ARCHIVES OF BIOCHEMISTRYAND BIOPHYSICS Vol. 198, No. 2, December, pp. 379-385, 1979
Immobilization
of Glycoenzymes through Carbohydrate HUMG-YU HSIAO AND GARFIELD
Department
of Biochemistry,
The Ohio State University,
Side Chains’
P. ROYER2
484 W. 12th Ave., Columbus, Ohio 43210
Received June 19, 1979 Glucoamylase, peroxidase, glucose oxidase, and carboxypeptidase Y were covalently bound to water-insoluble supports through their carbohydrate side chains. Two approaches were used. First, the carbohydrate portions of the enzymes were oxidized with periodate to generate aldehyde groups. Treatment with amines (ethylenediamine or glycyltyrosine) and borohydride provided groups through which the protein could be immobilized. Ethylenediamine was attached to glucoamylase, peroxidase, glucose oxidase, and carboxypeptidase Y to the extent of 24,20,30, and 15moYmo1of enzyme, respectively. These derivatives were coupled to an aminocaproate adduct of CL-Sepharose via an Nhydroxysuccinimide ester or to CNBr-activated Sepharose. Coupling yields were in the range of 3’7-50%. Retained activities of the bound aminoalkyl-enzymes were 41% (glucoamylase), 79% (peroxidase), 71% (glucose oxidase), 83% (carboxypeptidase Y). A glycyltyrosine derivative of carboxypeptidase Y was bound to diazotized arylamine-glass. Coupling yield was 42% and retained esterase activity was 84%. In the second approach, the enzyme was adsorbed to immobilized concanavalin A and the complex was crosslinked. Adsorption of carboxypeptidase Y on immobilized concanavalin A followed by crosslinking with glutaraldehyde was also effective. The bound enzyme retained 96% of the native esterase activity and showed very good operational stability.
Immobilization of enzymes and other biochemicals is of interest because of research and industrial applications of the solid-phase products (1, 2). Covalent coupling has been the most popular approach to immobilization presumably because of the accessibility and stability of the bound enzyme product. Usually the carboxyl groups, phenolic groups, or amino groups on the enzyme surface are reacted with a solid support. In our experience glycoproteins are difficult to immobilize possibly because the amino acid side chains are shielded by carbohydrate spines which project from the surface of the enzyme molecule. One such example is carboxypeptidase Y (CPY>,3 a carbohydrate con’ This work was supported by a grant from the National Institutes of Health (GM 19507). G.P.R. is the recipient of Public Health Service Career Development Award l-K4-GM-00051. * To whom correspondence should be sent. 3 Abbreviations used: CPY, carboxypeptidase Y; Con A, concanavalin A; AcOH, acetic acid; BuOH, butanol; Bicine, N,N-bis(2-hydroxyethyl)glycine; tic, thin layer chromatography; SDS, sodium dodecyl sulfate; APM, aminopeptidase M; EDA, ethylenediamine; Cbz, benzyloxycarbonyl. 379
taining exopeptidase from yeast. After many unsuccessful attempts to immobilize CPY, we prepared a bound derivative using water-soluble carbodiimide, hexamethylenediamine-agarose, and enzyme (3). Whitesides and co-workers have reported an effective procedure for enzyme immobilization which is based on the use of a polymeric N-hydroxysuccinimide ester (4). In this case also glycoenzymes proved recalcitrant (4,5). Zaborsky and Ogletree showed that glucose oxidase could be adsorbed to water-insoluble p-aminostyrene after periodate oxidation of the carbohydrate of the enzyme (6). Sulkowski and Laskowski showed that venom exonuclease retained activity while adsorbed on Con A-Sepharose (7). It seemed reasonable to us to extend these studies by making stable, covalently immobilized enzyme adducts in which the carbohydrate side chains provide the points of attachment between the enzyme and the solid support. The first approach is shown in Fig. la, the glycoenzymes are oxidized with periodate and coupled to an amine such as ethylenediamine or glycyltyrosine with NaBH, or NaBH,CN. In the second approach (Fig. 0003-9861/79/14037907$02.00/0 Copyright 0 1979by AcademicPress, Inc. All rights of reproductionin any form reserved.
380
HSIAO AND ROYER
FIG. 1. Immobilization of glycoproteins. (a) After oxidation with periodate, glycyltyrosine or ethylenediamine was attached to the enzyme by treatment with borohydride. The aminoalkyl groups provide additional points of attachment in the immobilization process. In (b) the glycoenzyme was first adsorbed on immobilized concanavalin A. The complex thus formed can be stabilized by crosslinking with glutaraldehyde.
lb) the glycoenzyme is adsorbed to insolubilized Con A and the complex is stabilized with glutaraldehyde. Both approaches gave stable covalently immobilized enzymes with good activity. MATERIALS
AND METHODS
Materials CPY from baker’s yeast (Anheuser Busch) was activated according to Kuhn et al. (8) and purified by affinity chromatography (9). Con A, glucoamylase, and hexokinase were purchased from BoehringerMannheim. Glucose oxidase (type II), ribonuclease A, NADP, and ATP were the products of Sigma. Sodium borohydride, sodium cyanoborohydride, and 4-aminoantipyrine were supplied by Aldrich. Vega-Fox provided Z-L-Leu-L-Phe. N-Ethylmorpholine was refluxed with ninhydrin and distilled. Ethylenediamine was distilled after drying over sodium metal. Both amines were stored in small brown bottles under nitrogen.
Methods Preparation
of ethyknediumine-enzyme
adduets.
