[5] Immobilization of enzymes to various acrylic copolymer

[5]

ACRYLIC COPOLYMERS

53

Properties of Cellulose-Bound Enzymes Some examples of cellulose-bound immobilized enzymes are given in Table If. The usual amounts of enzymes that can be immobilized are about 100 m g per gram of modified cellulose.The retentions of activity given in Table II are only approximate, since many of the preparations are sufficientlyactive to be subject to internal diffusional limitation.21 Shifts in the p H activity profilesof enzymes on immobilization have been observed with ion-exchange celluloses when assayed at low ionic strength.rag,22 Similarly, changes in K m due to electrostatic interaction have been observedY ~,23 T w o enzymes, ficin~2 and bromelain,23 had enhanced resistance to oxidation on immobilization. M a n y of the enzymes have increased thermal stabilitieson immobilization. ~1D. L. Regan, M. D. Lilly, and P. Dunnill, Biotechnol. Bioeng. 16, 1081 (1974). W. E. Hornby, M. D. Lilly, and E. M. Crook, Biochem. J. 98, 420 (1966). C. W. Wharton, E. M. Crook, and K. Brocklehurst, Eur. J. Biochem. 6, 565, 572 (1968).

[5] Immobilization of E n z y m e s

to Various Acrylic C o p o l y m e r s

By ROLF MOSBACH, ANN-CHRISTIN KOcH-SCHMIDT, and KLAUS MOSBACH Among the large number of supports available for the immobilization of enzymes, the synthetic carriers, in particular the hydrophilic crosslinked polyacrylic polymers, are of great interest. They show good mechanical and chemical stability and are inert to microbial degradation. In one respect they are outstanding: that is, in the ease of preparing various polymers "tailor-made" for a particular enzyme and application, e.g., with regard to stability and specific activity. Parameters that can easily be varied to this end include (1) the degree of porosity and (2) the chemical composition, which can be achieved by either copolymerization of different monomers among the large number available or by chemical modification of preformed polymers. Acrylic polymers have been used primarily as supports for the covalent attachment of enzymes (see this chapter and Chapters [6][8]), but they have also been used for the entrapment of enzymes (see in particular Chapter [12] and also Chapters [13], [14], [50], and [62]). The intermediate situation, i.e., where enzymes are both covalently bound and entrapped in acrylic gels, is treated in Chapter [12] and, in particular, in Chapter [15]. In Table I are listed a number of acrylic polymers that have been applied in the covalent attachment of enzymes.

54

IMMOBILIZATION TECHNIQUES

[5]

TABLE I VARIOUS SYNTHETIC POLYMERS BASED ON ACRYLAMIDE OR ACRYLATE TO WHICH ENZYMES HAVE BEEN COVALENTLY BOUND

Original matrix with functional group

Coupling or activating agents

Intermediate matrix

R t

R t

1

I

N

NH

II H+ ~-coo-; [

Acrylamide/acrylic acid

NIl

Acrylamide/maleic acid

R"

I

--OH

11

--NH2 --SH

---@o. a,b

Rn

¢

--NH 2

--O~c=NH

--OH Acry lamide/hydroxyethylmethacrylate Hydroxyalkylmethacrylate

~

Refs.

+IN[HI

I

BrCN

Major reacting groups of proteins

--CONH 2

_o /

OHC(CH~)sCHO

~

-- CON---- C H ( C H 2 ) , C H O

Acrylamide Acrylamide/methylacrylate

~

--CONH,

-@OH

--CONHz

coNH '

H,N. NH,

--CONH,

Acrylamtde Acrylamide/methylacrylate

--NH 2 -- C O O H

H2N. (CI-I~),NH~

--COOH

H2N' NH2, HNO,

g,h

--N~

Acrylamide

~

a, d, e f

Acrylamide

~

--NH~

~

-- C O N s

g, h

--NH~

--SH

--@OH

g,h

J

[5]

ACRYLIC COPOLYMERS

55

TABLE I (Contin~d) Original matrix with functional group

Coupling or activating agents

Intermediate matrix

Major reacting groups of proteins

Refs.

