[25] Assay procedures for immobilized enzymes

[2S]

ASSAY PROCEDURES FOR IMMOBILIZED ENZYMES

335

[25] A s s a y P r o c e d u r e s for I m m o b i l i z e d E n z y m e s

By Bo ~{ATTIASSON and KLAL'S MOSBACH Immobilized enzyme systems (matrix-bound or support-bound) are usually particulate in nature. For this reason conventional, soluble enzyme assay procedures can rarely be applied without modification. It is the aim of this chapter to summarize and exemplify various procedures that are suited for such preparations. It may very well be that the methods that have been developed for the assay of immobilized enzymes may also find applications in the future in the study of naturally occurring particulate cell components, such as membranes and organelles. The mere fact of immobilizing enzymes on particulate systems renders them easier to handle; such preparations can, for instance, be packed into columns. There are at least two fundamentally different methods that may be used in the assay of particulate enzymes, one alternative involving columns, the other the use of homogeneous suspensions of the enzymesupport in the incubation medium. By having a homogeneous suspension, the substrate concentration is kept uniform around all the beads, each of which is thus in a milieu equivalent to that of any other in the suspension. In the column alternative, the enzyme beads at the top of the column are in contact with pure substrate, whereas farther down the column the substrate concentration decreases and the product concentration increases. This causes a concentration gradient in the column resulting in a range of different microenvironmental conditions between the enzymes at the top and those at the bottom. Columns operating either at a high percentage conversion or in systems with product inhibition or in multistep enzyme-catalyzed processes may suffer from the problem of heterogeneity, whereas at a low percentage conversion of the added substrate, such heterogeneity may be negligible, at least for single enzyme-catalyzed processes with no product inhibition or stimulation. Once again there are two principal approaches to the problem of assaying immobilized enzymes. In cases where a discontinuous measurement is permissible, it suffices to remove aliquots at intervals then, after filtration, assay according to conventional techniques. For continuous measurements, however, complications occur, such as inhomogeneity and light scattering caused by the support necessitating the modification of conventional procedures before they can be applied. It is essential to carry out the assay of an immobilized enzyme under well-defined conditions if the kinetic data obtained are to provide useful information in the overall characterization of a bound enzyme. For exam-

336

ASSAY PROCEDURES

[25]

pie, in a stirred-batch assay, care must be taken to obtain optimal stirring rates to avoid diffusional hindrance of the substrate or product. In other chapters of this volume, dealing with enzyme membranes [63] and in particular enzyme technology [49]-[57] and analysis [41][45], the reader will find several of the assay procedures given here which provide useful additional information and therefore arc not covered here. The principal difference is that whereas in the present chapter the immobilized enzyme per se is in focus, in the others the interest lies mainly in the conversion of substrate or analysis.

Spectrophotometric Methods The most common and often the easiest way of following an enzyme reaction is to use spectrophotometry, thereby studying changes in absorbance caused by consumption of substrate or generation of product. The systems to which spectrophotometric measurements can be applied may be conveniently divided into two main groups: that in which the enzyme-matrix is in the light path and that in which it is not. E n z y m e - M a t r i x in the Light Path The activity of enzymes immobilized on matrices that are not too optically dense can be measured in a cuvette provided the particles are kept in suspension by means of stirring during the assay procedure. Stirring in the Cuvette during the Assay A homogeneous suspension of immobilized enzyme may be obtained in the cuvette if the particles are adequately stirred. It is then possible to measure the activity almost as easily as that of the free enzyme. The method was introduced by Weliky et al. for determining the activity of peroxidase immobilized on carboxymethyl cellulose l and has since been developed further by Mort et al. 2 Equipment. Besides a photometer and recorder facilities, the method requires a thermostated stirring device. Such units can be obtained as accessories to some of the more elaborate commercially available instruments. ~ Example. In the paper presented by Mort et al. ~ aldolase bound to Sepharose was studied using a Beckman Acta V double-beam spectro1N. Weliky, F. S. Brown, and E. C. Dale, Arch. Biochem. Biophys. 131, I (1969). ~J. S. Mort, D. K. K. Chong, and W. W.-C. Chan, Anal. Biochem. 52, 162 (1973). * In this context we would like to stress that whenever in the following a specific commercially available model of equipment is given, this should be considered as an example taken usually out of several possible.

