griseus trypsin was separated from Pronase by affinophoresis.
Introduction Affinophoresis [1], a new approach for the separation of biomolecules, is based on the specific affinity of a biomolecule to be separated and on the electric interaction as a driving force of separation. This technique requires a carrier molecule, the affinophore, which is a macromolecular polyelectrolyte containing affinity ligands. In an electric field, the affinophore rapidly migrates due to its charges, and carries with it molecules which have specific affinity for the ligand. Two main distinctive features of affinophoresis are apparent from its principle. First, afflnophore-
Abbreviation: TLCK, heptanone.
L-l-chloro-3-tosylamido-7-amino-2-
0304-4165/84/$03.00 © 1984 Elsevier Science Publishers B.V.
sis is effective for neutral substances or substances which have poor effective charges for electrophoresis. Second, no insoluble support to immobilize the affinity ligands is necessary, whereas it is indispensable for so-called 'affinity electrophoresis' [2,3] or affinity chromatography [4]. We have reported the first application of this technique [1] in which we prepared a cationic affinophore for trypsins: a dextran derivative containing diethylaminoethyl groups as cationic groups and m-aminobenzamidine as the affinity ligand. In this paper, we describe the affinophoresis of trypsins with a newly synthesized anionic affinophore, which contains sulfonate groups as ionic groups and p-aminobenzamidine [5] as the affinity ligand on a matrix of polyacrylyl-fl-alanylfl-alanine.
136 Materials and Methods
The following materials were obtained from commercial sources: p-aminobenzamidine monohydrochloride, L-l-chloro-3-tosylamido-7-amino2-heptanone (TLCK) (Sigma, St. Louis, MO, U.S.A.); Sepharose CL-6B (Pharmacia Fine Chemicals, Uppsala, Sweden); agarose GP-36 (Nakarai Chemicals Co., Kyoto); Pronase P [6] (Kaken Chemical Co., Tokyo); 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide [7] (Dojin Laboratories, Kumamoto, Japan). fl-Alanine methyl ester hydrochloride was synthesized from fl-alanine in methanol using thionyl chloride. It was hygroscopic. Purified Streptomyces erythreus trypsin [8] and Streptomyces griseus trypsin [9] were gifts from Professor F. Sakiyama (Protein Research Institute, Osaka University) and Dr. M. Nakano (Faculty of Pharmaceutical Sciences, Hokkaido University), respectively. Preparation of TLCK-treated trypsins (TLCK-trypsins) [10] was described previously [1]. Other chemicals of the purest grade available were obtained from Wako Pure Chemical Industries (Osaka).
Synthesis of acrylyl-fl-alanine In a flask shielded from light, fl-alanine (17.8 g) and hydroquinone (20 mg) were dissolved in 50 ml of 2 M NaOH. Acrylyl chloride (25 g) was added in small portions over 30 min to the solution on an ice bath with vigorous stirring. The pH of the solution was maintained at 9-10 by the addition of 2 M NaOH. After another 5 min, the solution was acidified to pH 2 with 6 M HCI. The solution was frozen at - 8 0 ° C and dried over 3 days in the dark by evaporation, using a vacuum pump without heating. Acrylyl-fl-alanine was extracted from the solid three times, each time with 100 ml acetone, and the extract was applied to a column of silica gel (50 g, 10 x 3.5 cm i.d.). The column was eluted with acetone. The effluent containing acrylyl-flalanine (350 ml), detected by ultraviolet absorption measurement after thin-layer chromatography on silica gel (R F = 0.35, chloroform/acetic acid (4 : 1, v/v)), was concentrated in a rotary evaporator and then left to crystallize. The crystals were collected by filtration, washed twice with 20 ml of acetone/diethyl ether (1 : 1, v / v ) and dried in air.
The product was recrystallized from ethyl acetate by the addition of petroleum ether. Yield: 15.7 g (55%). M.p.: 94-98°C. Elemental analysis. Calc. for C6H9NO3: C, 50.34; H, 6.35; N, 9.80. Found: C, 49.97; H, 6.38; N, 9.71.