Acrylamide/malelc acid anhydride Methacrylic actd anhydride

c k

H O, CONH~

--SH

Acrylamide/ anilinoarylamide

~ O H Histidine/ argtnine

l

F

--NH~ Methacrylic acid/ 3- or 4-fluorostyrene

m

F

F

HNOs Methacrylic acid Methacrylic acid/ m-fluoroaniltde

~--CO--NH--~

--NH2 NO2

m

This chapter. This volume, Chapter [6]. c H. Lang, N. Hennrich, H. D. Orth, W. Briimmer, and M. Klockow, Chem.-Ztg. 96, 595 (1972). d p. O. Weston and S. Avrameas, Biochem. Biophys. Res. Commun. 45, 1574 (1971). T. Ternynck and S. Avrameas, F E B 8 Le.tt. 28, 24 (1972). / A.-C. Johansson and K. Mosbach, Biochim. Biophys. Ada 370, 348 (1974). g J. K. Inman and H. M. Dintzis, Biochemistry 8, 4074 (1969). h j. K. Inman, this series Vol. 34 [3]. Requiring additional coupling agent. i y. Ohno and M. A. Stahmann, Macromolecules 4, 350 (1971). k A. Conte and K. Lehmann, Hoppe-Seyler's Z. Physiol. Chem. $§2, 533 (1971). This volume, Chapter [7]. "~This volume, Chapter [8]. I n the first p a r t of this chapter procedures are given for the p r e p a r a tion of various acrylic copolymers in either bead form or as granules. I n the second part, a n u m b e r of examples are given on the binding of en-

56

IMMOBILIZATION TECHNIQUES

[5]

zymes to such preparations; and in the third, the "tailor-made" approach is illustrated with two examples. P r e p a r a t i o n of Various Acrylic Copolymers

Acrylic copolymers are available from commercial sources and also relatively easily prepared in the laboratory. Commercially available crosslinked polymers include Bio-Gel P (polyacrylamide), Bio-Gel CM2 (acrylic lattice containing carboxyl groups), aminoethyl Bio-Gel P and hydrazide Bio-Gel P (all from Bio-Rad Laboratories Ltd., Richmond, California 94804, USA), Enzacryl AA and AH (polyacrylamide containing aromatic amines and hydrazide residues, respectively; Koch-Light Laboratories Ltd., Colnbrook S L 3 0 B Z , Buckinghamshire, England), Spherons [poly (hydroxyalkylmethacrylate)] (see this volume [6]). Below are given two general procedures for the preparation of various acrylic copolymers from the great variety of available monomers. By choosing the ratios of the participating monomers, it is possible to prepare a polymer with the desired amount of a specific functional group. The polymerization is carried out in a liquid medium as bulk, or directly as bead, polymerization. The procedure in liquid medium has the effect that porous polymers of defined structure are obtained in contrast to polymerization in substance. The structure, and thus the size and distribution of porosity within the gel, is dependent besides on polymerization kinetics and other conditions, mainly on the total monomer concentration T (w/v), and the relative concentration, or the proportion by weight, C, (w/w) of cross-linking agent (such as N,N'-methylenebisacrylamide, Bis). Increasing the total monomer concentration, T, while keeping the concentration, C, of the cross-linking agent constant, decreases the resolution of proteins (as tested in gel chromatographyl-'~). T thus determines the pore size of the gel. Conversely, C will also influence pore size. Thus it was found that polyacrylamide with C = 5% yields the minimal average pore size (using constant total concentration). Above and below approximately 5%, pore size will be larger; such transition points are a characteristic of crosslinked polymers. 4 Preparations with C values above the transition point, again keeping T constant, turn more and more turbid indicating microprecipitation and organization leading to larger pores. 4,5 However, gel 1 S. Hjert~n and R. Mosbach, Anal. Biochem. 3, 109 (1962). sS. Hjert~n, Arch. Biochem. Biophys. Suppl. 1, 147 (1962). s R. Mosbach, U ~ . Patent No. 3,298,925 (1967). 6$. S. Fawcett and C. J. O. R. Morris, 8cp. 8ci. 1, 9 (1966). aE. G. Richards and C. J. Temple, Nature (London) Phys. 8ci. 230, 92 (1971).