[2S]

ASSAY PROCEDURES FOR IMMOBILIZED ENZYMES

337

photometer equipped with stirring facilities and a Beckman spherical stirrer. For differentiation between soluble and immobilized enzymes, fines were removed from Sepharose 4B by several decantations. A typical incubation mixture (final volume 2.0 ml), containing 50 mM triethanolamine, pH 7.4, 10 mM EDTA, 0.1 mM fructose 1,6-diphosphate, 0.15 mM NADH, triosephosphate isomerase (10 gg/ml), and a-glycerophosphate dehydrogenase (10 gg/ml), was stirred with a Teflon-coated bead. The reaction was started by addition of an aliquot of gel. The aldolase-Sepharose sample was suspended in buffer in a beaker, and, while stirring was maintained, the aliquot for assay was removed rapidly using a Biopet (Schwarz-Mann). In general the gel was diluted 10-fold with 10 mM sodium phosphate at pH 7, containing 1 mM EDTA. Aliquots of 0.1 ml were assayed. When greater amounts of gel suspension were used, assay mixtures were made more concentrated and diluted to give a constant final concentration. The enzymic activity was registered on a recorder. As shown in Fig. 1, a continuous, linear reaction rate was observed as long as stirring continued. However, when stirring ceased, the reaction rate dropped to zero, indicating that all the enzyme activity was associated with the matrix, which had sedimented, leaving no soluble enzyme in the supernatant. Resumption of stirring led to a linear decrease in absorbance at the same rate as that observed during the previous period of stirring. Discussion. That the light scattering of the enzyme beads in the light beam causes small perturbations iS apparent from the noise on the recorder diagram. These disturbances in the registered signal are more pronounced the bigger the particles used. 0.8

E

Stirring

0.7

restarted

"6 8 c Duplicote filtered after IO min

I~0

1'5

20

Time (rain)

Fro. 1. Spectrophotometric recording of the reaction catalyzed by matrix-bound aldolase (1.7 mU, 0.1 ml of 1-in-10 gel suspension), with and without stirring. Reproduced with permission from J. S. Mort, D. K. K. Chong, and W. W.-C. Chan, Anal. Biochem. 52, 162 (1973).

338

ASSAY PROCEDURES

[25]

The use of this assay technique is restricted to those matrices that are, a~ most, only slightly fragmented by the stirring action. A Sepharosebound enzyme, as is shown by Mort et al., 2 is suitable for measurement, whereas more fragile matrices, such as porous glass beads, give a "background activity" due to increased absorbance and light scattering from the ground particles. Cessation of stirring during the incubation gives a fairly strong indication of whether any free enzyme activity or soluble matrix-fragments containing bound enzyme are present. The method is simple and gives accurate measurements of the activity of immobilized enzymes, provided the support is not too fragile. Enzyme-Matrix Outside the Light Path Keeping the enzyme-matrix outside the light path circumvents the problems of light scattering. This is achieved by running the reaction outside the photometer and pumping only the clear product-containing solution through a flow-cuvette for spectrophotometric analysis. Two maim approaches may be distinguished: analysis of an eluate from a packed bed; recirculation of the supernatant from a stirred-batch procedure. Method 1. Continuous Spectrophotometric Analysis o] the Eluate ]rom a Packed Bed o] Immobilized Enzyme This method has been one of the most frequently used of the continuous methods for assaying the activity of immobilized enzymes. A kinetic evaluation of this method was presented by Lilly et al. ~ (see also this volume [49]). Equipment. The equipment needed comprises a spectrophotometer, a flow-cuvette, a column, and a pump. The enzyme-matrix is packed in the column, through which fresh substrate solution is pumped for subsequent analysis in the photometer. Example. Lilly et al2 studied ficin immobilized to CM-cellulose. The final preparation, containing 4.2% of protein, was packed into columns 1.0 cm in diameter and of various lengths, which, like the perfusion solutions, were thermostated at 25 °. The columns were equilibrated by pumping phosphate-saline, I - - 0 . 4 . The esterolytic activity was determined by following the hydrolysis of N-a-benzoylarginine ethyl ester (BAEE) hydrochloride (see the Table), measuring the absorbance of the eluate at 251 nm. The effect of variation in flow rate on the degree of hydrolysis as well as on the Km,app was studied (see Fig. 2). z M. D. Lilly, W. E. Hornby, and E. M. Crook, Biochem J. I00, 718 (1966).

[25]

ASSAY PROCEDURES FOR IMMOBILIZED ENZYMES

339

VALUES OF THE MICHAELIS CONSTANT FOR N-~-BENzOYLARGININE ETHYL

ESTER (BAEE) HYDROLYZEDBY FICIN ATTACHEDTO CM-CELLULOSEa CM-cellulose-70-ficin (I = 0.5) Flow Km,tpp 30 ml/hr 140 ml/hr Very fast

10.0 5.4 ~-~5.2

Reproduced with permission from M. D. Lilly, W. E. Hornby, and E. M. Crook, Biochem. J. 100, (1966).

Discussion. The method is straightforward in that it is easy to handle, but as Lilly et al. stated, all kinetic constants obtained are severely influenced by the flow rate. A high flow rate results in a thin Nernst diffusion layer and hence improved diffusional conditions for the entrance of substrate and the exit of product as compared to a situation with a slower flow rate. Thus when kinetic data such as Km and V~ax are discussed, care should be taken to specify the exact assay conditions used. 1.0

O.S

O,S

P 0.4

0.2

3'o

~o

~o"

,~o

,Ko

O (rnl/hr)

Fro. 2. Relationship between degree of hydrolysis, P, of N-a-benzoylarginine ethyl ester (BAEE) and flow rate, Q, for a column (4.0 c m x 1.0 cm diameter) packed with 670 mg of CM-cellulose-90-ficin containing 42% of protein (Kin 25 mM and K~ 0.18 mole of BAEE hydrolyzed per second per mole of bound ficin) with initial substrate concentrations 0.5 mM (O), 5 mM (A), and 15 mM (El) in phosphateNaCl, I ffi 0.4. Reproduced with permission from M. D. Lilly, W. E. Horaby, and E. M. Crook, Biochem. J. 100, 718 (1966).