Unless otherwise noted, all the reactions were carried
out at 4°C. CPY (1.4 mg/ml) in water was treated with sodium metaperiodate (0.02 M final concentration) for 1 h in the dark. Iodate was removed by dialysis against water (five changes, 1 liter per change) for 2.5 h. Periodate-treated enzyme was further modified by mixing with ethylenediamine (0.05 M final concentration at pH 8.0) followed by the addition of 0.1 M NaBH, (10 4, in 0.1 N NaOH) in two portions at 30- and 60-min intervals. After 90 min the derivative was purified by either dialysis or chromatography on Sephadex G-25 column (0.01 M phosphate-O.5 M NaCl buffer, pH 8.0). High concentration of salt was needed to prevent the precipitation of the aminoalkylated CPY. The absence of free ethylenediamine in the derivatized enzyme preparations was ascertained by thin layer chromatography (silica gel, n-BuOH-AcOH-pyridine-H,O, 15:3:12:10).Peroxidase was modified using similar procedures. Due to sensitivity of enzymes to alkaline conditions the derivatization of glucoamylase and glucose oxidase was performed in phosphate buffer (0.1 M, pH 6.0) containing NaBH,CN (0.1 mg/ml of reaction mixture) and ethylenediamine (0.05 M). The reaction time was 3 h. Immobilization of ethylenediamine-enzyme derivatives. Aminocaproic acid was bound to CL-Sepharose according to Liberatore et al. (3). TheN-hydroxy-
succinimide ester derivative of the gel was prepared as described by Cuatrecasas and Parikh (10). Glycoamylase, glucose oxidase, and peroxidase (1 mg/ml) were immobilized at pH 6.0, 6.3 (0.1 M phosphate buffer) and 8.5 (0.1 M Na-Bicine buffer) using the N-hydroxysuccinimide ester of the CL-Sepharose-aminocaproate derivative (1 ml of enzyme solution/ml of settled gel). The CPY derivative (2 mg/ml) was immobilized on CNBr-activated gel (1 ml of enzyme solution/ml of settled gel) at pH 8.0 (0.1 M Bicine-0.5 M NaCl) following the procedure of March et al. (11). Preparation of glycyltyrosine-CPY-glass. Glycyltyrosine was linked to oxidized CPY in a manner analogous to the preparation of the ethylenediamineCPY adduct. Glycyltryosine (0.1 M in 0.1 M Bicine buffer) was coupled to the oxidized CPY at pH 8 with NaBH, added as before. The derivative thus formed was purified by chromatography on Sephadex G-25 equilibrated with 0.01 M phosphate buffer (pH 7.8) which contained 0.15 M NaCl. Absence of free glycyltyrosine was verified by tic (silica gel; n-butanol-acetic acid-water, 4:1:5). Glycyltyrosine-CPY was then coupled to diazotized arylamine-glass according to the procedure of Royer and Andrews (12). The coupling reaction was carried out in 0.1 M phosphate buffer, 0.15 M NaCl, pH 7.8. Preparation of CL-Sepharose-Con A-CPY. Con A was reacted with N-hydroxysuccinimide ester of CLSepharose under the following conditions: 0.1 M phosphate buffer, pH 7.4, 4 mg Con A/2 ml buffer/g damp gel activated as previously described (10). The bound Con A was thoroughly washed with 1 M KC1 and ace-
IMMOBILIZATION
381
OF GLYCOENZYMES TABLE I
IMMOBILIZATIONOFETHYLENEDIAMINE ADDIJCTSOFGLYCOENZYMESONCARBOXYALKYLAGAROSE Glucoamylase Hexose remained after oxidation (o/o) Moles of ethylenediamine incorporated Protein on gel (mg/ml of settled gel) Yield (W) Specific activity of native form Specific activity of soluble ethylenediamine adduct Specific activity of bound enzyme
Peroxidase
Glucose oxidase
CPY
48
45
50
50
24
20
30
17
0.56 43
0.48 50
0.30 37
0.36 40
10
1100
63
64*
10 (100)
1090 (99)
55 (87)
56b (87)
4.1 (41)
870 (79)
45 (71)
53* (83)
n Percentage of protein bound on gel, determined by amino acid analysis. * Esterase activity. tate buffer (0.01 M, pH 5) (13). CPY (1 mg/ml, 3.3 ml) was stirred with immobilized Con A (1 g damp gel) for 30 min at 4°C in phosphate buffer (0.02 M, 0.15 M NaCl, pH 7.4). The gel was washed with 300 ml of 1 M ECl. Crosslinking of the Con A-CPY conjugate with glutaraldehyde was accomplished under the following conditions: 0.1 M phosphate buffer, pH 8, 2 ml buffer/l ml settled gel, 0.5% glutaraldehyde, 1 h. Enzyme assays and characterization. Peroxidase activity was determined by the procedure of Trinder (14). Glucoamylase activity was determined by measuring the amount of glucose released from glycogen (15). Glucose oxidase was assayed in the following procedure. An appropriate concentration of glucose oxidase enzyme solution (0.1 ml) was added to a mixture of phenol-4-aminoantipyrlne solution (1.4 ml, 0.170.0025 M), glucose solution (0.3 ml, 18% previously allowed to stand for 12 h), phosphate buffer (1.2 ml, 0.1 M, pH 6.0), and peroxidase (10 ~1, 1 mg/ml). Its activity was followed by measuring the increase in the absorbance at 510 nm. Esterase activity of CPY was followed titrimetrically using N-acetyltyrosine ethyl ester as substrate (3). Peptidase activity against Z-Leu-Phe was followed by ninhydrin analysis (3). Proteolytic digestion of ribonuclease A by immobilized CPY was followed by amino acid analysis at timed intervals. Digestions were carried out with ribonuclease A (0.29 PmoYml) in N-ethylmorpholine-acetate buffer (0.1 N, pH 6) containing 0.5% SDS. Amine content of the ethylenediamine-enzyme adducts was determined with the ninhydrin reagent of Moore (16). The incorporation of glycyltyrosine into CPY was found by amino acid analysis for tyrosine. Protein content of insoluble enzyme conjugates was also determined by amino acid analysis. Samples were hydrolyzed for 24 h with 6 N HCl at 110°C.The residues
were extracted twice with fresh HC1; the combined extracts were then dried using the flash evaporator. Hexose content of proteins was determined by the phenol-sulfuric acid method (17). RESULTS AND DISCUSSION
Ethylenediamine conjugates of glucoamylase, peroxidase, glucose oxidase, and CPY were prepared. In the four cases studied about 50% of the hexose was altered by periodate oxidation. The soluble aminoalkyl derivatives were quite active (Table I); glucoamylase containing 24 mol of ethylenediamine/mol was as active as the native molecule. Coupling yields were in the range of 37-50%, which is satisfactory for the conditions employed. The amount of protein bound could probably be increased by using more solid support per given amount of enzyme. The insoluble derivatives of peroxidase, glucose oxidase, and CPY showed good activity (U-83% of native). The activity of immobilized glucoamylase was 41% of the activity for the native enzyme. The level of retained activity of glucoamylase is below those of the other enzymes discussed here probably because of a substrate exclusion effect. Glucoamylase was assayed using glycogen which is a high polymer with a molecular weight in the millions. Stability studies on the immobilized aminoalkyl enzymes were encouraging (Fig.
