Proceedings of the National Academy of Sciences Vol. 68, No. 2, pp. 395-398, February 1971

Rapid and Complete Purification of of Electric Eel and Erythrocyte by Affinity Chromatography

JONATHAN DEMIBITZ BERMAN AND MICHAEL YOUNG Departments of Biological Chemistry and Medicine, Harvard Medical School, Boston, Massachu- setts 02114; and the Massachusetts General Hospital, Boston, Mass. 02114 Communicated by Francis 0. Schmitt, November 23, 1970

ABSTRACT Affinity chromatography has been used to 0.04 M MgC12, 0.1 M NaCl, 4 mM chloride. 1 purify both from the electric tissue of unit of activity represents 1 ,umol of acetylcholine Electrophorus electricus and from bovine erythrocyte membranes. For this purpose, several specific enzymic cleaved per minute. Measurements of pH utilized a Radiometer inhibitors of each were synthesized and joined model 4 pH-meter and buret attachment. The impure eel covalently to an insoluble support resin. AchE is selec- AchE preparations used in this study were obtained from tively retained by such inhibitor-resins when highly im- Sigma Chemical Co. and from Worthington Biochemical pure solutions are chromatographed upon them. After to 100 removal from the resin, both are electrophoreti- Corp.; their specific activities were close units/mg cally homogeneous and they may be recovered in yields of protein. Crude bovine erythrocyte AchE preparations were 75% or more. purchased from Mann Research Laboratories. All manipulations, unless otherwise noted, were carried out In the study to be presented below, we have used affinity with a solvent 0.04 M in MgCl2 and in 0.1 M in NaCi, ad- chromatography to purify acetylcholinesterase (AchE) from justed to pH 7.8 with NaHCO3. Protein concentrations were both the electric tissue of Electrophorus electricus and from measured by the Lowry et al. procedure (7). bovine erythrocyte membranes; only a single purification step is required. The central feature of this technique depends upon Synthesis and characterization of inhibitors coupling a specific inhibitor of the enzyme to an inert, insol- For these studies, the nonionic resin agarose (Bio-Rad) was uble support resin. Under appropriate solvent conditions, used as the insoluble support. 2% agarose (molecular weight chromatography of the enzyme upon the inhibitor-resin com- exclusion limit = 5 X 107 g/mol) was selected so that AchE pound selectively retains AchE and thus eliminates contam- (MW = 250,000) (3) would potentially be able to interact inating that possess no affinity for the inhibitor with inhibitor molecules bound on both the inside and the out- . Subsequently, AchE is removed from the column by side of the gel matrix. elution with another specific inhibitor whose inhibition con- Cuatrecasas (2) has recently presented chemical methods stant (Ki) is less than that of the inhibitor-resin compound. for attachment of small organic molecules to agarose. The Purification by affinity chromatography therefore requires several inhibitor ligands used in this study are illustrated in (a) synthesis of an inhibitor that can be covalently bound Table 1 and their syntheses are described below. to a solid support; (b) attachment of the inhibitor Trimethyl (p-aminophenyl)ammonium chloride hydro- to resin of the support by a chain atoms long enough chloride (I) was prepared essentially according to the method to enable the inhibitor to interact readily with the binding of Traylor and Singer (8) as follows. N,N-dimethyl-p-phe- site(s) of the enzyme; and (c) establishing conditions under nylenediamine dihydrochloride (Eastman) was recrystallized which the protein can be selectively removed from the from methanol. The purified diamine and a few drops of con- column. (For a summary of recent studies on affinity chroma- centrated sulfuric acid were added to a 10-fold excess of acetic tography, see refs. 1 and 2.) mixture on In anhydride, and the was heated gently a steam bath earlier studies, Leuzinger and Baker (3) have used con- until solid diamine dissolved. This solution was then cooled to ventional procedures to purify eel AchE. Erythrocyte AchE room temperature, and 4 vol of water was added with gentle has never been purified, although partial purification pro- warming to hydrolyze unreacted acetic anhydride. The solu- cedures have been presented (4-6). The work outlined here tion was into 30 of 5 N was to poured vol NaOH, whereupon p- initiated facilitate complete purification of both pro- dimethylaminoacetanilide crystallized. Crystals were collected teins prior to comparative studies of their molecular structures by filtration, washed with water, and methylated with excess and function. methyl iodide (or [14C]methyl iodide obtained from New MATERIALS AND METHODS England Nuclear Co.) in refluxing acetone. Trimethyl Measurements of enzyme activity (p-acetamidophenyl)ammonium iodide (II) crystallized from AchE activity was measured from the amountof 0.01 NNaOH the reaction mixture after about 15 min. Compound II was needed to maintain constant pH during hydrolysis of acetyl- added to 50% (v/v) ethanol-concd HCl, and this solution was at pH 7.5 and 22°C. The incubation solvent contained refluxed for 2 hr. Compound I crystallized upon addition of acetone (9). Trimethyl (m-aminophenyl)ammonium chloride Abbreviation: AchE, acetylcholinesterase. hydrochloride (III) was synthesized from N,N-dimethyl-m- 395 Downloaded by guest on September 26, 2021 396 : Berman and Young Proc. Nat. Acad. Sci. USA