340

ASSAY PROCEDURES

.er,sto,.,ot%

[25]

Differential "Grodientless" Reactor

% y/1/1/i/i///////////] ~h

,//,///,~:,~!s#//,~ Ill g////////,;'/A Spectrophotometer 4 L_:J ti2/ffS,,:~}~A

i Immobilized ~'fEnzyme

Reservoir

"/ll~lllH//2r]rn'fl'f/,g'/7/lll/ll/i

Flow-Through Cuvette

Fz6. 3. Reeirculation packed-bed reactor. Reproduced with permission from J. R. Ford, A. H. Lambert, W. Cohen, and R. P. Chambers, Biotech;zol. Bioeng. Syrup. 3, 267 (1972).

Method 2. Continuous Enzymic Assay Using a "Gradientless" Recirculation Packed-Bed Reactor Circulating a substrate solution through a column of immobilized enzyme, under conditions of very low conversion of added substratc on each cycle, gives rise to a continuous enzymic process. With shortening of the column bed and increase of the flow rate, the system gets more and more identical to the stirred-batch procedure. This reactor type was to the best of our knowledge first described by Ford et al. 4 and has later been used, among others, by Gould and Goheer2 Equipment. A small column, spectrophotometcr, peristaltic pump, magnetic stirrer, tubing, and glassware. Example. The equipment was arranged according to Fig. 3 for assay of glucose-6-phosphate dehydrogenase immobilized on Sepharose2 The incubation solution contains glucose 6-phosphate (2 mM), NADP + (0.08 raM), MgCI._, (10 raM), and glycylglycine buffer, pH 7.4 (42 mM), final pH 7.2, total volume 20 ml, flow rate 1-40 ml/hr. The amount of immobilized enzyme selected ensures that the conversion rate never exceeds 2% per pass. The effect of recirculation flow rate on activity of immobilized glucose-6-phosphate dehydrogenase is shown in Fig. 4. Discussion. The method is easy to handle. The enzyme preparation may be used repeatedly. Solutions can be changed easily in the recirculation reactor system. ~J. R. Ford, A. H. Lambert, W. Cohen, and R. P. Chambers, Biotechnol. Bioeng. Symp. 3, 267 (1972). B. J. Gould and M. A. Goheer, Biochem. Sac. Trans. I, 1284 (1973).

[25]

ASSAY PROCEDURES FOR IMMOBILIZED ENZYMES ioo

f-"

:

_=

341

e

.= a

~ 5o

,~

2'o

~

Zo

Flow rate (ml/min)

Fro. 4. Effect of increase in recirculation flow rate on activity of immobilized glucose-f-phosphate dehydrogenase (O ~). Reproduced with permission from B. J. Gould and M. A. Goheer, Biochem. Soe. Trans. 1, 1284 (1973). M e t h o d 3. The Fluidized-Bed Reactor

The fluidized bed, a modification of the packed-bed reactor, was recently introduced into the field of immobilized enzymes; it improves the homogeneity throughout the column. Instead of pumping the substrate solution down through the packed bed, the substrate flow is applied from below. This results in a "bed" in which the enzyme particles are suspended in the medium by the pressure of the flow, but are retained in the column by the force of gravity. In many cases improved efficiency is obtained with this reactor type because of both better mixing and the avoidance of channels in the reactor bed. Additionally, the risk of precipitation of protein or lipids in a packed bed, which can lead to clogging, is reduced. 6,7 In order to run such fluidized beds with good flow rate, high-density particles have to be used; recent developments on the binding of enzymes to magnetic supports (stainless steel and iron oxide) have in part been stimulated by this need s,9 (see also this volume [24] ). To date no reports directed toward the kinetic analysis of such immobilized enzymes have appeared. The use of low enzyme activities in reactors permitting good mixing, however, creates conditions quite similar to those in the stirred batch reactor (to be discussed below), thus permitting kinetic studies. The eluate from such a fluidized bed m a y then be analyzed using a flow cuvette placed in a spectrophotometer. e S. A. Barker and R. Epton, Process Biochem. Aug., p. 14 (1970). J. M. Novais, Ph.D. thesis, University of Birmingham, Birmingham, U.K., 1971. 8G. Gellf and J. Boudrant, Biochim. Biophys. Acta 334, 467 (1974). 9F. X. Hasselberger, B. Allen, E. K. Rarachuri, M. Charles, and R. W. Coughlin, Biochem. Biophys. Res. Commun. 57, 1054 (1974).