382
HSIAO AND ROYER
5w
50
I I
1 2 NUMBER
I I 3 4 OF ASSAY
I 5
I 6
FIG. 2. Stability test of immobilized enzymes. Glucoamylase (01, glucose oxidase (A), carboxypeptidase Y (O), and peroxidase (0) were immobilized, following the procedure shown in Fig. la, to N-hydroxysuccinimide activated CL-Sepharose. These insoluble enzymes were repeatedly assayed in the period indicated.
2). After six assays in a stirred suspension, the immobilized forms of glucoamylase, glucose oxidase, and CPY retained full activity. In the peroxidase assay very small amounts of gel were used. Part or all of the loss of activity of peroxidase shown in Fig. 2 could be ascribed to the loss of gel particles in the separation and washing steps between
assays. Since much larger amounts of enzyme could be used for the other immobilized enzyme assays, physical loss of small amounts of enzyme gel was not significant. CPY is a valuable tool for carboxyl-terminal sequencing because of its very broad specificity (18-19). Proline is normally released smoothly. Also, we have demonstrated that immobilized CPY is useful for deblocking in sequential synthesis of polypeptides (20). In this approach the carboxyl terminus is extended using water-soluble carbodiimide and amino acid ethyl esters. The deblocking is accomplished by hydrolysis of the peptide ester with immobilized CPY at pH 8.5. At this pH value the peptidase activity is barely detectable and the esterase activity is maximal. Because of the use of CPY in sequencing and synthesis, we immobilized CPY by four different methods and characterized the derivatives in terms of peptidase and esterase activity (Table II). All four methods are superior to our previously reported procedure for immobilization. The Con A-CPY is quite active in both esterase and peptidase as-
TABLE II IMMOBILIZATION OF CPY
EDA-CPY” (CNBr-agarose)
EDA-CPY (Carboxy-agarose)
Gly-Tyr-CPY (arylamine-glass)
48
50
48
-
15
17
-
-
-
Con A-CPY Hexose remaining after oxidation (%o) Estimated moles of EDA incorporated Addition of Gly-Tyr on CPY (mol/mol of enzyme) Enzyme on gel (mgiml of settled gel) Yield (%) Specific esterase activity determined at pH optimum Ratio of specific esterase activity between bound and native form (X 100) Specific peptidase activity Ratio of specific peptidase activity between bound and native form (x100)
8
2.8 85
0.9 45
0.36 40
1.46 42
62
60
53
54
96 20
94 9.1
83 8.3
84 8.2
90
41
37
37
a Ethylenediamine-CPY. * Determined as mg enzyme/g of damp glass.
IMMOBILIZATION
says. Levels of retained activity were 96 and 90% of native. The amount of protein immobilized in this case was 85% presumably because of the strong initial adsorption of CPY to the immobilized Con A. Considering yield and retained activities, the immobilized Con A-CPY preparation is one of the best immobilized enzyme derivatives we have seen. Aminopeptidase M attached to porous glass retains full activity (12). However, the stability of this bound APM was not as good as immobilized Con A-CPY. The immobilization of aminoalkyl derivatives of CPY was also successful. Yields were in the range of 40-45% (Table II). The retention of esterase activity (83-94%) was better than the retention of peptidase activity (37-41%). For deblocking peptide esters this characteristic is desirable, since peptidase activity should be low compared to esterase activity. The immobilized aminoalkyl CPY derivatives were tested for stability (Fig. 3). As expected the most stable preparation was prepared with aminocaproate-agarose. The linkage between agarose and the enzyme contains an alkylamine C-N bond and an amide bond, both of which are very stable. One would expect the loss of activity of the EDA-CPY on CNBr-activated agarose since the substituted isourea linkages formed with CNBractivated agarose are known to be unstable at alkaline pH (21-22). Also, the surface of porous glass is eroded under the condi-
FIG. 3. Stability tests of four types of immobilized CPY. Immobilized EDA-CPY coupled toN-hydroxysuccinimide-activated CL-Sepharose (0), immobilized Con A-CPY (O), immobilized EDA-CPY coupled to CNBr-activated Sepharose (A), and immobilized TyrGly-CPY (0). Activity against N-acetyltyrosine ethyl ester at pH 8.0 was used for this test.
383
OF GLYCOENZYMES
6.0
6.5
I
I
7.0
7.5
I
8.0
I
8.5
I
9.0
I
9.5
I
10.0
PH
FIG. 4. The pH dependencies of esterase activities. Native CPY (V), immobilized EDA-CPY coupled to N-hydroxysuccinimide-activated CL-Sepharose (0), and immobilized Con A-CPY (Cl), immobilized EDACPY coupled to CNBr-activated Sepharose (A), and immobilized Tyr-Gly-CPY (0) in 0.1 M KC1 with 10 mM N-acetyltyrosine ethyl ester. The esterase activity at each pH was normalized by its activity at pH 8.0 being used as indicator of enzyme concentration. The rate profile of native CPY was adopted from Liberatore et al. (3).
tions of the assay which explains the lack of stability of the Gly-Tyr-CPY bound to arylamine -glass. In order to make intelligent use of an immobilized enzyme preparation, the pH-rate profile must be determined. The perturbations of some pH optima can be quite significant (23). Shifts as great as two pH units have been observed. The pa-rate profiles of the four immobilized CPY were determined for peptide and ester substrates. The pH-rate profiles for ester hydrolysis appear in Fig. 4. The shifts of 0.5-l pH unit are to the alkaline side of the pH opti.mum of the native enzyme. The pH optima of the bound enzymes are shifted to the acid side of the native enzyme in the peptidase assays (Fig. 5). Microenvironmental effects, diffusional effects, and chemical modification of the enzyme may be invoked to explain the changes in pH dependence curves for reactions catalyzed by immobilized enzymes. The alkaline shifts of the pa-rate profiles
384
HSIAO AND ROYER
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
6.0
9.5
9.0
DH
FIG. 5. The pa-rate profiles of peptidase activities. Immobilized EDA-CPY coupled toN-hydroxysuccinimide-activated CL-Sepharose (O), immobilized EDACPY coupled to CNBr-activated Sepharose (A), immobilized Con A-CPY (O), immobilized Tyr-GlyCPY (0), and native CPY (V). Activity was measured with Cbz-Leu-Phe. Buffers and assay procedure are described in the text. The rate profile of native CPY was adoped from Liberatore et al. (3).