[5]

ACRYLIC COPOLYMERS

57

structure can also still be found in the microparticles of this coherent disperse system. These porous bodies (also called macroreticular gels) are characterized by having interparticular pores which have different physicochemical properties from the pores normally found in gels. There exists a continuous transition, depending on the method of preparation, from gels to hollow-space systems. Another method of preparing bodies of varying porosity involves polymerization in different solvent systems (see Chapter [6] on Spheron gels).

Preparation o/ Acrylic Copolymers as Granules (T:C = 10:5) through Bulk-Polymerization Followed by Physical Fragmentation Reagents Acrylic monomer(s) such as acrylamide, 2-hydroxyethyl methacrylate, methylacrylate, acrylic acid (Eastman Organic Chemicals, New York. Solid monomers are preferably recrystallized and liquid ones distilled before use) N~V'-Methylenebisacrylamide, Bis (Eastman, once recrystallized in acetone, 45 ° ) Tris-HC1 buffer, 0.1 M, pH 7.0 N,N,N',N'-Tetramethylethylenediamine, T E M E D (Eastman) Ammonium persulfate (peroxido sulfate) (Merck, Darmstadt, West Germany) Monomer(s) 2.375 g (neutralized to pH 7.0), and Bis, 0.125 g, are dissolved in 0.1 M Tris-HC1 buffer, pH 7.0, to a volume of 24 ml in a beaker. After addition of the catalyst system system consisting of 0.5 ml of T E M E D and 0.5 ml of ammonium persulfate solution (0.5 g/ml), the solution is placed under slight vacuum (02 inhibits the polymerization process) for about 1 hr. The gel block is cut into 1-mm slices and pressed through a 30-mesh sieve. The granules are then thoroughly washed with water in order to remove nonpolymerized monomers (toxic!) and the catalyst system.

Preparation o/ Spherical Acrylic Copolymers (T: C = I0:5) by Bead Polymerization 6,~ Reagents Hydrophilic phase: Acrylic monomer (s) (Eastman)

N,N'-Methylenebisacrylamide, Bis (Eastman) 6It. Nilsson, R. Mosbach, and K. Mosbach, Biochim. B~ophys.Acta 268, 253 (1972). v A.-C. Johansson and K. Mosbach, Biochim. Biophys. Acta 370, 339 (1974).

58

IMMOBILIZATION TECHNIQUES

[5]