342

ASSAY PROCEDURES

[25]

Method .4. Continuous Spectrophotometric Analysis of the Supernatant from a Stirred Batch Reactor Principle. The reaction is carried out in a stirred vessel containing the enzyme beads suspended in the incubation solution. With a peristaltic pump, this solution is continuously pumped out of the reactor, passed through a flow euvette in a speetrophotometer, and recycled through the reaction vessel. The method was first developed by Mosbach and Mattiasson TM to determine the enzyme activities in an immobilized two-step enzyme system (hexokinase-glucose-6-phosphate dehydrogenase) ; several similar reports have since been published 1~,12 (see also this volume [31] ). Equipment. Magnetic stirrer, magnetic bar (ideally the length of the Teflon-coated magnetic bar should correspond to the inner diameter of the reaction vessel to ensure homogeneity), peristaltic pump, flow cuvette, photometer, water bath, Teflon and silicon tubing with an inlet covered with nylon net (200 mesh), E-flask 25 ml, or beaker. Example. Reagents for the assay of immobilized hexokinase: 11.6 ml of 0.05 M Tris-HC1 buffer with 7 mM MgC12, pH 7,6, containing enzyme polymer (bound to Sepharose, polyacrylamide, or acrylic acid-aerylamide copolymer) (equivalent to 20 mg of dry polymer containing about 10-e enzyme units), 26.64 ~moles of glucose, 5.23 tLmoles of NAD1~, and 3.3 U of soluble glueose-6-phosphate dehydrogenase. The enzymic test was started by addition of 7.26 t~moles of ATP dissolved in 400 td of the TrisHC1-MgC12 buffer. The experiment was arranged according to Fig. 5. Prior to starting the assay by the addition of ATP, all reagents were well mixed and circulating through the pumping system. Discussion. The "dead" volume of the reeireulating system, i.e., tubing and flow cuvette has to be minimized to be only a small fraction of the total. In the experiments cited, the total volume was 12 ml whereas the recireulating volume was 2 ml, but this has since been halved. In cases of large excess of substrate and relatively low enzymic activity, no effects were observed that were due to isolation of a fraction of the incubation solution from the site of catalysis---the enzyme matrix--during the time of recireulation. By maintaining a high flow rate, any such effects should be even further reduced. As in the case of the column situation, the enzyme-catalyzed reaction is highly diffusion sensitive unless very little enzyme activity is present, 1, K. Mosbach and B. Mattiasson, Aeta Chem. ~eand. 24, 2093 (1970). ~1D. L. Marshall, J. L. Walter, and R. D. Falb, Bioteehnol. Bioen#. Syrup. 3, 195 (1972). n I. C. Cho and H. E. Swaisgood, Biochim. Biophys. Acta 258, 675 (1972).

[25]

343

ASSAY PROCEDURES FOR IMMOBILIZED ENZYMES

Pump Thermostated

waterbath

--~

,~ •

~

•

d-

~~

Reaction vessel Filter Stirring

ber

/A FIG. 5. The stirred-batch reactor with auxiliary pump and flow system. z_ 0.50 ~) LU

w ~

0.4(1

8 _J tD i,i J 0

o.~

I

o

50

I I00

I 150

/~ 200

REVOLUTIONS PER MINUTE (RPM)

F~G. 6. The effect of rotation rate (rpm) of a 1.3-cm stirring bar on activity of fl-galactosidase at 37 °. Little or no change was observed at stirring rates between 50 and 125 rpm. Reproduced with permission from G. O. Hustad, T. Richardson, and N. F. Olson, J. Dairy Sci. 56, 1111 (1973). and thus it is very dependent upon the flow rates used. On increasing the stirring speed, the thickness of the unstirred layer is gradually decreased. This leads to a higher mass transfer in the diffusion-restricted system and hence to more rapid catalysis. The effect due to increased stirring gradually plateaus, resulting in a constant rate of catalysis above a certain stirring speed (see Fig. 6).1a "G. O. Hustad, T. Richardson, and N. F. Olson, J. Dairy Scl. 56, 1111 (1973).

ASSAY PROCEDURES

344

[25]

J

STOP PUMP

!

,,,

U Z

t

¢v 0 m

<

t

START PUMP

SUBSIRAT

I

I

I

I

I

I

I

I

2

h

6

8

10

12

11,

16

i,

18 min

Fro. 7. A generalized recorder diagram obtained from a stirred-batch procedure. Arrows indicate the addition of initiating substrate, stopping of the pump, and restarting the pump. This approach presents an alternative method with great accuracy, high reproducibility, and good possibilities of obtaining homogeneity in the system. A further advantage lies in the possibility of using the same gel-enzyme preparation for many assays with washings in between the measurements without removing the matrix from the reaction vessel. Control assays for the presence of soluble enzyme are readily carried out by stopping the pump and monitoring any free enzyme in the flow cell 14,15 (Fig. 7). Drawbacks include the grinding of fragile supports, e.g., glass, and also in some cases a short time delay (see Fig. 7) before the results of addition of substrate or other reactants to the incubation solution can be observed. Recently Horton et al. ~ developed a modified assay system for analysis of enzymes suited for fragile matrices, such as glass beads. Instead of being stirred, the incubation solution was kept as a homogeneous suspension by vibrating on a Vortex mixer according to Widmer et al. ~4 (see also this volume [34] ). Intermittent Assay of Immobilized E n z y m e s in a Stirred Batch (Fig. 8) Example. The reaction is carried out in a thermostated vessel. The incubation solution is kept homogeneous by means of a Vortex mixer. 14

