Eur. J. Biochcm. 59, 567-572 (1975)

Reversible, Covalent Immobilization of Enzymes by Thiol-Disulphide Interchange

Jan CARLSSON, Rolf AXEN, and Torsten UNGE

Institute of Biochemistry, University of Uppsala

(Received May 28:August 16, 1975)

1. a-Amylase and a-chymotrypsin have been immobilized by covalent attachment to mercapto- hydroxypropyl ether agarose gel. The technique involves two steps : (a) thiolation of the enzymes by methyl 3-mercaptopropioimidate, (b) coupling of the thiolated enzymes to a mixed disulphide derivative of agarose obtained by reacting mercaptohydroxypropyl ether agarose with 2,2’-dipyridyl disulphide.

2. The immobilization technique can be performed so that most of the inherent activity of the enzymes is conserved. However, diffusion limitations and steric factors prevent full manifestation of the immobilized activities.

3. Immobilized a-amylase was used in a packed-bed reactor for the continuous hydrolysis of starch. When the enzymically active gel had lost its activity it could be regenerated in situ by reductive un- coupling of the inactive protein and attachment of a new portion of thiolated a-amylase.

A wide range of techniques have been developed for the covalent attachment of enzymes to water- insoluble supports [l]. Most methods at present in use give irreversible immobilization of the enzymes. Although methods have been described for func- tionalization of support materials with the purpose of achieving reversible covalent immobilization, the potential advantages of such techniques have scarcely been demonstrated. By means of reversible covalent attachment it is feasible to detach the enzyme which has lost its catalytic properties, regenerate the support and recharge with a new portion of enzyme.

A previous paper described a technique for the reversible immobilization of urease to a reactive, mixed disulphide, derivative of agarose [2]. The thiol- group containing urease was immobilized via disul- phide linkages. The immobilized enzyme was very active and could be removed by reduction. The mixed disulphide derivative was obtained by reacting agarose- bound glutathione with 2,2’-dipyridyl disulphide as described by Brocklehurst efal. [3]. The coupling of a thiol component such as urease to agarose - gluta- thione - 2-pyridyl disulphide proceeds by a thiol- disulphide interchange reaction. Undesired inter- and intramolecular reactions of the thiol-component in the solvent phase could be avoided.

The preparation of a thiolated agarose gel espe- cially designed for the immobilization of enzymes, was subsequently described [4]. The thiol-gel has increased

chemical stability through cross-linking and is equip- ped with mercaptohydroxypropyl ether constituents. The thiol-groups are activated by dipyridyl disulphide.

Immobilization via disulphide linkages is also of interest for enzymes not containing thiol-groups, or containing masked or otherwise unreactive thiol- groups if a sufficiently mild thiolation method can be found. This paper describes reversible immobiliza- tion of a-chymotrypsin and a-amylase. The thiolation was performed using methyl 3-mercaptopropioimi- date. Procedures are given for the attachment of an enzyme to support material retained in a column, continuous use of the bed reactor, reductive detach- ment, chemical reactivation and reattachment of the catalyst.

MATERIALS AND METHODS Muter ials

cc-Chymotrypsin (3 x crystallized, lyophilized) was purchased from Worthington Biochem. Corp. (Free- hold, N.J., U.S.A.). a-Amylase (Hog pancreas type 1-A) was obtained from Sigma Chemical Co. (St. Louis, Mo., U.S.A.).

Sepharose 2B and Sephadex G-25 were from Pharmacia Fine Chemicals AB (Uppsala, Sweden). Dithiothreitol and N-acetyl-L-tyrosine ethyl ester were obtained from Sigma Chemical Co. (St. Louis, Mo., U.S.A.). Soluble starch (Zulkowski), 3J-dinitro-

568 Reversible, Covalent Immobilization of Enzymes

salicylic acid (DNS), and sodium thiosulphate were products of Merck AG (Darmstadt, West-Germany). 2,2'-Dipyridyl disulphide was purchased from Aldrich-Europe (B 2340 Beerse, Belgium). Epichloro- hydrin (l-chloro-2,3-epoxypropane) was obtained from Kabo AB (Stockholm, Sweden). Methyl 3-mer- captopropioimidate was synthesized essentially as described by Perham and Thomas [5 ] and was pro- tected against hydrolysis by storage under nitrogen at - 25 "C. Epichlorohydrin cross-linked, desulphated agarose was prepared from Sepharose 2B by the method of Porath et ul. [6 ] .