of the esterolytic reactions of the bound CPY derivatives could be accounted for by rate-limiting efflux of protons from the gel. The shift to a lower pH optimum is typical when enzymes are bound to positively charged supports. Such a microenvironmental effect is probably operating in the acid region for the peptidase activities. The aminoalkylated derivatives are themselves more positively charged than native enzymes in the acid pH range. The perturbation of the pH-rate profile for the peptidase activity of Con A-CPY would not be explainable by this argument in a direct way. Conjugates of CNBr-activated agarose are positively charged. However, it is doubtful that such an electrostatic effect would be acting in this case because the Con A molecule is positioned between the support and CPY. Evidently the Con A itself provides an environment which resembles a positively charged matrix. The digestion of protein substrates by immobilized derivatives of CPY is of interest in protein structure studies. Immobilized Con A-CPY was used in a buffer containing 0.5% SDS for digestion of ribonuclease A. The time course of release of amino acids
appears in Fig. 6. The sequence of the C-terminal hexapeptide, -His-Phe-AspAla-Ser-Val-OH, is suggested by the rates of amino acid release. This sequence is identical to that reported by Smyth et al. (24). The bound derivatives of EDA-CPY and Gly-Tyr-CPY showed reduced activity with ribonuclease A as a substrate in buffers containing 0.5% SDS. Esterase activities of these preparations were reduced by only 10% by exposure to 0.5% SDS solution. It appears that chemical modification of the carbohydrate side chain has an effect on peptidase activity, especially with substrates of high molecular weight, but esterase activity is not significantly changed. Means and others have studied reductive alkylation of proteins using aldehydes and sodium borohydride (25). Many enzymes are stable to the conditions required for the introduction of methyl groups. Our system is related to reductive alkylation with the important differences that the periodate oxidation step precedes the reductive alkylation step and that the aldehyde is on the protein. In the cases studied (Table I> coupling of ethylenediamine to enzymes has limited effect on catalytic activity. In other experiments we have reduced the periodatetreated enzymes in the absence of amine z a;
0%
1
.. .. His-Phe-Asp-Ala-Ser-Vol-COOH
Time (min)
FIG. 6. Rate of release of amino acids from ribonuclease A by immobilized Con A-CPY. The reaction was carried out at 23°C with substrate concentration of 0.29 PmoYml and substrate to enzyme ratio (mol/ mol) of 440 to 1 in 0.1 N N-ethylmorpholine-acetate0.5% SDS buffer (pH 6.0).
IMMOBILIZATION
OF GLYCOENZYMES
to find only small changes in activity even when 50% of the original hexose was lost. We believe, therefore, that the method of immobilization introduced here should be generally applicable to other glycoproteins including some antibodies. Another extension would involve other solid supports. Hydroxylic matrices activated by carbonyl diimidazole would appear as likely supports for ethylenediamine-enzyme adducts. Finally, the procedures used here could be employed for the introduction of other functional groups into the carbohydrate portions of glycoproteins in order to alter surface charge and/or polarity. REFERENCES 1. GOLDSTEIN, L., AND KATCHALSKI-KATZIR, E. (1976) in Immobilized Enzyme Principles (Wingard, L. B., Katchalski-Katyir, E., and Goldstein, L., eds.), Vol. 1, pp. l-22, Academic Press, New York. 2. ROYER, G. P. (1979) Catal. Rev., in press. 3. LIBERATORE, F. A., MCISAAC, J. E., JR., AND ROYER, G. P. (1976) FEBS Lett. 68, 45-48. 4. POLLAK, A., BAUGHN, R. L., ADALSTEINSSON, ii., AND WHITESIDES, G. M. (1978) J. Amer. Chem. Sot. 100, 302-306. 5. G. M. WHITESIDES, personal communication. 6. ZABORSKY, 0. R., AND OGLETREE, J. (1974) Bio&em. Biophys. Res. Commun. 61, 210-216. 7. SULKOWSKI, E., AND LASKOWSKI, M., SR. (1974) Biochem. Biophys. Res. Commun. 57, 463468. 8. KUHN, R. W., WALSH, K. A., AND NEURATH, H. (1974) Biochemistry 13, 3871-3877. 9. JOHANSEN, J. I., BREDDAM, K., AND OTTESEN, M. (1976) Carlberg Res. Commun. 41, 1-15.
385
10. CUATRECASAS, P., AND PARIKH, I. (1972) Biochemistry 11, 2291-2298. 11. MARCH, J. C., PARIKH, I., AND CUATRECASAS, P. (1974) Anal. Biochem. 60, 149-152. 12. ROYER, G. P., ANDANDREWS, J. P. (1973)J. Biol. Chem. 248, 1807- 1812. 13. HARDMAN, K. D., WOOD, M. K., SCHUFFER, M., EDMUNDSON, A. B., AND AINSWORTH, C. F. (1971) Proc. Nut. Acad. Sci. USA 68, 13931397. 14. TRINDER, P. (1969) Ann. Clin. Biochem. 6, 24-ZI. 15. BERGMEYER, H. U., BERNT, E., SCHMIDT, F., AND STORK, H. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), p. 1196, Verlag Chemie, New York. 16. MOORE, S. (1968) J. Biol. Chem. 243,6281-6283. 17. ASHWELL, G. (1966) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), vol. 8, pp. 85-95, Academic Press, New York. 18. HAYASHI, R., MOORE, S., AND STEIN, W. H. (1973) J. Biol. Chem. 248, 8366-8369. 19. HAYASHI, R. (1976) in Methods in Enzymology (Lorand, L., ed.), vol. 45, pp. 568-587, Academic Press, New York. 20. ROYER, G. P., AND ANANTHARAMAIAH, G. M. (1979) J. Amer. Chem. Sot. 101, 3394-3396, 21. WILCHEK, M., OKA, T., ANDTOPPER, Y. J. (1979) Proc. Nat. Acad. Sci. USA 72, 1055-1059. 22. LASCH, J., AND KOELSCH, R. (1978) Eur. J. Biothem. 82, 181-187. 23. ROYER, G. P. (1975) in Immobilized Enzymes, Antigens, and Peptides (Weetall, H. H., ed.), pp. 49-51, Dekker, New York. 24. SMYTH, D. G., STEIN, W. H., AND MOORE, S. (1963) J. Biol. C&m. 238, 227-234. 25. MEANS, G. E. (1977) in Methods in Enzymology (Hirs, C. H. W., ed.), vol. 47, pp. 469-478, Academic Press, New York. 26. BETHELL, G. S., AYERS, J. S., HANCOCK, W. S., AND HEARN, M. T. W. (1979) J. Biol. Chem. 254, 2572-2574.