14F. Widmer, J. E. Dixon, and N. O. Kaplan, Anal. Biochem. 55, 282 (1973). ~5p. A. Stere, B. Mattiasson, and K. Mosbach, Proc. Natl. Acad. Sci. U.S.A. 70, 2534 (1973). ~eR. Horton, H. E. Swaisgood, and K. Mosbach, Biophys. Biochem. Res. Commun. 61, 1118 (1974).

[25]

ASSAY PROCEDURES FOR IMMOBILIZED ENZYMES

r I I i

I I I

345

®

SPECTRO PHOTOMETER ®

®®

I-I PEN-RECORDER I I I

L

[ VORTEX-MIX1ER

FIa. 8. Arrangement of the equipment for intermittent assay of immobilized enzymes. A, Reaction vessel; B, water-jacketed holder; C, reaction medium; D, airdriven syringe; E, flow cell; F, recording chart; G, needle with nylon strainer; H, rubber septum cap; I, plastic tubing. Reproduced with permission from F. Widmer, J. E. Dixon, and N. O. Kaplan, Anal. Biochem. 55, 282 (1973).

To the reaction vessel inlet is connected an empty syringe; the outlet leads through a nylon mesh to a flow cuvette placed in a spectrophotometer. Intermittently the syringe is actuated, thereby displacing a small volume of the reaction medium into the flow cell. After the absorbance has been read, the syringe piston is released, withdrawing the aliquot into the reaction vessel. The reaction is performed in 0.1 M potassium phosphate pH 7.5 containing 0.1 mM NADH and varying concentrations of pyruvate. Discussion. The syringe-driven assay method is simple to handle, permitting the repeated use of a gel preparation (after washings) without removing it from the reaction vessel. This minimizes the experimental errors, since the weighing out of the enzyme gel is usually a difficult step to reproduce. The mixing technique almost eliminates grinding and fracturing of the support. There are, however, some drawbacks. The intermittent mode of operation necessitates either manual operation or the development of some auxiliary automatic unit to govern the pushing and withdrawal of the reaction medium to and from the flow cuvette. The problem of having part of the incubation solution out of contact with the enzyme has been discussed in relation to the continuous stirred-batch procedure (see above). This source of error seems also to be negligible in this case. Adaptation of these spectrophotometric assay methods to a fluorimetric procedure using, for example, the flow cell described in this volume [36], seems an obvious extension to make.

346

ASSAY PROCEDURES

[25]

Titrimetric Methods Many enzyme-catalyzed reactions produce or consume protons. Such reactions dominated research in the field of immobilized enzymes until the recent development of spectrophotometric methods. This may have been due to various factors, for example, the availability of esterolytic enzymes, but also the simplicity of assaying these enzymes when immobilized. In one of the first studies, Levin et al. 17 studied trypsin immobilized on copolymers based on maleic anhydride and ethylene. Equipment. An automatic titrator (TTT1C, Radiometer, Copenhagen), a recorder, and a thermostated reaction vessel. Example. The enzymic activity of the immobilized trypsin was determined at pH 9.5 whereas the free enzyme was assayed at pH 7.5. This because the immobilized trypsin showed maximum esterase activity in the pH range 9.5-10. (See further the section General Considerations at the end of this article and, in particular, chapter [29].) The reaction mixture (5 ml) was 0.01 M in phosphate and 5.8 mM in substrate, N-a-benzoyl-L-arginine ethyl ester (BAEE) hydrochloride; 0.1 M N a O H was used as titrant. Trypsin at a concentration of 2.0-20 ~g per milliliter of reaction mixture gave a specific activity of 35 ~moles per minute per milligrams of enzyme per milliliter. The rate registered was obtained from the rate of addition of titrant to the reaction mixture. Disucssion. The assay is easy to handle, accurate, and reproducible. Since, however, it is sensitive to exchange of carbon dioxide with the surroundings, the reaction should preferentially be run under nitrogen. In the presence of strong buffering substances, for example, charged matrices or buffers, titration is impossible. The sensitivity is lower than, for example, that obtained from using spectrophotometric methods when a suitable chromophore is present. Titrimetry is a realistic alternative in cases with excess of substrate and good enzyme activities, provided the reaction consumes or produces protons. Electrode-Monitored Reactions

Photometric method of assaying immobilized enzymes have to be modified versions of the standard procedures. All methods, however, that are based on the use of specific electrodes to follow depletion of substrate or generation of products, can be used almost unmodified. ~'Y. Levin, M. Pecht, L. Goldstein, and E. I~atchalski, Biochemistry 3, 1905 (1964).