Preparation of Thiol Polymer

Preparation of mercaptohydroxypropyl ether aga- rose gel was carried out essentially as described by Axtn et al. [4].

Preprution of Epoxide-Activated Axarose Gel

Epichlorohydrin cross-linked and desulphated agarose was washed on a glass filter with distilled watcr and sucked free from interstitial water. 50 g of the gel was suspended in 40 ml 1 M hydroxide solution. 1.4 ml 1 -chloro-2,3-epoxypropane (epichloro- hydrin) was added slowly under stirring at room temperature, whereupon the temperature was in- creased to 60 "C and maintained for 1 h. The activated gel was washed on a glass filter until neutral with distilled water. The product is not stable to storage.

Preparation of S-AIkyl- Thiosulphate Agarose Gel

Epoxide activated agarose (50 g) was washed on a glass filter with 0.5 M phosphate buffer (4.1 g NaH,PO,. H 2 0 + 2.8 g Na2HP04. 2 H 2 0 dissolved in 100 ml distilled water, pH 6.3). The gel was sucked free from interstitial buffer and suspended in the same buffer to a final volume of 100 ml. A solution of 2 M sodium thiosulphate (50 ml) was added and the reaction mixture was shaken for 6 h at room temper- ature. The gel was washed free from sodium thio- sulphate with distilled water. The thiosulphate ester gel is stable to storage as suspension in distilled water.

Reduction of S-AIkyl-Thiosulphate Gel

The thiosulphate ester gel (50 g) was suspended in 0.1 M sodium bicarbonate solution (1 mM EDTA) to a total volume of 100 ml. Dithiothreitol (60 mg) dissolved in 4 ml 1 mM EDTA-solution was added to the suspension. The reaction time was 30 min at room temperature. The gel was washed on a glass filter with 0.1 M sodium bicarbonate solution (1 M in sodium chloride and 1 mM in EDTA) and finally with 1 mM EDTA-solution. The thiol content of the mercapto-

hydroxypropyl ether agarose gel was determined by means of 2-pyridyl disulphide according to Brockle- hurst etuf . [3] but 0.1 M sodium bicarbonate buffer was used instead of Tris-buffer.

The thiolated gel was normally used directly. However, for a few days the gel can be stored in 0.01 M deaereated sodium acetate buffer of pH 4 (1 mM in EDTA).

Activation of Thiol Polymer

This was performed essentially as described by Brocklehurst etal. [3]. The thiol agarose (50 g) was washed on a glass filter with 1 mM EDTA. The washed gel was rapidly added to 200 ml 2,2'-dipyridyl disul- phide solution (1.5 mM in 0.1 M NaHCO,). The mixture was stirred during the reaction which was allowed to proceed for 30 min at room temperature. The product was washed with 0.1 M NaHCO,, 1 M NaCl and finally with 1 mM EDTA solution. The degree of substitution was determined by nitrogen determination according to Kjeldahl. The product called activated thiol agarose is stable to storage.

Thiol Enrichment of Enzymes

30 mg enzyme (a-chymotrypsin of a-amylase) was dissolved in 5 ml 0.1 M NaHC0, pH 8.2 (in the case of a-chymotrypsin the solution also contained 30 mg of the inhibitor N-acetyl-D-tryptophan to prevent autodigestion of cr-chymotrypsin). The solution was deaereated in nitrogen atmosphere for 15 min and 0.1 - 2 mg methyl 3-mercaptopropioimidate was added. The thiolation was carried out at room temper- ature (+ 23°C) in nitrogen atmosphere (glove bag) for 60 min. Excess imidate was removed by gel filtration on Sephadex G-25 using 0.1 M NaHCO, as eluant. To prevent oxidation of the thiolated enzymes, dithiothreitol (1 mM final conc.) was added to the solution just before the gel filtration.