0.1 M Tris-HC1 buffer, pH 7.0

N,N,N',N'-Tetramethylethylenediamine, TEMED (Eastman) Ammonium persulfate (Merck) Hydrophobic phase: Chloroform (analytical grade) Toluene (analytical grade) Sorbitan sesquioleate (Pierce Chemical Company, Rockford, Illinois) The bead-polymerization process is carried out in a l-liter roundbottom flask equipped with a stirrer, an inlet and outlet for N2, and a tap funnel for admission of the monomer solution (see Fig. la). The flask is cooled in ice water, and contains 400 ml of the hydrophobic phase, toluene/chloroform (290/110, v/v), in order to achieve the same density as the aqueous phase. This phase also contains 1-3 ml of the suspension stabilizing agent, Sorbitan sesquioleate. Prior to the polymerization, the hydrophobic phase is stirred under N2 at about 260 rpm; the stirring rate determines the bead size formed. To the cold monomer solution, consisting of 5.7 g of monomer(s) and 0.3 g Bis dissolved in Tris-HC1 buffer to a total volume of 58.5 ml, 0.5 ml of TEMED and 1.0 ml ammonium persulfate solution (0.6 g/ml) are added. When the resulting mixture begins to polymerize slightly (as indicated by an increasing viscosity), it is run into the flask. The polymerization of the aqueous phase, when performed at 1-5 ° , is usually complete within 30 min. The beads are washed on a glass filter, first with toluene to remove the chloroform and then with several liters of distilled water prior to freeze-drying. It is important that the aqueous and organic phases have the same density. Instead of using toluene it is possible to use other organic solvents, e.g., n-butanol. By changing the stirring rate and/or the amount of stabilizer, different bead diameters (10-500 ~m) can be prepared. Hydrophobic monomers, if employed, must be first dissolved in organic solvents (methanol) (to a maximum volume of 50% of the total volume of the hydrophilic phase) and then added to the buffer solution. The purpose of the alcohol is to retain the monomer in the aqueous phase during the polymerization process. A much simpler alternative arrangement is given in Fig. lb. This procedure, in which polymerization is carried out in a beaker with magnetic stirring, but otherwise under identical conditions, as outlined above, also yields polymer beads. Whether the bead diameter distribution is as good as when the former technique is used remains to be studied. It should be added that the above (Fig. la) bead-polymerization techniques have been used in the entrapment of enzymes~,7 and

[5]

ACRYLIC COPOLYMERS

50

-ICE

(b)

N2 ,=~

,,,,

,,,

•"

,%-%

~, N 2

BEAKER, 2S0 ml

ICE STIRRING BAR

HAGNETIC

STIRRER

I

Fro. I. Apparatus used for the preparation of bead-formed acrylic copolymers.

60

IMMOBILIZATION TECHNIQUES

[5]

the latter alternative (Fig. lb) recently also to whole cells of Streptomyces albus (A.-C. Koch-Schmidt and K. Mosbach, to be published). In some cases, it was found advantageous, in order to avoid leakage, to entrap the enzyme in the presence of a carbodiimide in the acrylic acid-acrylamide monomer solution, leading to a preparation in which the enzyme was both entrapped and covalently bound. 8 Examples of Covalent Binding of Enzymes to Bead-Formed Acrylic Copolymers Below are given methods suitable for the covalent binding of enzymes and other ligands to various acrylic copolymers, which have been prepared in bead form (as outlined here) except for one example using a commercially available polymer. The methods given involve (a) glutaraldehyde treatment of polyacrylamide first reported in 1971,9 (b) CNBr activation of acrylamide/hydroxyethylmethaerylate polymers, first reported in 19707,8,1° (see also Chapter [6] ), and (c) coupling by the carbodiimide method 7,8,11,12 (see also Chapters [6] and [34]) to acrylamide/acrylic acid polymers. The properties of these preparations are summarized in Table II.

Covalent Binding to G lutaraldeh yde- Treated P olyacrylamide Beads (T:C -- 10:5) Example: Im~nobilization o] Trypsin and Ribonuclease A to Polyacrylamide Beads. Dry polymer, 100 mg, prepared in the setup given in Fig. la, is allowed to swell in water and then activated for 14 hr at 37 ° using 4 ml of a 6% solution of glutaraldehyde dissolved in 0.2 M phosphate buffer, pH 7.4. The glutaraldehyde-treated beads are thoroughly washed with distilled water, at 4-hr intervals, for 1 day at 4 °. Filtered beads are transferred to a tube containing 2 mg of trypsin (Sigma, bovine pancreas, 10,000 BzArg-OEt units/mg) or ribonuclease A (Sigma, bovine pancreas, type IA, 60 Kunitz units/mg) dissolved in 2 ml of 0.1 M phosphate buffer, pH 7.5. The coupling is allowed to proceed for about 12 hr at 4% The beads are then washed with 0.5 M NaCl, 0.1 M NaHCOs, and distilled water, successively. In a control experiment run to investigate the binding capacity of 8 K. Mosbach, Acta Chem. 8cand. 24, 2084 (1970). g P. O. Weston and S. Avrameas, Biochem. Biophys. Res. Commun. 45, 1574 (1971). 10j. Turkov£, O. Hub£1kov~t, M. Kriv~kov£, and J. (~oupek, Biochim. Biophys. Acta 322, 1 (1973). 11K. M~.rtensson and K. Mosbach, Bio~echnol. Bioeng. 14, 715 (1972). 12K. M~.rtensson, Biotechnol. Bioeno. 16, 579 (1974).