[25]

ASSAY PROCEDURES FOR IMMOBILIZED ENZYMES

347

Oxygen Electrodes Any reaction that consumes or produces oxygen may be followed using oxygen electrodes. From in vitro experiments on the metabolism of mitochondria, it is known that oxygen-sensitive polarographic electrodes may be used with great success. TM In the particulate suspensions, any change in oxygen concentration is registered. This method turned out to be convenient in the study of immobilized enzymes. Weibel and Bright 19 studied the kinetics of glucose oxidase immobilized on porous glass. Equipment. Clark electrode, electrode assembly and power supply, an electrode chamber designed to allow rapid stirring (necessary to uniformly suspend the glass beads) (Yellow Springs Instrument Co., Model 5301 bath assembly), and a recorder. Example. Typically 0.4-0.6 ml of the immobilized enzyme preparation was used in each assay. Buffer, 0.1 M potassium acetate, pH 5.5, containing 0.5 mM EDTA and 0.1 mM KCN, was added up to 5 ml; the reaction was started by the addition of an aqueous solution of glucose (25-200 ~moles). Oxygen was recorded as a function of time. Discussion. In this case, in which a glass-bound enzyme was used, grinding of the support took place, necessitating the replacement of the enzyme preparation for each assay. However, fracturing of the beads was not accompanied by destruction of active enzyme. The method may be used also for optically dense suspensions.

Ion-Selective Electrodes The combination of ion-specific electrodes and enzymes has been used in the development of enzyme electrodes, i.e., instruments used in routine analysis of substrate levels (see this volume [41] ). The ion-selective electrodes have also been applied to the determination of the activity of soluble enzymes. 2° The methods have been found to be less sensitive than the photofluorimetric methods where these are available, but it presents an alternative method for especially opaque solutions. Example. An NH4+-sensitive monovalent cation electrode has been used for assaying the activity of urease immobilized on glassy 1 Equipment required includes a pH meter, NH~+-sensitive monovalent cation R. W. Estabrook, this series Vol. 10, p. 41 (1967). ~' M. K. Weibel and H. J. Bright, Biochem. Y. 124, 801 (1971). G. G. Guilbault, R. K. Smith, and J. G. Montalvo, Jr., Anal. Ohem. 41, 600 (1969). H. H. Weetall and L. S. Hersh, Biochim. Biophys. Acta 185, 464 (1969).

348

ASSAY PROCEDURES

[25]

electrode (Corning Glass Works, Cat. :No. 476220), pump, column, and tubing. The preparation of immobilized urease was packed in a column 1 X 10 cm. Urea, dissolved in 0.5 M Tris-HCl, at various concentrations and pH, was passed through at a constant flow rate of 2.5 ml/min. The eluate was assayed for :NH4+ ions. Discussion. The electrode (calibrated against increasing concentrations of NH4C1 and NH4HC03) gave a nearly Nernstian response over the range 10-4 to 10-1 M. Maximum velocity of the enzyme-catalyzed reaction was registered for 0.17 M urea, whereas at concentrations higher than 0.34 M substrate inhibition was observed.

Warburg Apparatus The laborious Warburg apparatus may also be used to measure reactions consuming or producing gases. It was originally demonstrated using immobilized orsellinic acid decarboxylaseY2 Examples. Orsellinic acid, 36 ~moles dissolved in 3 ml of 0.02 M phosphate buffer at pH 6.2, was placed in the main chamber of the Warburg vessel. The side arm contained 0.4 g of the polyacrylamide-entrapped enzyme together with 0.1 ml of the same buffer. After equilibration and mixing, readings were made for a total of 10 min and enzymic activity was calculated as micromoles of C02 evolved per minute. Discussion. The apparatus is laborious to use, but accurate. In most cases, however, pC02 or p02 electrodes will fulfill the same purpose.

Differential Conductivity The change in conductivity of a reaction mixture during enzyme catalysis provides a good method for monitoring the process. In two recent reports, 2s,24 Messing demonstrated the method on the system glucose oxidase-catalase. A conductivity flow cell, Model 219-020, having a cell constant K - - 80, was obtained from Wescan Instruments Inc. Equipment. Differential conductivity meter (Wescan Instruments Inc., Model 211), recorder, magnetic stirrer, pump, column, flasks, and tubing. The equipment was arranged as shown in Fig. 9. = K. Mosbach and R. Mosbaeh, Aeta Chem. Scand. 20, 2807 (1966). R. A. Messing, Biotechnol. Bioeng. 16, 525 (1974). 2~R. A. Messing, Bioteehnol. Bioeng. 16, 897 (1974).

[25]

ASSAY PROCEDURES FOR IMMOBILIZED ENZYMES

GLUCO

• EVE. - ~

T--~\

IMMOBILIZED hi II

l

349

\~

MAGNETIC STIRRER

__

0

DIFFERENTIAL

x

c~%~:.,,,,.