Immobilization of Thioluted Enzyme

After removal of excess thiolating agent at pH 8.2 (see above), the thiolated enzyme (1 -20 mg) in 10-15 ml 0.1 M NaHCO, was mixed (within 10-40 min) with 3.0 ml sedimented activated thiol agarose (prewashed with 0.1 M NaHCO,). The reac- tion, performed in a 25-ml stoppered plastic bottle slowly rotated end-over-end to ensure good mixing, was run for 24 h at + 23 "C.

Some of the immobilized a-amylase derivatives were prepared by passing 5 - 20 mg thiolated a-amylase in 20 mlO.1 M NaHCO, through a column (1.4 x 2 cm) containing 3 ml activated thiol agarose (flow rate 10 ml/h).

The conjugates were washed with 0.1 M NaHCO, (100 ml) on a sintered glass funnel (when the batch

J. Carlsson. R. Axcn, and T. Unge

A to,, Eplchlorohydrin b #O-CH2-CY2CH,

569

c ~O-CHI-CH(OH)-CHI-S20~N; Dithiothrrito', 0-CH2- CHIOHI-CHZ-SH

Fig. 1. Prepurution qfutfivuted thiol ugurose. (A) Activation of epichlorohydrin cross-linkcd and desulphated agarose gel by cpichlorohydrin ; (B) preparation of S-alkyl-thiosulphate agarose gel from epoxide-activated agarose with sodium thiosulphatc: (C) reduction of S-alkyl-thio- sulphate gel by dithiothreitol; (D) activation of thiol-agarose gel with 2,2'-dipyridyl disulphide

procedure was used) and then in a column (1.4 x 3 cm) by passing the following solutions at a flow rate of 10 ml/h; (1) 0.1 M NaHCO, containing 0.2 M NaCl (24 h); (2) 0.1 M sodium acetate buffer pH 5.4 contain- ing 0.2 M NaCl (24 h) and (3) 0.2 M NaCl (24 h). When the a-amylase conjugates were washed, 1% starch was included in solution 2.

Recharging of a-Amylase Column

The inactivated a-amylase was removed by pass- ing 50 ml 20 mM dithiothreitol in 0.1 M NaHC03 through the column (0.5 ml) (flow rate: 20 ml/h). The reduced support was washed with 150 ml 1 M NaCl and activated by passing 100 ml 1.5 mM 2,2'- dipyridyl disulphide in 0.1 M NaHCO, through t h e column. The activated thiol gel was washed with 100 ml 1 M NaCl and 100 mlO.1 M NaHCO,. Freshly thiol-enriched a-amylase was then immobilized on the column material as described above.

The immobilized enzyme conjugates were stored as suspensions in 0.2 M NaCl at + 4 "C.

Activity Measurements

a-Chymotrypsin Conjugates. Activity towards N-acetyl-L-tyrosine ethyl ester was determined titri- metrically at 23 "C. The assay solution was 25 mM in N-acetyl-L-tyrosine ethyl ester and 0.2 in sodium chloride with a total volume of 2 ml. The enzyme conjugates and the free enzyme were added as sus- pensions or solution in 0.2 M sodium chloride. Optimal activity was obtained at pH 8 for free a-chymotrypsin and at pH 9.5 for the a-chymotrypsin-agarose con- jugates.

a-Amylase Conjugates. To I ml stirred enzyme solution or suspension in 0.02 M sodium phosphate buffer pH 6.9 (this pH was optimal both for free and immobilized a-amylase) containing 0.01 M NaCl was added 1 ml 1 starch in the same buffer. The reaction was allowed to proceed for 3 min. The reaction was then interrupted and the content of reducing sugar was determined as described by Bernfeld by reaction with 3,5-dinitrosalicylate at 100 "C [7]. Absorption

at 500 nm was measured with a Beckman DB-60 spectrophotometer and the activity was calculated from a maltose standard curve.