Immobilization
of Glycoenzymes through Carbohydrate HUMG-YU HSIAO AND GARFIELD
Department
of Biochemistry,
The Ohio State University,
Side Chains’
P. ROYER2
484 W. 12th Ave., Columbus, Ohio 43210
Received June 19, 1979 Glucoamylase, peroxidase, glucose oxidase, and carboxypeptidase Y were covalently bound to water-insoluble supports through their carbohydrate side chains. Two approaches were used. First, the carbohydrate portions of the enzymes were oxidized with periodate to generate aldehyde groups. Treatment with amines (ethylenediamine or glycyltyrosine) and borohydride provided groups through which the protein could be immobilized. Ethylenediamine was attached to glucoamylase, peroxidase, glucose oxidase, and carboxypeptidase Y to the extent of 24,20,30, and 15moYmo1of enzyme, respectively. These derivatives were coupled to an aminocaproate adduct of CL-Sepharose via an Nhydroxysuccinimide ester or to CNBr-activated Sepharose. Coupling yields were in the range of 3’7-50%. Retained activities of the bound aminoalkyl-enzymes were 41% (glucoamylase), 79% (peroxidase), 71% (glucose oxidase), 83% (carboxypeptidase Y). A glycyltyrosine derivative of carboxypeptidase Y was bound to diazotized arylamine-glass. Coupling yield was 42% and retained esterase activity was 84%. In the second approach, the enzyme was adsorbed to immobilized concanavalin A and the complex was crosslinked. Adsorption of carboxypeptidase Y on immobilized concanavalin A followed by crosslinking with glutaraldehyde was also effective. The bound enzyme retained 96% of the native esterase activity and showed very good operational stability.
Immobilization of enzymes and other biochemicals is of interest because of research and industrial applications of the solid-phase products (1, 2). Covalent coupling has been the most popular approach to immobilization presumably because of the accessibility and stability of the bound enzyme product. Usually the carboxyl groups, phenolic groups, or amino groups on the enzyme surface are reacted with a solid support. In our experience glycoproteins are difficult to immobilize possibly because the amino acid side chains are shielded by carbohydrate spines which project from the surface of the enzyme molecule. One such example is carboxypeptidase Y (CPY>,3 a carbohydrate con’ This work was supported by a grant from the National Institutes of Health (GM 19507). G.P.R. is the recipient of Public Health Service Career Development Award l-K4-GM-00051. * To whom correspondence should be sent. 3 Abbreviations used: CPY, carboxypeptidase Y; Con A, concanavalin A; AcOH, acetic acid; BuOH, butanol; Bicine, N,N-bis(2-hydroxyethyl)glycine; tic, thin layer chromatography; SDS, sodium dodecyl sulfate; APM, aminopeptidase M; EDA, ethylenediamine; Cbz, benzyloxycarbonyl. 379
taining exopeptidase from yeast. After many unsuccessful attempts to immobilize CPY, we prepared a bound derivative using water-soluble carbodiimide, hexamethylenediamine-agarose, and enzyme (3). Whitesides and co-workers have reported an effective procedure for enzyme immobilization which is based on the use of a polymeric N-hydroxysuccinimide ester (4). In this case also glycoenzymes proved recalcitrant (4,5). Zaborsky and Ogletree showed that glucose oxidase could be adsorbed to water-insoluble p-aminostyrene after periodate oxidation of the carbohydrate of the enzyme (6). Sulkowski and Laskowski showed that venom exonuclease retained activity while adsorbed on Con A-Sepharose (7). It seemed reasonable to us to extend these studies by making stable, covalently immobilized enzyme adducts in which the carbohydrate side chains provide the points of attachment between the enzyme and the solid support. The first approach is shown in Fig. la, the glycoenzymes are oxidized with periodate and coupled to an amine such as ethylenediamine or glycyltyrosine with NaBH, or NaBH,CN. In the second approach (Fig. 0003-9861/79/14037907$02.00/0 Copyright 0 1979by AcademicPress, Inc. All rights of reproductionin any form reserved.
380
HSIAO AND ROYER
FIG. 1. Immobilization of glycoproteins. (a) After oxidation with periodate, glycyltyrosine or ethylenediamine was attached to the enzyme by treatment with borohydride. The aminoalkyl groups provide additional points of attachment in the immobilization process. In (b) the glycoenzyme was first adsorbed on immobilized concanavalin A. The complex thus formed can be stabilized by crosslinking with glutaraldehyde.
lb) the glycoenzyme is adsorbed to insolubilized Con A and the complex is stabilized with glutaraldehyde. Both approaches gave stable covalently immobilized enzymes with good activity. MATERIALS
AND METHODS
Materials CPY from baker’s yeast (Anheuser Busch) was activated according to Kuhn et al. (8) and purified by affinity chromatography (9). Con A, glucoamylase, and hexokinase were purchased from BoehringerMannheim. Glucose oxidase (type II), ribonuclease A, NADP, and ATP were the products of Sigma. Sodium borohydride, sodium cyanoborohydride, and 4-aminoantipyrine were supplied by Aldrich. Vega-Fox provided Z-L-Leu-L-Phe. N-Ethylmorpholine was refluxed with ninhydrin and distilled. Ethylenediamine was distilled after drying over sodium metal. Both amines were stored in small brown bottles under nitrogen.
Methods Preparation
of ethyknediumine-enzyme
adduets.