[5]

ACRYLIC COPOLYMERS

61

TABLE II VARIOUS ENZYMES IMMOBILIZED TO DIFFERENT ACRYLIC COPOLYMER8 BY THREE IMMOBILIZATIONTECHNIQUES DESCRIBED

Specific mg % Bound activity enzyme/g enzyme of of bound dry added enzyme polymer enzyme as % of free Polyacrylamide (glutaraldehyde) Ribonuclease A Trypsin Trypsinb Hydroxyethyl methacrylateacrylamide copolymer (50/50, w/w) (CNBr) Ribonuclease A Trypsin Trypsin b Acrylic acid-acrylamide copolymer (50/50, w/w) (EDC) Ribonuclease A Trypsin Trypsinb Bio-Gel CM-100 (CMC) pullulanase

Units~/g dry polymer

20 11 18

98 63 90

10 14 69

60 67 541

20 15 17

50 85 87

36 20 57

216 131 422

40 20 26

100 57 65

9 49 65

108 429 736

19

34

43

1.70

The units given refer to international units. b Recently prepared acrylic gels identical to those given except using a ratio of T:C = 8:30 instead of T:C = 10:5 to increase the porosity of the gels. the polymer, glyeine was bound to the glutaraldehyde-treated beads in the amount of 54 ~moles per gram of dry polymer.

Covalent Binding to CNBr-Treated Copolymers o] Acrylamide ~-Hydroxyethylmethacrylate (50/50, w/w ; T: C ffi I0:5) Example: Immobilization of Trypsin and Ribonuclease A to an Acrylamide-$-hydroxyethylmethacrylate Copolymer. Swollen beads (100 mg dry weight) are activated in 4 ml of distilled water containing 80 mg of CNBr. T h e activation step proceeds for 6 min at 4 °, while the p H is maintained at 10.8 by the addition of 1 M N a O H . Activated beads are thoroughly washed on glass filter with ice cold 0.1 M NaHCO3 and then transferred to the enzyme solution containing 2 mg of trypsin or ribonuclease A dissolved in 2 ml 0.1 M of NaHCOs. The coupling reaction proceeds for about 12 hr at 4 °. The beads are then washed in 0.1 M N a H C 0 8 , 0.5 M NaC1, 1 m M HC1, and distilled water.

62

IMMOBILIZATION TECHNIQUES

[5]

It appears that the concentration of CNBr used for activation of the copolymer has a critical bearing on the amount of enzymic activity recovered. A concentration of about 80 mg of CNBr per 100 mg of dry polymer appears to be optimal for several enzymes. If the concentration of the activating agent is too high, there is a drastic decrease in the swelling capacity of the polymer. When coupling glycine to this copolymer and using 500 mg of CNBr per 100 mg of dry polymer in the activation step, 540 ~moles of glycine are bound per gram of dry polymer.