Continuous Digestion of Starch by Immobilized a-Amylase

This was performed as described in Fig. 4.

Other Methods

The Protein Contents of the Gel Suspensions and the concentrations of the solutions of free enzymes were determined by total amino acid analysis after hydro- lysis in 6 M HCl for 24 h at 110°C [8].

Disintegration of Enzyme-Sepharose Gel Purticles was performed by ultra sonication (20 kHz, pulsed 50 x 5 s).

The Thiol Contents of the thiolated enzymes were determined spectrophotometrically by titration with 2,2'-dipyridyl disulphide [9].

RESULTS AND DISCUSSION

Agarose gel beads have been found to possess valuable properties as carriers for enzymes. Agarose- enzyme conjugates may be stabilized against leakage of enzyme activity if the gel carrier is cross-linked by epichlorohydrin before attachment of the enzyme [lo]. The cross-linked gel can subsequently be sub- stituted with highly reactive epoxide-structures [l 1 ] by a second epichlorohydrin treatment. The epoxide structures can be utilized for the preparation of thiol agarose gels according to Fig. 1 A-C [4]. Treatment with a water solution of sodium thiosulphate converts the epoxide-structures to S-alkylthiosulphate struc- tures and subsequent reduction with dithiothreitol gives epichlorohydrin cross-linked mercaptohydroxy- propyl ether agarose. The thiol gel used in the present paper contained about 80 pmol of thiol groups per g dried polymer. The experiments showed that a higher degree of substitution was unnecessary or even incon- venient. Sepharose 2 B was used as the starting material

570 Reversible, Covalent Immobilization of Enzymes

Tablc 1. Immohilizution of u-chymolrypsin on thiolated ugurose by thiol-disulphide interchange. Mercaptohydroxypropyl ether agarosc was reacted with 2.2'- dipyridyl disulphide. Chymotrypsin was thiolated with methyl 3-mercaptopropioimidate. The thiolated chymotrypsin was immo- bilized by reaction with the activated thiol agarose in 0.1 NHCO, at pH 8.2 and 23 "C. Catalytic activity was determined titrimetrically towards N-acetyl-L-tyrosine ethyl estcr

Catalyst Enzyme pH- Activity contcnt optimum

mgig pmol min-' conjugate mgprotein-'

Chymotrypsin - 8.0 370 Thiolated chymotrypsin - 8.0 360- 370

(1.5 SH/mol) Chymotrypsin-S-S-agarose 12 9.5 260 Chymotrypsin-S-S-agarose 30 9.5 250 Chymotrypsin-S-S-agarose 55 9.5 220 Chymotrypsin-S-S-agarose 55 8.5 355

(sonicated)

for the preparation of thiol agarose. For the im- mobilization of thiol group containing enzymes ac- cording to the thiol disulphide interchange technique the thiol gel was treated with 2,2'-dipyridyl disulphide (Fig. 1D). This treatment gives an activated thiol gel containing 40 pmol of 2-pyridyl disulphide structures per g dry polymer. During the activation process, unreacted thiol groups underwent a thiol disulphide interchange with gel-bound 2-pyridyl disulphide struc- tures with the formation of stable internal disulphide bridges. This explains why only 50% of the thiol groups are converted into reactive 2-pyridyl disulphide structures. The activated thiol gel can be stored as a suspension in distilled water for five months a + 4 "C without significant decrease in the concentration of mixed disulphide structures.

Pancreatic hog a-amylase is reported to contain thiol groups required for high activity [12]. There was, however, no thiol disulphide interchange between the used enzyme-preparation and 2,2'-dipyridyl disul- phide or the 2-pyridyl disulphide-agarose derivative. a-Chymotrypsin lacks thiol groups.