TABLE 1. Inhibitor ligands and related derivatives for affinity ml of water and succinylated with 4 mmol of succinic an- chromatography of AchE hydride (Eastman) exactly as described by Cuatrecasas (2). An inhibitor could now be coupled to the free carboxylate I: H2N-c N(CH3)3CIHCI III: H2N /HC1 groups of the succinylated gel by reaction of 2 ml of gel with 0.2 mmol of inhibitor in the presence of 1.5 mmol of carbo- N (CH3)3C1 diimide at pH 5.0. These reaction mixtures were stirred over- night at 220C. 0 0 Alternatively, the inhibitor could be further extended from II: CH3C-NHi--{JN(CH3)3I IV: the gel. For this purpose, succinylated derivatives prepared as CH3C-NHNH described above were allowed to react further with 4 mmol of 3,3'-diaminodipropylamine and 1.5 mmol of carbodiimide. The N(CH3)3I resultant derivative was again succinylated with 4 mmol of 0 succinic anhydride and then coupled to the inhibitor with CH31- -1 V: N-C-0 carbodiimide. In all cases, excess reagents were eliminated from the gel by filtration. To establish the degree of coupling of inhibitor to agarose, N(CH)3CI '4C-labeled compounds I and III were prepared with [14C]- H2N CH3I. These derivatives were then attached to agarose that had been aminated, succinylated, again aminated and suc- cinylated as described above. Scintillation counting (Triton fluor, Packard revealed phenylenediamine (Eastman) exactly as described for com- X-100-toluene scintillation counter) pound I. that about 9 umol of ligand was bound to 1 ml of packed gel. Inhibition constants of the inhibitors for both eel and Electrophoresis erythrocyte were obtained by the Augustinsson method (10) of plotting rate (minus inhibitor)/rate (plus in- Preparations of AchE were examined by electrophoresis on 5% polyacrylamide gels with the Tris-glycine system described by hibitor) against inhibitor concentration. If we assume that II Davis (11). Stacking gels were omitted. Samples dissolved in acts as a purely competitive inhibitor of AchE, then Ki is close to to 1 10-4 M. The for compound II for bovine erythrocyte 50-100 AI of 0.025 M Tris phosphate, pH 6.9, were applied X Ki each gel. Bromophenol blue served as a tracking dye and a AchE (Mann) was found to be 1.5 X 10-4 M, and Ki for com- constant current of 2.5 mA per gel was applied for 1.5 hr at pound IV for the same enzyme is 0.9 X 10-4 M. Values of Ki 220C. Gels were stained with 0.25% for II and IV (rather than forI and III) were determined, since and were destained electrophoretically (12). these inhibitors exist as acetamido derivatives on the gel. Another inhibitor with a free amino group, the N-methyl- RESULTS AND DISCUSSION N-(p-aminophenyl) ester of m-(trimethylamino)- The eel enzyme phenol (V), was also prepared (to be published); Ki for this Fig. 1 presents several elution profiles of eel AchE chromato- reagent, an analog of , is about 10-6 M. graphed on agarose coupled to the ligands described above. To Tensilon m-(trimethylamino)phenol chloride was a gift from Dr. W. E. Scott, Hoffman-LaRoche Co., Nutley, N.J. ensure that these gels did not adsorb protein nonspecifically, we chromatographed bovine serum albumin oneach gel deriva- tive. Nonspecific adsorption was slight, since more than 90% of to agarose Attachment inhibitors of the BSA applied to any gel was unretarded. In initial studies, several of the inhibitors described above Fig. 1A demonstrates that AchE does not bind to a deriva- were coupled directly to agarose. However, such derivatives tive of agarose that does not contain an AcbE-inhibitor--40 failed to retard the esterases. Consequently, in light of the enzyme units were applied to this gel and all of it emerged un- possibility that these ligands lay spatially too close to the gel retarded. However, the studies of Cuatrecasas (2) demonstrate matrix to interact with the enzyme, side chains terminating in that simply coupling an inhibitor to a gel does not necessarily free carboxylate groups were attached to agarose by the meth- cause the desired enzyme to bind. Thus, although compound ods described by Cuatrecasas (2). Inhibitors containing free (V) is a strong inhibitor of AchE (Ki ~-' 10-6 M), this reagent, amino groups were then bound to the carboxylate derivatives when joined to agarose as shown in Fig. 1B, only slightly re- via a water-soluble carbodiimide, 1-cyclohexyl-3-(2-morpho- tards the enzyme. linoethyl)-carbodiimide methyl-p-toluenesulfonate (Aldrich These studies suggested that the inhibitor might be steri- Chemical Co.). cally shielded from interaction with the enzymatic site(s) of For a typical synthesis, 0.5 g of BrCN (Eastman) was added AchE. Accordingly, inhibitors (I) and (V) were even further to 2 ml of 2% agarose suspended in 20 ml of water, and pH was extended from the gel matrix; Fig. 1C and D demonstrate that maintained at 11.0 with 5 N NaOH. After 30 min, reaction these gels bound 94-100% of the eel applied to them. was essentially complete. The gel was washed on a sintered Under these conditions, AchE is not merely retarded but re- glass filter with 300 ml of cold 0.1 M bicarbonate buffer (pH tained; the enzyme remainsfirmly bound to resin even after 30 10.0). The washed gel was then added to a solution containing column volumes of solvent have passed through the column. 4 mmol of 3,3'-diaminodipropylamine (Eastman) in 20 ml of (Fig. ID reveals that <1% of AchE activity was not bound to cold water previously adjusted to pH 10.0 with 6 N NCl. This the resin.) solution was stirred overnight at 4°C and excess amine was Gel-bound AchE was removed by elution with a strong removed by filtration. The aminated gel was dispersed in 20 enzyme inhibitor, Tensilon (Ki = 10-6 M). Ten column Downloaded by guest on September 26, 2021 Vol.VAffinity68,1]971 Chromatography of Acetylcholinesterase 397