Covalent Binding to Copolymers o] Acrylamide and Acrylic Acid (50/50, w/w; T:C = 10:5) Example: Immobilization o] Trypsin and Ribonuclease A to an Acrylamide-Acrylic Acid Copolymer. Swollen beads (100 mg dry weight) are washed in 0.2 M NaHC03 after which 2 ml of a solution of 1-ethyl3 (3-dimethylaminopropyl) carbodiimide (EDC) .HCI (Sigma Chemical Company, 2 mg of EDC per milliliter of distilled water) and 2 ml of enzyme solution (2 mg of trypsin or ribonuclease A per milliliter of 0.2 M NaHCOs) are added. (Alternatively, the coupling can be performed at pH 3.5 using diluted HC1.) The coupling proceeds for 2-4 hr at 4 °. The beads are then washed in 0.1 M NaHC03, 0.5 M NaCl, distilled water, 1 mM HC1, and buffer. When coupling glycine and using equimolar amounts of EDC, the coupling yield is 86% resulting in 120 ~moles of bound glycine per gram of dry polymer. Covalent Binding o! Pullulanase to Bio-Gel CM 100 Using Carbodiimide 11 One hundred milligrams of Dry Bio-Gel CM-100 (high capacity, 100-200 mesh, Bio-Rad Lab.) is treated with 0.5 M HC1 and then thoroughly washed in distilled water. The beads are transferred to the enzyme solution, containing 5.5 mg of pullulanase (Aerobacter aerogenes) dissolved in 10 ml of distilled water, which also contains 60 mg of the substrate pullulan. After addition of 50 mg of 1-eyelohexyl-3-(2morpholinoethyl) carbodiimide metho-p-toluenesulfonate (CMC) (Aldrich Chemical Company), the mixture is gently shaken for 18 hr at 4 °. The enzyme beads are then washed in 0.1 M NaHCO~, distilled water 1 mM HC1, and 0.5 M NaC1. Per gram of dry polymer 19 mg (1.70 units) of pullulanase are bound (coupling yield of 34%), and the specific activity of bound enzyme as percent of free is 43.

[5]

ACRYLIC COPOLYMERS

63

The same carrier, Bio-Gel CM-100, was also used when coupling the sequentially working enzymes fl-amylase and pullulanase (Aerobacter aerogenes) on the same matrix TM (see Table II). The EDC-mediated coupling reaction when tested with trypsin was found to be more efficient than CMC.

Examples of "Tailor-Made" Acrylic Polymer Preparations The use of immobilized enzymes as biological model systems has in recent years attracted great interest (see also Chapters [29], [31], and [63] ). In order to obtain a relevant model that will mimic the polyelectrolytic nature of biological membranes charged groups can be introduced in the polymer preparation. Furthermore, the high content of lipids in biological membranes indicates the lipid requirement for enzyme action; for example, a hydrophobic medium has been shown to be of importance in the biosynthesis of phospholipids. This would seem to indicate the need to introduce hydrophobic groups in order to obtain relevant models. Ideally, to be able to correlate the influence of charge or hydrophobicity exerted by a membrane on enzymic activity, it would be necessary to prepare a series of "polymer membranes" in which only one parameter is changing. This possibility is also of interest from a practical point of view. For instance, when forced to carry out enzymic reactions at low substrate concentrations, it should be advantageous to prepare matrices that "attract" substrate creating a higher substrate concentration around the immobilized enzyme than that found in the bulk solution. This then will lead to a lower Km~pp~, and thereby increase the efficiency of the immobilized enzyme preparation. Example. When optimizing the hydrolysis of the positively charged substrate a-N-benzoylarginine ethyl ester catalyzed by immobilized trypsin, the negatively charged acrylamide/acrylic acid copolymer (50/50, w/w) is a more suitable carrier than the neutral polyacrylamide, since the former immobilized enzyme derivative shows a decrease in Km~p,~ for this substrate by a factor of 4 over the latter when assayed at pH 8.3 in 0.1 M Tris-HC1 buffer, 0.1 M in NaC1. (The Km~pp~values found were 0.58 mM and 2.4 mM, respectively.) Example. The influence of hydrophobicity was studied varying only one parameter, i.e., the amount of the hydrophobic component,, methylacrylate, in an acrylic copolymer.13 The enzyme chosen was liver alcohol dehydrogenase known to have a broad substrate spectrum for alcohols with different degrees of hydrophobicity. The matrices studied, polyacryl~3A.-C. Johansson and K. Mosbach, Biochim. Biophys. Acta 370, 348 (1974).