The introduction of auxiliary thiol groups in u- amylase and a-chymotrypsin was originally attempted using the commonly recommended N-acetyl-homo- cysteine thiolactone; the results, however, were un- satisfactory. It was found that methyl 3-mercapto- propioimidate was a suitable reagent for introducing de novo thiol groups. This reagent, which has been used for thiol-enrichment of tobacco mosaic virus [ 5 ] , reacts under slightly alkaline conditions with amino- functions with the formation of amidine-linkages

The thiolation of a-amylase with methyl 3-mercapto- propioimidate could be performed with practically no loss of activity, on introduction of up to about 5 thiol groups per mole enzyme. A preparation con- taining 3 thiol groups per mole enzyme was used for the immobilization studies. The introduction of 2 - 3 thiol groups per mole chymotrypsin led to a decrease in activity of about 20% and the introduction of about 1 - 1.5 thiol groups per mole interferred little with the catalytic efficiency (Table 1). To minimize formation of disulphide by oxidation of the thiolated enzyme and methyl 3-mercaptopropioimidate, the thiolation reac- tions were always carried out under a nitrogen atmosphere and dithiothreitol(1 mM) was added to the reaction mixture before removal of excess reagent by gel filtration.

The thiolated enzyme was then reacted with the activated polymer within 30- 60 min. Storing experi- ments with thiolated a-chymotrypsin, however, showed that the thiol titer remained constant for at least two days.

The immobilization of the thiol-enriched enzymes was either performed batchwisc or according to the column technique by pumping a solution of the thiol- enriched enzyme through a column charged with activated thiol gel material. In the latter case the coupling reaction was monitored by absorption determinations of the effluent at 343 and 280 nm. The quotient A,,,/A,,, equals 1.25 for the formed 2-thio- pyridone [9] (Fig. 2A) and an increased quotient shows the passage of protein. It should be noted that the rate of coupling is so high that if the column is not exposed to saturating amounts of thiolated enzyme one obtained nonuniform coupling along the column. Saturation of the column with the thiolated enzyme preparations gave uptakes of protein on 250 mg/g dried conjugate as determined by amino acid analysis [8]. On exposing a column to an amount of thiolated a-amylase corresponding to 10 :g of the saturating amount, it was found that 15 '%, of the added protein passed through, and that increasing the passage time did not change the result. Analogues immobilization experiments with thiolated chymotrypsin showed that when applying a preparation characterized by about 1.5 thiol groups per mole, 40% of the added protein passed the column and by applying a preparation characterized by about 3 thiol groups per mole about 35% passed through. It seems reasonable that the thiolation reactions had not proceeded homogene- ously. The thiolated enzymes may consist of a popula- tion of variously modified enzyme molecules and espe- cially at low degrees of thiolation, some may not have

Nh, ,Nir, HS-CH,.CH,.C, + H,N -@ + @- NH. * iH,-CH2-SH +CH30H

OCH,

J . Carlsson, R. Axkn, and T. Unge

-

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8000

7000 2 6000 '$ 5000P

4000 5

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A ~ o - C H 2 - C H I O H i - C H 2 - s - s ~ + -SH+ i f o - c H 2 - c H i o H i - c H , - s - s ~ +SQ

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been thiolated at all. However, it cannot be excluded that during the immobilization process concomitant reductive decoupling may occur as well, explaining at least in part the observations.

The enzyme-conjugates characterized in Table 1 and Fig.3 were prepared according to a batch tech- nique to ascertain that homogeneous coupling of the thiolated enzymes occurs in the gel. The coupling uptake during the batch procedure was about 60- 80 of the added amounts of protein. On treatment with 20 mM dithiothreitol solution, the immobilized enzyme was quantitatively removed from the carrier (Fig. 2B) indicating that the enzymes are attached by disul- phide bridges. Blank experiments employing the 2,2'- dipyridyl disulphide activated thiol gel ' with native enzyme preparations under coupling conditions and subsequent washing steps resulted in catalytically inactive gel materials

The specific activity of an enzyme is normally considerably decreased due to its conjugation with a water-insoluble carrier [I]. The chemical modification of an enzyme molecule usually interferes with the catalytic properties. Even if the enzyme has been immobilized without loss of inherent activity, the apparent activity of the insolubilized enzyme is usually considerably less than the internal activity since the presence of the carrier interferes with mass transport of substrates and products [13,14,15]. In the present cases it seems reasonable that the coupling reaction itself, namely the thiol/disulphide interchange reaction involving artificially introduced enzyme thiol groups, would not interfere with the catalytic properties.