Unless otherwise noted, all the reactions were carried
out at 4°C. CPY (1.4 mg/ml) in water was treated with sodium metaperiodate (0.02 M final concentration) for 1 h in the dark. Iodate was removed by dialysis against water (five changes, 1 liter per change) for 2.5 h. Periodate-treated enzyme was further modified by mixing with ethylenediamine (0.05 M final concentration at pH 8.0) followed by the addition of 0.1 M NaBH, (10 4, in 0.1 N NaOH) in two portions at 30- and 60-min intervals. After 90 min the derivative was purified by either dialysis or chromatography on Sephadex G-25 column (0.01 M phosphate-O.5 M NaCl buffer, pH 8.0). High concentration of salt was needed to prevent the precipitation of the aminoalkylated CPY. The absence of free ethylenediamine in the derivatized enzyme preparations was ascertained by thin layer chromatography (silica gel, n-BuOH-AcOH-pyridine-H,O, 15:3:12:10).Peroxidase was modified using similar procedures. Due to sensitivity of enzymes to alkaline conditions the derivatization of glucoamylase and glucose oxidase was performed in phosphate buffer (0.1 M, pH 6.0) containing NaBH,CN (0.1 mg/ml of reaction mixture) and ethylenediamine (0.05 M). The reaction time was 3 h. Immobilization of ethylenediamine-enzyme derivatives. Aminocaproic acid was bound to CL-Sepharose according to Liberatore et al. (3). TheN-hydroxy-
succinimide ester derivative of the gel was prepared as described by Cuatrecasas and Parikh (10). Glycoamylase, glucose oxidase, and peroxidase (1 mg/ml) were immobilized at pH 6.0, 6.3 (0.1 M phosphate buffer) and 8.5 (0.1 M Na-Bicine buffer) using the N-hydroxysuccinimide ester of the CL-Sepharose-aminocaproate derivative (1 ml of enzyme solution/ml of settled gel). The CPY derivative (2 mg/ml) was immobilized on CNBr-activated gel (1 ml of enzyme solution/ml of settled gel) at pH 8.0 (0.1 M Bicine-0.5 M NaCl) following the procedure of March et al. (11). Preparation of glycyltyrosine-CPY-glass. Glycyltyrosine was linked to oxidized CPY in a manner analogous to the preparation of the ethylenediamineCPY adduct. Glycyltryosine (0.1 M in 0.1 M Bicine buffer) was coupled to the oxidized CPY at pH 8 with NaBH, added as before. The derivative thus formed was purified by chromatography on Sephadex G-25 equilibrated with 0.01 M phosphate buffer (pH 7.8) which contained 0.15 M NaCl. Absence of free glycyltyrosine was verified by tic (silica gel; n-butanol-acetic acid-water, 4:1:5). Glycyltyrosine-CPY was then coupled to diazotized arylamine-glass according to the procedure of Royer and Andrews (12). The coupling reaction was carried out in 0.1 M phosphate buffer, 0.15 M NaCl, pH 7.8. Preparation of CL-Sepharose-Con A-CPY. Con A was reacted with N-hydroxysuccinimide ester of CLSepharose under the following conditions: 0.1 M phosphate buffer, pH 7.4, 4 mg Con A/2 ml buffer/g damp gel activated as previously described (10). The bound Con A was thoroughly washed with 1 M KC1 and ace-
IMMOBILIZATION
381
OF GLYCOENZYMES TABLE I
IMMOBILIZATIONOFETHYLENEDIAMINE ADDIJCTSOFGLYCOENZYMESONCARBOXYALKYLAGAROSE Glucoamylase Hexose remained after oxidation (o/o) Moles of ethylenediamine incorporated Protein on gel (mg/ml of settled gel) Yield (W) Specific activity of native form Specific activity of soluble ethylenediamine adduct Specific activity of bound enzyme
Peroxidase
Glucose oxidase
CPY
48
45
50
50
24
20
30
17
0.56 43
0.48 50
0.30 37
0.36 40
10
1100
63
64*
10 (100)
1090 (99)
55 (87)
56b (87)
4.1 (41)
870 (79)
45 (71)
53* (83)
n Percentage of protein bound on gel, determined by amino acid analysis. * Esterase activity. tate buffer (0.01 M, pH 5) (13). CPY (1 mg/ml, 3.3 ml) was stirred with immobilized Con A (1 g damp gel) for 30 min at 4°C in phosphate buffer (0.02 M, 0.15 M NaCl, pH 7.4). The gel was washed with 300 ml of 1 M ECl. Crosslinking of the Con A-CPY conjugate with glutaraldehyde was accomplished under the following conditions: 0.1 M phosphate buffer, pH 8, 2 ml buffer/l ml settled gel, 0.5% glutaraldehyde, 1 h. Enzyme assays and characterization. Peroxidase activity was determined by the procedure of Trinder (14). Glucoamylase activity was determined by measuring the amount of glucose released from glycogen (15). Glucose oxidase was assayed in the following procedure. An appropriate concentration of glucose oxidase enzyme solution (0.1 ml) was added to a mixture of phenol-4-aminoantipyrlne solution (1.4 ml, 0.170.0025 M), glucose solution (0.3 ml, 18% previously allowed to stand for 12 h), phosphate buffer (1.2 ml, 0.1 M, pH 6.0), and peroxidase (10 ~1, 1 mg/ml). Its activity was followed by measuring the increase in the absorbance at 510 nm. Esterase activity of CPY was followed titrimetrically using N-acetyltyrosine ethyl ester as substrate (3). Peptidase activity against Z-Leu-Phe was followed by ninhydrin analysis (3). Proteolytic digestion of ribonuclease A by immobilized CPY was followed by amino acid analysis at timed intervals. Digestions were carried out with ribonuclease A (0.29 PmoYml) in N-ethylmorpholine-acetate buffer (0.1 N, pH 6) containing 0.5% SDS. Amine content of the ethylenediamine-enzyme adducts was determined with the ninhydrin reagent of Moore (16). The incorporation of glycyltyrosine into CPY was found by amino acid analysis for tyrosine. Protein content of insoluble enzyme conjugates was also determined by amino acid analysis. Samples were hydrolyzed for 24 h with 6 N HCl at 110°C.The residues
were extracted twice with fresh HC1; the combined extracts were then dried using the flash evaporator. Hexose content of proteins was determined by the phenol-sulfuric acid method (17). RESULTS AND DISCUSSION
Ethylenediamine conjugates of glucoamylase, peroxidase, glucose oxidase, and CPY were prepared. In the four cases studied about 50% of the hexose was altered by periodate oxidation. The soluble aminoalkyl derivatives were quite active (Table I); glucoamylase containing 24 mol of ethylenediamine/mol was as active as the native molecule. Coupling yields were in the range of 37-50%, which is satisfactory for the conditions employed. The amount of protein bound could probably be increased by using more solid support per given amount of enzyme. The insoluble derivatives of peroxidase, glucose oxidase, and CPY showed good activity (U-83% of native). The activity of immobilized glucoamylase was 41% of the activity for the native enzyme. The level of retained activity of glucoamylase is below those of the other enzymes discussed here probably because of a substrate exclusion effect. Glucoamylase was assayed using glycogen which is a high polymer with a molecular weight in the millions. Stability studies on the immobilized aminoalkyl enzymes were encouraging (Fig.