0 0 0 0 1 11 11 -NH(CH2)3NH(CH2)3NHC(CH2)2COOH -NH(CH2)NH(CH2)3NHC(CH2)CNI NC0Q 1-8 A " 1.8[ Rw CH3 N(CH3)3 1.6/ 16 16 1.4 / 14 1.2 - I 12 / 1.0' 10 0.8- 8 8 - CZ 0.6 I 6 6 II% 0.4 / 4 4 co 0.2 j 0 7 2 2 1 0 f - 0 0 4- 0 0 0 0 0 -[NH(CH2)3NH(CH2NHC(CH2)2JHON9Cs -[NH(CHNH(CH2hNHC&CH2)2C] HO2N(CH3)3 A: 1.1 1.8 C CH3 N(CH3 1%% 1.6 16 1.4 14 %%a 1.21 12 1.01 10 8 0.8 8 6 0.6 6 4 0.4 4 2 0.2 2 IA&k ^ L I - of 0 0 I 0 1 2 3 4 5 10 Column Vo/umes FIG. 1. Affinity chromatographic elution profiles for eel acetylcholinesterase. Column dimensions: 0.9 cm X 1-7 cm, packed with derivatives of 2% agarose; flow rate = 3 ml/hr; temperature, 4VC; solvent: 0.1 M NaCl-0.04 M MgC12, adjusted to pH = 7.8 with NaHCO3; enzyme activity, (0-0); absorbance, (0-4). Inhibitor Migands together with "side arm" extensions are shown for each figure. Bovine serum albumin was added to each enzyme sample as carrier protein. A, 3.2 mg of albumin (40 enzyme units) applied, column volume 0.5 ml; B, same as A, column volume 3 ml; C, 4.5 mg of albumin (110 units), column volume 1.5 ml; D, 7.5 mg of albumin (2000 units), column volume 4 ml.

volumes of 10-2 M Tensilon dissolved in 0.04 M MgCl2-O.1 M geneous, but only a single electrophoretic component was NaCl, pH 7.8, were applied to the column illustrated in Fig. detected after chromatography (Fig. 2B). iD at a flow rate of 3 ml/hr. The eluate was dialyzed to reduce the free Tensilon concentration below 1 X 10-9 M. Subse- TABLE 2. Percentage recovery of AchE after affinity quent enzyme analyses revealed that 70-100% of the gel- chromatography* bound AchE is released by Tensilon (see Table 2), and more than 80% of the activity is recovered in the first three Units recovered column volumes of Tensilon-containing solvent. Enzyme units Units not from column Preparation added to bound to with Tensilon The purity of the chromatographed enzyme was estimated and source column by specific activity measurements and by polyacrylamide gel column (%)t (%) electrophoresis. Typically, the specific activity of the eel 1. Eel 1520 0(0%) 1064 (70%) AchE preparations increased from 460 to 16,000 units/mg 2. Eel 134 8 (6%) 128 (94%) protein after affinity chromatography. Leuzinger and Baker 3. Erythrocyte 72 0 (0%) 55 (76%) have shown that their crystallized enzyme preparation has a * specific activity of about 12,500 units/mg protein (3). We Enzyme activity unit represents 1 psmol of acetylcholine hydrolyzed per minute. attach no significance to this difference since the t Table presents total eel enzyme activity units applied to the procedures are somewhat different. inhibitor-resin depicted in Fig. iD, together with percent re- Fig. 2A and B presents polyacrylamide gel electrophoretic covery. For the red enzyme, inhibitor compound III (see patterns of enzyme preparations before and after affinity Table 1) was attached to the "side arm" extension shown in chromatography. The crude preparation is grossly hetero- Fig. iD. Downloaded by guest on September 26, 2021 398 Biochemistry: Berman and Young Proc. Nat. Acad. Sci. USA