The activity of immobilized chymotrypsin (Table 1) was assayed towards N-acetyl-L-tyrosine ethyl ester in an unbuffered medium. An increase in the pH- optimum of about 1.5 pH-units was noticed as a result of the immobilization compared with the free enzyme. The activity of the insolubilized enzyme decreases with increasing protein content in the gel-carrier.

Disintegration of the enzyme-agarose beads to smaller sizes by ultrasonication increased the activity and also resulted in a displacement of the apparent pH-optimum backwards by about one pH-unit

(Table 1). These findings indicate that the enzyme gel carrier/substrate/product system is diffusion control- led [14,15]. Essentially no activity decrease was observed when immobilized a-chymotrypsin was stored as a suspension in 1 mM EDTA for 1 month at + 4°C.

250

200

150

= 100

:Z 50

0

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h - - U

Q O 0 100 2 00 Enzyme content (mg/g conjugate)

Fig. 3. Activiiy of the conjugate us a junction of en:)ime content Jor immobilized a-umylase given us units per mg enzyme (0 ~ 0) and units per g conjugate (w). One unit of activity liberates an amount of reducing groups from starch equivalent to 1 mg maltose in 3 min at pH 6.9 at 23 "C

~~

) ) 4 5 " c ( l r ~ e enzyme) 0 10 20 30

Time (days) Fig. 4. Continuous digestion of sturch by immobilized a-amylase. 17; starch (in 0.02 M sodium phosphate buffer. 0.01 M NaCI, pH 6.9) was passed through columns containing 0.5 ml of a-amy- lase-agarose (containing 50 mg a-amylase/g dry conjugate) at a constant flow rate of 10 rnl/h. At regular intervals, aliquots were withdrawn and their contents of reducing sugar determined by reac- tion with 3,5-dinitrosalicylate as described by Bernfeld [7]. During the sampling procedure the Row rate was adjusted to 120 ml/h. This gave 80% conversion in the first pass. The experiments were carricd out at + 2 3 T and +45"C.

Catalytic activities of free and immobilized a- amylase were assayed towards starch at pH 6.9; this pH gave optimal activity for both free and immobilized a-amylase. Fig. 3 summarizes the results regarding the activity as a function of the enzyme content of the conjugate. The figure shows that the activity, expressed as U/mg bound enzyme, decreases with increasing amount of bound enzyme. It is interesting to notice that when the activity is expressed as U/g conjugate an optimum in activity is obtained at an enzyme content of the conjugate of about 100 mg/g conjugate, and at a higher loading of enzyme, the conjugate becomes less efficient as a catalyst. Steric hindrance

5 72 J. Carlsson, R. Axtn. and T. Unge: Reversible, Covalent Immobilization of Enzymes

3 0 2 ~ 0 2 L 0 2 L O 2 L Time (days)

Fig. 5 . Repeated reactivation in situ OJ a-urnjhw column. 0.5 ml a-amylase - agarose (containing 70 mg a-amylase/g dry conjugate) in a small column was allowed to digest 1 "/, starch (in 0.02 M sodium phosphate buffer, 0.01 M NaCl pH 6.9) passing at 10 ml/h. Aliquots of the eluate were assayed. During the sampling procedure the flow rate was adjusted to give 90% of maximum conversion. The experi- ment was carried out at +45"C. Arter 5 days lO-15% of the original activity ofthe a-amylase column remained. The column was then recharged. Inactive a-amylase was detached by dithiothreitol (20 mM in 0.1 M NaHCO,). After reactivation of the reduced sup- port with 2,2'-dipyridyl disulphide (1.5 mM), a new batch of freshly thiolated a-amylase was attached. The whole recharging procedure, which was performed in situ by passing the different solutions through the column, was repeated three times

for substrate moleciilcs reaching active sites when the enzyme molecules are too highly crowded could provide an explanation.