Synthesis of acrylyl-fl-alanyl-fl-alanine methyl ester fl-Alanine methyl ester hydrochloride (5.38 g), triethylamine (5.37 ml), hydroquinone (8 mg), acrylyl-fl-alanine (5.51 g) and dicyclohexylcarbodiimide (7.94 g) were added and dissolved consecutively in dichloromethane (38.5 ml), and the solution was left overnight at room temperature in the dark. Methanol (38.5 ml) was added, and the resultant slurry was stirred for 1 h at room temperature and then filtered. The filtrate was concentrated in a rotary evaporator, and the residue was dissolved in methanol (58 ml) containing 0.02% hydroquinone and filtered once more. The filtrate was passed through a column of Amberlite MB-1 (140 ml, 25 x 2.5 cm i.d., equilibrated with methanol) at a flow rate of 4 ml per min with methanol containing 0.02% hydroquinone as a solvent. The effluent containing the product (120 ml), detected by ultraviolet absorption measurement after thin-layer chromatography on silica gel (R v = 0.45, chloroform/methanol (8 : 1, v/v)), was evaporated and the residue was dissolved in chloroform (20 ml). The solution was applied to a column of silica gel (200 g, 36 x 3.4 cm, i.d.) equilibrated with chloroform/methanol (8 : 1, v / v ) containing 0.02% hydroquinone and eluted with the solvent at a flow rate of 2.5 ml per rain. The effluent containing the product (220 ml) was evaporated and the residue was crystallized from hot ethyl acetate (150 ml). Yield: 4.21 g (48%). M.p.: 129-131°C. Elemental analysis. Calc. for C10H16N204: C, 52.62; H, 7.07; N, 12.27. Found: C, 52.74; H, 7.11; N, 12.35.
Preparation of the affinophore (Fig. 1) Acrylyl-fl-alanyl-fl-alanine methyl ester (0.5 g, 2.2 mmol) was dissolved in water (4 ml) and mixed with 0.4 ml of 6 M NaOH. The solution was allowed to stand for 30 min on an ice bath to hydrolyze the ester. After neutralization with 6 M HC1, 50 ~tl of 10% (v/v) thioglycolic acid, which controls the degree of polymerization, and 100/~1 of 5% (w/v) ammonium persulfate were added.
137 The container was sealed after evacuation with a water-jet pump, and the monomer was polymerized at 70°C for 20 h. p-Aminobenzamidine hydrochloride [5] (75.5 mg, 0.44 mmol) was added to the solution of the polymer, and the pH was brought to 5 with 6 M HC1. 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride [7] (170 mg) was added in 3 portions over 5 min. After 1 h, almost all the p-aminobenzamidine was bound to the polymer (checked by ultraviolet absorption measurement after thin-layer chromatography on silica gel, n-butanol/acetic a c i d / w a t e r ( 4 : 1 : 2 , v/v)). A 1 ml portion of the reaction mixture was removed, diluted with water (4 ml), dialyzed against water and freeze-dried after neutralization with 1 M N a O H (product I, 0.1 g). To the residual solution, aminomethanesulfonic acid (220 mg) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (420 mg) were added in three portions over 5 rain. The pH was maintained between 4.5 and 5.0 with 6 M N a O H for 1 h. The solution was diluted with water (16 ml), dialyzed against water (5 1 x 3), neutralized with 1 M N a O H and freeze-dried (product II, 0.55 g).
a cold room (4°C). Proteins were stained according to the literature [11] except that a staining solution of 0.5% Coomassie brilliant blue R250 in methanol/acetic a c i d / w a t e r (4 : 1 : 5, v / v ) and a destaining solution of methanol/acetic a c i d / w a t e r (2 : 1 : 7, v / v ) were used.
Analysis of the affinophore The affinophore solutions (1%, 0.1 ml) were evaporated, and the residues were hydrolyzed in 6 M HC1 at l l 0 ° C for 16 h in evacuated and sealed tubes. The content of p-aminobenzamidine was determined by spectrophotometry (X max = 292 nm, e = 1.54.10 4 M - ] • c m - 1 in 50 mM sodium phosphate buffer (pH 7.0)) with correction for the recovery during the hydrolysis (79%). The content of fl-alanine was determined with an amino acid analyzer. Results
Preparation of the anionic affinophore for trypsin Analysis of the affinophore (product II) showed 0
0
0
Affinophoresis Agarose powder (0.1 g), 1 ml of 1 M sodium phosphate buffer (pH 7.0) and water (8-9 ml) were mixed and heated on a boiling water bath. After dissolution, 1% ( w / v ) a f f i n o p h o r e solution was added to make a total volume of 10 ml, and the solution was spread on a Gel Bond film (12.5 × 8 cm; FMC, Marine Colloids Division, Rockland, ME, U.S.A.). The gel plate was placed on the cooling block (with circulating ice-cold water) of an electrophoretic apparatus (ATTO, SJ-1073, Tokyo) with its shorter edges facing the electrodes. A minimum amount of water was spread between the support film and the cooling block. Samples (2 /~1), which consisted of 4 ~g of trypsin (16 ~tg in the case of Pronase) or TLCK-treated trypsin and 8 ttg of the affinophore in 0.1 M sodium phosphate buffer (pH 7.2), were applied on the middle of the plate perpendicular to the longitudinal axis, using Sample Application Foil (LKB, 2117-206, Bromma, Sweden). The plate was bridged to the electrode solution, 0.1 M sodium phosphate buffer (pH 7.2). Electrophoresis was carried out at a constant current of 100 mA per plate for 40 min in
(5~q.)