BALANCE

°0

Fro. 9. Differential conductivity equipment with immobilized enzyme column. Reproduced with permission from R. A. Messing, Biotechnol. Bioeng. 16, 897 (1974).

Example. Glucose solution, 6%, containing 0.0045% hydrogen peroxide, pH 5.7-6.4, unbuffered. Volume of reference and reaction mixture was 25 and 100 ml, respectively. The flow rate was 145 or 390 ml/hr. The column assay was performed by recirculating the reaction mixture as well as the blank solution and thereby passing conductivity flow cells. The difference in conductivity between the two sensors was registered on the recorder. The results were then corrected for cell constants and dilution effects. Discussion. The differential conductivity assay method offers an alternative that in some cases may add to the total knowledge of the system studied details that are not measurable by any of the conventional techniques. In the cited case, glucose oxidase was studied in the presence of eatalase. Hydrogen peroxide added in the medium produced oxygen, which is necessary for the glucose oxidase-catalyzed reaction. Most other methods for glucose oxidase assay are based either on 0~ consumption or on the conversion of hydrogen peroxide using peroxidase. An advantage of using the conductivity method is that the reaction per se may be followed without any additions of auxiliary enzymes, etc. This fact may be even more stressed in connection with practical applications.

350

ASSAY P R O C E D U R E S

[25]

Polarimetry In a recent study ~5 of a-galactosidase, polarimetry was applied to continuous monitoring of the reaction. Example. To follow the development of products by assaying the specific rotation of the eluate from a packed-bed reactor requires a polarimeter with high sensitivity, e.g., a Perkin Elmer 141, flow cell, recorder, column, pump, and tubings. A solution of 1.85% raffinose in 0.1 M phosphate buffer at pH 7.0 was pumped through the column filled with ,-galactosidase immobilized on nylon microfibrils. The enzyme catalyzes the conversion of raffinose to galactose and sucrose. The initial rotation (ao) of the raffinose solution was 2.279% At 100% conversion to galactose (37%) and sucrose (63%), the rotation was 1.351 ° . The approximate percent conversions was calculated from the ratio (2.279 - a)/(2.279 - 1.351) X 100 = % hydrolysis

Discussion. The polarimetric assay is not very accurate because of mutarotation. The experimental errors inherent in this fact may be markedly reduced either by using long retention times to complete mutarotation or, preferentially, retention times short enough to allow the assumption of zero mutarotation. Simultaneous Registration o/More Than One Enzymic Activity The above-discussed assay procedures for immobilized enzymes may be used in combination with each other for the simultaneous registration of more than one enzymic activity. This is achieved by recirculating the incubation solution through two or more different monitoring systems. For example, the esterolytic activity of trypsin has been assayed titrimetrically in a stirred-batch procedure with the simultaneous spectrophotometric assay of glucose oxidase activity 8~. In an extended system, two different enzymic activities (glucose oxidase and hexokinase) were registered simultaneously spectrophotometrically with the titrimetric assay of trypsin using a double-beam spectrophotometer equipped with a repetitive scanning device. General Considerations

The kinetic behavior of immobilized enzymes is, in many respects, different from that of free enzymes in solution. uJ. H. Reynolds, Biolechnol. Bioeng. 16, 135 (1974).

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Di]]usional Restrictions Diffusional problems are pronounced in all particulate enzyme preparations-naturally membrane bound as well as artificially immobilized. This matter is discussed in more detail in this volume by Goldstein [29], but the importance of creating homogeneous conditions for the assay of immobilized enzymes cannot be overstressed. There are several categories of diffusional problems of which the diffusion from the external solution into the particle has already been mentioned. The thickness of the diffusional layer (Nernst layer) governs the mass transfer of substrates and products and thereby also the observed catalytic rate. The thickness of the diffusion layer is influenced by changes in the stirring and flow rate. An increase in the latter reduces the thickness, and thus the effect of this diffusional hindrance. Internal diffusional restrictions may be observed on immobilized enzyme preparations with high catalytic activities per volume and also when the gel beads are large enough to generate pronounced diffusional gradients. In kinetic analysis of immobilized enzymes, therefore, preparations containing rather low enzyme activities per matrix volume should be used. The matrix beads in the preparation should be homogeneous--or at least within a narrow size distribution--and not too big. Diffusional hindrance---external as well as internal--will influence all kinetic parameters determined for the immobilized enzyme. Km,~,p and K~,app values will increase as compared to the corresponding values for the soluble enzymes. 26 On decreasing the diffusional hindrance by increasing the flow around the particles (increased stirring or flow rate) the discrepancy between the apparent values and those obtained in free solution will decrease. On studying extremely heavily loaded enzyme-matrices it must be realized that the characteristics measured relate only to the operating fraction of all enzyme molecules. The reaction rate measured is in such cases strictly dependent upon diffusion of substrate. A system with such an excess of latent catalytic capacity will behave as though it were almost insensitive to changes in the external medium. On changing pH so that the activity of these "effective" enzyme molecules decreases, substrate will diffuse into the matrix, thus coming into the proximity of the "latent" enzyme molecules. This "buffering" effect of the overloaded gels may be observed as an enzyme activity almost independent of pH. What is being studied is pH dependence of diffusion. 2,$. Carlsson, D. Gabel, and R. Ax~n, Hoppe Seyler'8 Z. Physiol. Chem. 353, 1850 (1973).