The usefulness of affinity chromatography for rapidly purifying highly inhomogeneous enzyme preparations is demonstrated by isolation of erythrocyte . Thus, 18 mg of crude material (specific activity 4 units/mg protein) yielded 55 units of pure AchE. This amount of pro- tein is too low to estimate accurately, but the intensity of staining of the single polyacrylamide gel band (Fig. 2D) in- dicates that 55 enzyme units constitute no more than 6 ,gg of protein. This would mean that the crude preparation has been purified about 2500-fold. This work was supported by National Institutes of Health Research grant AM-09404, by a grant from The John A. Hart- ford Foundation, and by N.I.H. Career Development Award No. 5K3-AM18,565 (M.Y.). J.D.B. was supported by U.S.P.H.S. Training grant 2TlGM782-11 to the Biophysical Laboratory of A B C D Harvard Medical School. We are grateful to Dr. A. Stempel FIG. 2. Gel electrophoretic patterns of eel and erythrocyte (Hoffman La-Roche) and particularly to Drs. J. D. White and M. J. Dimsdale (Harvard University) for their advice on the AchE before and after affinity chromatography. Migration synthesis of some of the compounds used in this study. direction is from top to bottom (see text for detailed conditions). A and B, crude and chromatographed eel AchE, respectively; 1. Cuatrecasas, P., M. Wilchek, and C. B. Anfinsen, Proc. C and D, crude and chromatographed red cell AchE, respectively. Nat. Acad. Sci., USA 61, 636 (1968). Bromophenol blue was added to each gel as a marker. Quanti- 2. Cuatrecasas, P., J. Biol. Chem., 245, 3059 (1970). ties of protein and activity added to each gel were A, 190 ug, 3. Leuzinger, W., and A. L. Baker, Proc. Nat. Acad. Sci., 88 units; B, 8 ug, 128 units; C, 170 ug, 0.7 unit; D, approxi- USA 57, 446 (1967). mately 2 ug, 25 units. 4. Zittle, C. A., E. S. DellaMonica, J. H. Custer, and R. Krikorian, Arch. Biochem. Biophys., 56, 469 (1955). 5. Warringa, M. G. P. J., and J. A. Cohen, Biochim. Biophys. Acta, 16, 300 (1955). Bovine erythrocyte AchE 6. Wright, D. L., and D. T. Plummer, Biochem. J., 118, 21 (1970). Initial chromatographic studies with inhibitor compound I 7. Bailey, J. L., Techniques in Protein Chemistry (Elsevier, yielded variable elution profiles. Only in some cases did the Amsterdam, 1967), p. 340. erythrocyte enzyme bind to the agarose inhibitor complex. 8. Traylor, P. S., and S. J. Singer, Biochemistry, 6, 881 Consequently, the meta analog of I (compound III), with (1967). 9. This reagent (compound I) was used by Changeux et al., smaller Ki, was joined to agarose via the same "side arm" in their studies on the electroplax protein. [Changeux, extension illustrated in Fig. 1C and D. Table 2 reveals that J. P., T. R. Podleski, and L. Wofsy, Proc. Nat. Acad. Sci. USA, 58, the gel fully bound the esterase and that 76% was recovered 2063 (1967)]. upon elution with 0.01 M Tensilon. Electrophoretic analyses 10. Augustinsson, K. B., Acta Physiol. Scand. Suppl. 15, of the crude and are 52, 1 (1948). chromatographed protein shown in 11. Davis, B. J., Ann. N. Y. Acad. Sci., 121, 404 (1964). Fig. 2C and D. After chromatography, the preparation is 12. Weber, K., and M. Osborn, J. Biol. Chem., 244, 4406 electrophoretically homogeneous. (1969). Downloaded by guest on September 26, 2021