I polyrnerizotion o
o
1) pABA(leq.)/WSC 2) H3NCH2SO~'/WSC ÷
NHz I'+
O
O /~
c~NH2 (leq.) (4eq.)
affinophore for trypsin Fig. 1. P r e p a r a t i o n of the a n i o n i c affinophore.
138
I
TGf
BD
20
30 40 tube number
50
Fig. 2. Gel chromatography of the affinophore. Product II (0.17 mg in 0.1 ml) was applied to a column of Sepharose CL-6B (50X 1 cm id.) equilibrated with 0.1 M sodium phosphate buffer (pH 7.0) and eluted with the buffer at a rate of 5.5 ml per h. Effluent was collected in fractions of 1 ml per tube, and the concentration of the affinophore was determined by ultraviolet absorption measurement. The arrows indicate the positions at which the following substances were eluted: BD, Blue dextran; TG, thyroglobulin (porcine, IU, (6.3-6.5).10’); IG, immunoglobuhn G (bovine, M, (1.551.55).10’); A, serum albumin (bovine, M, 6.6.104); G, glucose.
that 1% solution of the affinophore consists of 47 mM /3-alanine residue and 4.6 mM p-aminobenzamidine residue. This means that the ratio of the incorporated p-aminobenzamidine and the monomer in the final product was the same as that used in the reaction (Fig. 1). Titration of product I and product II showed that 95% of the carboxyl groups of product I was coupled with aminomethanesulfonic acid in product II (data not shown). Product II was applied to a column of Sepharose CLdB (Fig. 2). Almost all the affinophore was eluted behind thyroglobulin (M, 6.4. 105). Thus, we can expect that the affinophore would migrate practically freely in the 1% agarose gel used in the present experiments, because it is known that 1% agarose gel has a very low level of sieving effect for proteins with an M, lower than 5.105 [ll]. Zone electrophoresis of the affinophores was carried out under the same conditions as the affinophoresis described below. As shown in Fig. 3, product I (containing carboxyl groups) was heavily stained with the dye (lane 2). On the other hand, product II (containing sulfonate groups) was not stained (lane 3). In a separate experiment, in which the affinophores were detected by ultraviolet absorption measurements, product II migrated
Fig. 3. Agarose gel electrophoresis of the affinophores and trypsins. 2 pl each of S. griseus trypsin (4 pg, lane I), product 1 (20 pg. lane 2). product II (20 pg, lane 3) and S. erythreus trypsin (4 pg, lane 4) were applied to an agarose gel plate (12.5 x 8 cm) at the position marked as 0. Electrophoresis was carried out at 100 mA for 40 min. Dotted lines indicate the positions of the edges of the bridging sponges.
with a velocity of 90% of that of product I under the same conditions as in the experiment in Fig. 3 (data not shown). Product II was preferred in the present work, because it does not interfere with the detection of proteins with Coomassie blue dye.
a3 5
-_ .__..(l..._._...___._.*____ . . . . .. .i
3 cm 0
-3
; Fig. 4. Affinophoresis of trypsins with the anionic affinophore (product II). Electrophoresis of trypsins and trypsins treated with TLCK (4 pg each) was carried out in the absence (A) or presence (B) of the affinophore (0.1%). Details of the procedure were as described in the text. Lane 1, S. griseus trypsin; lane 2, TLCK-S. grisew trypsin; lane 3, S. etythreus trypsin; lane 4, TLCK-S. etythreus trypsin; lane 5, bovine trypsin; lane 6, TLCK-bovine trypsin.
139
E~
!
!
i
I
!
I
A
B
4
i
I
IC
!
i
!
~3 ~2 o=
o
--~ 0 E -I
@
Ii
|
*
i
I
|11
0.05 0.1 0.05 0.1 0 0.05 0.1 0 concentration of affinophore ( w / v % )
Fig. 5. Dependency of the migration of trypsins on the concentration of the affinophore (product II). A, S. griseus trypsin; B, S. erythreus trypsin; C. bovine trypsin. O, native trypsins; O, TLCK-trypsins.