382
HSIAO AND ROYER
5w
50
I I
1 2 NUMBER
I I 3 4 OF ASSAY
I 5
I 6
FIG. 2. Stability test of immobilized enzymes. Glucoamylase (01, glucose oxidase (A), carboxypeptidase Y (O), and peroxidase (0) were immobilized, following the procedure shown in Fig. la, to N-hydroxysuccinimide activated CL-Sepharose. These insoluble enzymes were repeatedly assayed in the period indicated.
2). After six assays in a stirred suspension, the immobilized forms of glucoamylase, glucose oxidase, and CPY retained full activity. In the peroxidase assay very small amounts of gel were used. Part or all of the loss of activity of peroxidase shown in Fig. 2 could be ascribed to the loss of gel particles in the separation and washing steps between
assays. Since much larger amounts of enzyme could be used for the other immobilized enzyme assays, physical loss of small amounts of enzyme gel was not significant. CPY is a valuable tool for carboxyl-terminal sequencing because of its very broad specificity (18-19). Proline is normally released smoothly. Also, we have demonstrated that immobilized CPY is useful for deblocking in sequential synthesis of polypeptides (20). In this approach the carboxyl terminus is extended using water-soluble carbodiimide and amino acid ethyl esters. The deblocking is accomplished by hydrolysis of the peptide ester with immobilized CPY at pH 8.5. At this pH value the peptidase activity is barely detectable and the esterase activity is maximal. Because of the use of CPY in sequencing and synthesis, we immobilized CPY by four different methods and characterized the derivatives in terms of peptidase and esterase activity (Table II). All four methods are superior to our previously reported procedure for immobilization. The Con A-CPY is quite active in both esterase and peptidase as-
TABLE II IMMOBILIZATION OF CPY
EDA-CPY” (CNBr-agarose)
EDA-CPY (Carboxy-agarose)
Gly-Tyr-CPY (arylamine-glass)
48
50
48
-
15
17
-
-
-
Con A-CPY Hexose remaining after oxidation (%o) Estimated moles of EDA incorporated Addition of Gly-Tyr on CPY (mol/mol of enzyme) Enzyme on gel (mgiml of settled gel) Yield (%) Specific esterase activity determined at pH optimum Ratio of specific esterase activity between bound and native form (X 100) Specific peptidase activity Ratio of specific peptidase activity between bound and native form (x100)
8
2.8 85
0.9 45
0.36 40
1.46 42
62
60
53
54
96 20
94 9.1
83 8.3
84 8.2
90
41
37
37
a Ethylenediamine-CPY. * Determined as mg enzyme/g of damp glass.
IMMOBILIZATION
says. Levels of retained activity were 96 and 90% of native. The amount of protein immobilized in this case was 85% presumably because of the strong initial adsorption of CPY to the immobilized Con A. Considering yield and retained activities, the immobilized Con A-CPY preparation is one of the best immobilized enzyme derivatives we have seen. Aminopeptidase M attached to porous glass retains full activity (12). However, the stability of this bound APM was not as good as immobilized Con A-CPY. The immobilization of aminoalkyl derivatives of CPY was also successful. Yields were in the range of 40-45% (Table II). The retention of esterase activity (83-94%) was better than the retention of peptidase activity (37-41%). For deblocking peptide esters this characteristic is desirable, since peptidase activity should be low compared to esterase activity. The immobilized aminoalkyl CPY derivatives were tested for stability (Fig. 3). As expected the most stable preparation was prepared with aminocaproate-agarose. The linkage between agarose and the enzyme contains an alkylamine C-N bond and an amide bond, both of which are very stable. One would expect the loss of activity of the EDA-CPY on CNBr-activated agarose since the substituted isourea linkages formed with CNBractivated agarose are known to be unstable at alkaline pH (21-22). Also, the surface of porous glass is eroded under the condi-
FIG. 3. Stability tests of four types of immobilized CPY. Immobilized EDA-CPY coupled toN-hydroxysuccinimide-activated CL-Sepharose (0), immobilized Con A-CPY (O), immobilized EDA-CPY coupled to CNBr-activated Sepharose (A), and immobilized TyrGly-CPY (0). Activity against N-acetyltyrosine ethyl ester at pH 8.0 was used for this test.
383
OF GLYCOENZYMES
6.0
6.5
I
I
7.0
7.5
I
8.0
I
8.5
I
9.0
I
9.5
I
10.0
PH
FIG. 4. The pH dependencies of esterase activities. Native CPY (V), immobilized EDA-CPY coupled to N-hydroxysuccinimide-activated CL-Sepharose (0), and immobilized Con A-CPY (Cl), immobilized EDACPY coupled to CNBr-activated Sepharose (A), and immobilized Tyr-Gly-CPY (0) in 0.1 M KC1 with 10 mM N-acetyltyrosine ethyl ester. The esterase activity at each pH was normalized by its activity at pH 8.0 being used as indicator of enzyme concentration. The rate profile of native CPY was adopted from Liberatore et al. (3).
tions of the assay which explains the lack of stability of the Gly-Tyr-CPY bound to arylamine -glass. In order to make intelligent use of an immobilized enzyme preparation, the pH-rate profile must be determined. The perturbations of some pH optima can be quite significant (23). Shifts as great as two pH units have been observed. The pa-rate profiles of the four immobilized CPY were determined for peptide and ester substrates. The pH-rate profiles for ester hydrolysis appear in Fig. 4. The shifts of 0.5-l pH unit are to the alkaline side of the pH opti.mum of the native enzyme. The pH optima of the bound enzymes are shifted to the acid side of the native enzyme in the peptidase assays (Fig. 5). Microenvironmental effects, diffusional effects, and chemical modification of the enzyme may be invoked to explain the changes in pH dependence curves for reactions catalyzed by immobilized enzymes. The alkaline shifts of the pa-rate profiles
384
HSIAO AND ROYER
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
6.0
9.5
9.0
DH
FIG. 5. The pa-rate profiles of peptidase activities. Immobilized EDA-CPY coupled toN-hydroxysuccinimide-activated CL-Sepharose (O), immobilized EDACPY coupled to CNBr-activated Sepharose (A), immobilized Con A-CPY (O), immobilized Tyr-GlyCPY (0), and native CPY (V). Activity was measured with Cbz-Leu-Phe. Buffers and assay procedure are described in the text. The rate profile of native CPY was adoped from Liberatore et al. (3).