64

IMMOBILIZATION TECHNIQUES

[5]

.E E 0

5

Z w

[

I

I

2

3

//

I 6

104M"1 n-butono{ Fro. 2. Mich&elis-Menten plots of liver alcohol dehydrogenase immobilized to

poly(acrylamide) (Q 0 ) and to copoly(acrylamide/methylacrylate, 75/25) ((D O) using n-butanol as substrate at pH 8.0. The NAD ÷ concentration was 2 raM.

amide and methylacrylate-acrylamide copolymer (25/75, w/w), were prepared by bulk polymerization as described in the methods section and 4 mg of the enzyme was coupled to the glutaraldehyde-treated polymers (300 mg dry weight) as described above. When the enzyme was bound to the more hydrophobie matrix, methylacrylate-acrylamide copolymer, the Km~app) (40 ~ / ) for the more hydrophobic substrate, n-butanol, decreased by a factor of 4 compared to Km~app) (160 ~ / ) obtained when the enzyme was bound to the hydrophilic polyacrylamide, whereas the Km~app) of ethanol was about the same (0.55 raM) in both cases. Equilibration studies with 14C-labeled alcohols and the respective polymers showed an enrichment of 6 times of n-butanol to the hydrophobic matrix compared to the hydrophilic one, while such preferential absorption was not found for ethanol. Figure 2 shows Michaelis-Menten plots for the substrate n-butanol of the enzyme immobilized to both types of copolymers. At a concentration of, for example, 50 ~M, the rate of reaction was found to be about 75% higher with the more hydrophobic methylacrylate-acrylamide copolymer compared to that of polyacrylamide. Thus preparations with "substrate attracting" carriers permit enzymic reactions to be carried out more efficiently at a rather low substrate concentration. Conclusion The procedures given for the preparation of various acrylic copolymers in bead form are simple to carry out in the laboratory. Usually

[S]

ACRYLIC COPOLYMERS

65

the simplified procedure (Fig. lb), where polymerization is carried out in a beaker, should suffice. (In addition, as mentioned, some of the acrylic copolymers are commercially available.) Obviously either bead polymerizaticm process appears to be advantageous compared to the bulk polymerization process, including physical fragmentation, because of the well-defined form of beads per se as well as improved mechanical stability. Further beads are better suited for quantitative measurements. By changing stirring rate or the hydrophobic phase in which the monomers are dispersed, different bead diameters can be prepared (10500 ~m). In general it can be assumed that the various monomers are present in the formed copolymer roughly in the same ratio as when administered. However, for further characterization infrared spectroscopy is a recommended technique (see also Chapter [28]). The data for the coupling procedures on the three supports given in Table II for ribonuclease A and trypsin show fairly good binding yields (no optimization has been attempted though) and specific activities of bound versus free, thus providing a general idea of the efficiency of these procedures. In this context, we would like again to draw the attention of the reader who wishes to apply these polymers, for example, in the medical area, to the toxicity of nonpolymerized monomers. For further reading on the use of CNBr and carbodiimide as coupling aid to acrylic polymers, see in particular Chapter [6]. In addition to the techniques reported here, polyacrylamide beads have been derivatized (see Table I) to yield functional groups, such as amines suitable for subsequent coupling14,15 (see also Chapter [30]). It is difficult at the present stage of development to recommend any one particular support or coupling procedure of the three major types described. Not enough enzymes have been tested, and it is likely that even then no such general recommendation can be made. (One recommendation is to use water-soluble carbodiimides when enzyme coupling has to be carried out at low pH.) However, the general techniques given here should provide the investigator with the means of preparing his own "tailor-made" support material. ~4j. K. Inman and H. M. Dintzis, Biochemistry 8, 4074 (1969). J. K. Inman, this series, Vol. 34 [3].