Affinophoresis of trypsins Trypsins and their inactive derivatives prepared by affinity labeling with T L C K [10] were subjected to affinophoresis (Fig. 4). Compared with the electrophoretogram without affinophore (plate A), the enhanced migration of S. griseus and bovine trypsin toward anode in the presence of the affinophore is evident (plate B). Such an effect was not observed for TLCK-treated S. griseus or bovine trypsin. This indicates that the active sites of the enzymes play a principal role in the formation of the trypsin-affiniphore complexes. For S. erythreus trypsin, an anionic trypsin (pI 4.0) [8], however,
Fig. 6. Separation of S. griseus trypsin from Pronase P by affinophoresis. Electrophoresis of S. griseus trypsin (4/Lg, lane 1), Pronase P (16 #g, lane 2) and TLCK-S. griseus trypsin (4 #g, lane 3) was carried out in the absence (A) or presence (B) of the affinophore (product II, 0.02%).
this anionic affinophore was not effective. The dependency of the migration of trypsins on the concentration of the affinophore in the agarose gel plate is summarized in Fig. 5. S. griseus trypsin reached the maximum migration at a lower concentration of the affinophore than bovine trypsin. This situation is consistent with the observation that benzamidine inhibits S. griseus trypsin 10times more strongly than bovine trypsin [12]. The slight effect of the affinophore on TLCK-treated S. griseus and bovine trypsin could be considered to be a result of ionic interactions.
Separation of S. griseus trypsin from Pronase by affinophoresis Pronase [6] is a mixture of proteinases, including S. griseus trypsin, produced by a strain of S. griseus. Specific separation of S. griseus trypsin from Pronase was achieved by affinophoresis (Fig.
6). Discussion
In the previous paper, we described the principle of affinophoresis and its application to trypsins using a cationic affinophore [1]. In this report affinophoresis of trypsins using an anionic affinophore is presented. A new approach to preparation of affinophores proved to be successful. A hydrophilic polymer, polyacrylyl-fl-alanyl-fl-alanine, was chosen as the matrix of the affinophore. Its carboxyl group was readily coupled with amino ligands by the use of water-soluble carbodiimide. The density of the affinity ligand on the affinophore could be controlled by varying the amount of the affinity ligand used in the reaction as a limiting reactant. The hydrophilic dipeptide spacer, fl-alanyl-/3-alanine, which protrudes from the main chain of the polymer, should reduce not only steric hindrance but also ionic repulsion between the matrix of the affinophore and proteins. A considerable improvement was brought about by the conversion of the anionic groups of the affinophore from carboxyl to sulfonate groups, because product II was then no longer stainable by protein dyes. This greatly facilitated detection of the protein bands after affinophoresis. The anionic affinophore was ineffective for the
140
anionic trypsin, S. erythreus trypsin, in contrast to the cationic affinophore, which was most effective for S. erythreus trypsin as described previously [1]. A major cause of the ineffectiveness is presumably ionic repulsion between the affinophore and the enzyme. Therefore, use of an affinophore which has opposite charges to a protein to be separated by affinophoresis is recommended. One of the distinctive features of affinophoresis is that it can be carried out without any insoluble support. This feature is favorable for the electrophoretic separation of particles such as cells or organelles. Since each sub-set of lymphocytes has distinct surface markers, they should be separable with affinophores carrying specific binding proteins, for example, lectins or antibodies, etc. The scope for further development and application of affinophoresis seems considerable.
Acknowledgements We wish to acknowledge the generous gifts of purified S. erythreus trypsin from Prof. Fumio Sakiyama (Protein Research Institute, Osaka University) and purified S. griseus trypsin from Dr.
Michiharu Nakano (Faculty of Pharmaceutical Sciences, Hokkaido University). We are indebted to Ms. Mari Kubodera and Mr. Keishi Uemura for their technical assistance.
References 1 Shimura, K. and Kasai, K. (1982) J. Biochem. 92, 1615-1622 2 Takeo, K. and Nakamura, S. (1972) Arch. Biochem. Biophys. 153, 1-7 3 Ho~ej~i, V. and Kocourek, J. (1974) Biochim. Biophys. Acta 336, 338-343 4 Cuatrecasas, P., Wilchek, M. and Anfinsen, C.B. (1968) Proc. Natl. Acad. Sci. USA 61,636-643 5 Mares-Guia, M. and Show, E. (1965) J. Biol. Chem. 240, 1579-1585 6 Narahashi, Y. (1970) Methods Enzymol. 19, 651-664 7 Hoare, D.G. and Koshland, D.E., Jr. (1967) J. Biol. Chem. 242, 2447-2453 8 Yoshida, N., Sasaki, A. and Inoue, H. (1971) FEBS Lett. 15, 129-132 90lafson, R.W. and Smillie, L.B. (1975) Biochemistry 14, 1161-1167 10 Show, E. (1967) Methods Enzymol. 11,677-686 11 Miyake, J., Fehrnstr6m, H. and Wallenborg, B. (1977) LKB Application Note 310, LKB-Produkter, Bromma, Sweden 12 Kasai, K. and lshii, S. (1978) J. Biochem. 84, 1051-1060