of the esterolytic reactions of the bound CPY derivatives could be accounted for by rate-limiting efflux of protons from the gel. The shift to a lower pH optimum is typical when enzymes are bound to positively charged supports. Such a microenvironmental effect is probably operating in the acid region for the peptidase activities. The aminoalkylated derivatives are themselves more positively charged than native enzymes in the acid pH range. The perturbation of the pH-rate profile for the peptidase activity of Con A-CPY would not be explainable by this argument in a direct way. Conjugates of CNBr-activated agarose are positively charged. However, it is doubtful that such an electrostatic effect would be acting in this case because the Con A molecule is positioned between the support and CPY. Evidently the Con A itself provides an environment which resembles a positively charged matrix. The digestion of protein substrates by immobilized derivatives of CPY is of interest in protein structure studies. Immobilized Con A-CPY was used in a buffer containing 0.5% SDS for digestion of ribonuclease A. The time course of release of amino acids
appears in Fig. 6. The sequence of the C-terminal hexapeptide, -His-Phe-AspAla-Ser-Val-OH, is suggested by the rates of amino acid release. This sequence is identical to that reported by Smyth et al. (24). The bound derivatives of EDA-CPY and Gly-Tyr-CPY showed reduced activity with ribonuclease A as a substrate in buffers containing 0.5% SDS. Esterase activities of these preparations were reduced by only 10% by exposure to 0.5% SDS solution. It appears that chemical modification of the carbohydrate side chain has an effect on peptidase activity, especially with substrates of high molecular weight, but esterase activity is not significantly changed. Means and others have studied reductive alkylation of proteins using aldehydes and sodium borohydride (25). Many enzymes are stable to the conditions required for the introduction of methyl groups. Our system is related to reductive alkylation with the important differences that the periodate oxidation step precedes the reductive alkylation step and that the aldehyde is on the protein. In the cases studied (Table I> coupling of ethylenediamine to enzymes has limited effect on catalytic activity. In other experiments we have reduced the periodatetreated enzymes in the absence of amine z a;
0%
1
.. .. His-Phe-Asp-Ala-Ser-Vol-COOH
Time (min)
FIG. 6. Rate of release of amino acids from ribonuclease A by immobilized Con A-CPY. The reaction was carried out at 23°C with substrate concentration of 0.29 PmoYml and substrate to enzyme ratio (mol/ mol) of 440 to 1 in 0.1 N N-ethylmorpholine-acetate0.5% SDS buffer (pH 6.0).
IMMOBILIZATION
OF GLYCOENZYMES
to find only small changes in activity even when 50% of the original hexose was lost. We believe, therefore, that the method of immobilization introduced here should be generally applicable to other glycoproteins including some antibodies. Another extension would involve other solid supports. Hydroxylic matrices activated by carbonyl diimidazole would appear as likely supports for ethylenediamine-enzyme adducts. Finally, the procedures used here could be employed for the introduction of other functional groups into the carbohydrate portions of glycoproteins in order to alter surface charge and/or polarity. REFERENCES 1. GOLDSTEIN, L., AND KATCHALSKI-KATZIR, E. (1976) in Immobilized Enzyme Principles (Wingard, L. B., Katchalski-Katyir, E., and Goldstein, L., eds.), Vol. 1, pp. l-22, Academic Press, New York. 2. ROYER, G. P. (1979) Catal. Rev., in press. 3. LIBERATORE, F. A., MCISAAC, J. E., JR., AND ROYER, G. P. (1976) FEBS Lett. 68, 45-48. 4. POLLAK, A., BAUGHN, R. L., ADALSTEINSSON, ii., AND WHITESIDES, G. M. (1978) J. Amer. Chem. Sot. 100, 302-306. 5. G. M. WHITESIDES, personal communication. 6. ZABORSKY, 0. R., AND OGLETREE, J. (1974) Bio&em. Biophys. Res. Commun. 61, 210-216. 7. SULKOWSKI, E., AND LASKOWSKI, M., SR. (1974) Biochem. Biophys. Res. Commun. 57, 463468. 8. KUHN, R. W., WALSH, K. A., AND NEURATH, H. (1974) Biochemistry 13, 3871-3877. 9. JOHANSEN, J. I., BREDDAM, K., AND OTTESEN, M. (1976) Carlberg Res. Commun. 41, 1-15.
385
10. CUATRECASAS, P., AND PARIKH, I. (1972) Biochemistry 11, 2291-2298. 11. MARCH, J. C., PARIKH, I., AND CUATRECASAS, P. (1974) Anal. Biochem. 60, 149-152. 12. ROYER, G. P., ANDANDREWS, J. P. (1973)J. Biol. Chem. 248, 1807- 1812. 13. HARDMAN, K. D., WOOD, M. K., SCHUFFER, M., EDMUNDSON, A. B., AND AINSWORTH, C. F. (1971) Proc. Nut. Acad. Sci. USA 68, 13931397. 14. TRINDER, P. (1969) Ann. Clin. Biochem. 6, 24-ZI. 15. BERGMEYER, H. U., BERNT, E., SCHMIDT, F., AND STORK, H. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), p. 1196, Verlag Chemie, New York. 16. MOORE, S. (1968) J. Biol. Chem. 243,6281-6283. 17. ASHWELL, G. (1966) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), vol. 8, pp. 85-95, Academic Press, New York. 18. HAYASHI, R., MOORE, S., AND STEIN, W. H. (1973) J. Biol. Chem. 248, 8366-8369. 19. HAYASHI, R. (1976) in Methods in Enzymology (Lorand, L., ed.), vol. 45, pp. 568-587, Academic Press, New York. 20. ROYER, G. P., AND ANANTHARAMAIAH, G. M. (1979) J. Amer. Chem. Sot. 101, 3394-3396, 21. WILCHEK, M., OKA, T., ANDTOPPER, Y. J. (1979) Proc. Nat. Acad. Sci. USA 72, 1055-1059. 22. LASCH, J., AND KOELSCH, R. (1978) Eur. J. Biothem. 82, 181-187. 23. ROYER, G. P. (1975) in Immobilized Enzymes, Antigens, and Peptides (Weetall, H. H., ed.), pp. 49-51, Dekker, New York. 24. SMYTH, D. G., STEIN, W. H., AND MOORE, S. (1963) J. Biol. C&m. 238, 227-234. 25. MEANS, G. E. (1977) in Methods in Enzymology (Hirs, C. H. W., ed.), vol. 47, pp. 469-478, Academic Press, New York. 26. BETHELL, G. S., AYERS, J. S., HANCOCK, W. S., AND HEARN, M. T. W. (1979) J. Biol. Chem. 254, 2572-2574.