The operational stability of immobilized a-amylase, when used continuously in a packed bed reactor for hydrolyzing an aqueous starch solution (1 %,) at room temperature and at 45"C, is given in Fig.4. After 22 days of operation at room temperature, the activity was still unchanged but a column operated at 45°C showed only 10-20% of the original activity after 5 days of operation. The activity of the column in the latter case was regenerated by reductive decoupling of protein, whereupon the thiol agarose was activated by 2,2'-dipyridyl disulphide and charged with a new portion of thiolated a-amylase. These procedures were carried out in the column by pumping reagents and the enzyme through the column. At each recharging the resulting uptake of activity was practically the same as the original uptake (Fig.5).

The possibility of reusing the polymeric support after inactivation of the enzyme may be of interest

for the practical use of immobilized enzymes in large- scale processes in industry, where their use often has been hampered by the high cost of the support material. Regarding immobilized enzyme activities, together with a certain type of reactor system in which catalyst and support are retained, it would be advantageous to perform the attachment, detachment and reattachment of the catalyst in the closed system.

Analogous utilization of thiol disulphide inter- change reactions should also be useful for the prepara- tion of adsorbents for affinity chromatography and immunosorbents. The possibility of removing the whole ligand-adsorbent complex from the support by reductive decoupling may be of interest.

This work has been supported by grants from the Swedish Board for Technical Development and the Lennander Foundation.

REFERENCES

1. Zaborsky, 0. R. (1973) Immohilized Enzymes (Weast, R. C.,

2. Carlsson, J . , Axen, R., Brocklehurst, K. &Crook, E. M. (1974) ed.) CRC Press, Cleveland, Ohio 44128.

3.

4.

5.

6.

7. 8.

9.

10.

1 1 . 12. 13.

14.

15.

Eur. J . Biochem. 44, 189- 194.

E. M. (1973) Biochem. J . 133. 573-584.

in press.

415 - 418.

Brocklehurst, K., Carlsson, J. , Kierstan, M. P. J . & Crook,

Axen, R., Drevin, H. & Carlsson. J . (1975) Acra Chem. Scand.

Perham.N. R. & Thomas, J. 0. (1971) J . Mol. Biol. 62.

Porath. J., Janson, J.-C. & LBPs. T. (1971) J . Chromatogr. 60.

Bernfeld, P. (1955) Methods in Enzymol. I , 149- 158. Spackman. D. H., Stein, W. H. & Moore, S. (1958) And.

Grassetti, D. R. & Murray, J . F. (1967) Arch. Biuchum. Biuphys.

Axen, R., Carlsson, J., Janson, J.-C. & Porath, J. (1971) E n q -

Sundberg, L. & Porath. J. (1974) J . Chromutogr. 90.87-98. Schramm, M. (1964) Biochemisrrj: 3, 1231 - 1233. Sundaram, P. V., Tweedale, A. & Laidler, K. J. (1970) Can. J .

Axen, R., Myrin, P:A. & Janson, J.-C. (1970) Biopolymers, 9.

Carlsson, J. , Gabel, D. & Axen, R. (1972) Hoppe-Sejfer's

167-177.

C'hrm. 30, 1190.

IIY,41-49.

rnologiu, 41, 359- 364.

Clzem. 48, 1498 - 1504.

401-413.

Z . Physiol. Chem. 353, 1850- 1854.

J. Carlsson, R. Axen, and T. Unge, Biokemiska Institutionen, Uppsala Universitet, Box 531, S-751 21 Uppsala 1 , Sweden