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A similar effect is observed on using enzyme preparations overloaded with respect to enzyme stability, as has previously been pointed out?-~

Leakage [rom the Matrix The immobilization procedure is usually followed by a careful washing routine to eliminate all enzyme not tightly bound to the matrix. This is important, since otherwise some enzyme may leach out into the assay solution giving rise to a mixed system containing both bound and free enzyme and thus to misleading values on characterizing the preparation. To ensure that the enzyme is firmly bound to the matrix even during and after the assay, controls have to be run. In the case of recirculation systems using a flow cell, the flow can be stopped and any free enzyme activity present in the cell at that instant can be measured '4,15 (Fig. 7). In all other situations the incubation must be filtered through double filter paper and then tested for free enzyme activity. If the assay for free enzyme is positive, various possibilities must be checked: (a) insufficient washing of the enzyme gel after coupling; (b) fragmentation of the gel during the assay, thereby liberating either free enzyme or enzyme bound to small soluble fragments of the matrix; (c) instability of the coupling bonds under the assay conditions chosen.

Mechanical Stability and Grinding Effects The mechanical stability of the particles has been mentioned several times in connection with various methods described. In cases of high catalytic activity per gel volume, the particle size is extremely important to the expressed catalytic activity. Regan et al. 2s have shown that in reactor experiments using B-galactosidase attached to aminoethyl cellulose, the smaller particles had a higher specific activity. Also, on stirring for a prolonged time, attrition of the support material will decrease the particle size and simultaneously increase the specific enzymic activity. This means that if severe grinding takes place during the assay, the results obtained may be misleading. Therefore it is important to choose a method of suspending the enzyme beads in the medium to suit the fragility of the support.

Buffer Capacity and Ionic Strength o] the Assay Medium Enzymes immobilized on charged matrices show pronounced pH shifts at low ionic strength. 17 This effect is caused by the creation of a ~D. F. Ollis and R. Carter, Jr., in "Enzyme Engineering" (E. K. Pye and L. B. Wingard Jr., eds.), Vol. 2, pp. 271-278. Plenum, New York. D. L. Regan, P. Dunnill, and M. D. Lilly, Biotechnol. Bioena. 16, 333 (1974).

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microenvironment around the immobilized enzyme that differs from the conditions in the bulk solution. The effect may be considerably diminished by raising the ionic strength of the surrounding medium? 7,29 Proton production or consumption catalyzed by the immobilized enzyme itself may create local proton concentrations different from that of the bulk solution. The effect of these changes is controlled by diffusion of product between the site of catalysis and the surrounding medium and also depends on the pK value of the buffer used2 °,31 Such microenvironmental pH effects may result in altered pH activity profiles2 -~-34 The effect of locally generated proton concentrations may be reduced and even eliminated either by increasing the buffer capacity of the medium s3,~4 or by reducing the size of the enzyme particles2 °,~ Independently of which method is used, some or all of the following points should be observed: (a) stirring and flow rate, (b) activity loading on the gel, (c) particle size and particle size distribution, (d) control measurements of free enzymes in the assay mixture, (e) appropriate suspension method, (f) buffer capacity, (g) ionic strength. In summarizing, the following points should be stressed: 1. The choice of assay procedure will for obvious reasons be governed by the nature of the product formed, e.g., carbon dioxide gas, protons, etc. 2. Likewise the properties, such as fragility, of the matrix, should be taken into consideration. 3. On strict kinetic characterization of immobilized enzymes, all the above considerations should be taken into account. However, for practical applications the assay is often carried out under conditions identical to those of the process studied. Consequently the demand for saturating substrate concentration, avoidance of overloading the matrix with enzyme, etc., do not always have to be met. However, the assay conditions used should always be given completely, to permit proper comparison and evaluation. All the methods presented here represent possible ways of assaying the activity of immobilized enzymes. Which method is likely to be appropriate in a particular case is difficult to predict, since not only the enzymic reaction, but also the properties of the support, have to be considered. It may be that also other procedures not discussed here, such as the versatile thermoanalysis, may be useful in the assay of the immobilized enzyme p e r s e (see also this volume [44] and [45]). ~ W. E. Hornby, M. D. Lilly, and E. M. Crook, Biochem. J. 98, 420 (1966). I. H. Silman and A. Karlin, Proc. Natl. Acad. Sci. U~.A. ,~8, 1664 (1967). 3~R. Ax~n, P. A. Myrin, and J. C. Jansson, Biopolymers 9, 401 (1970). s~R. Ax~n and S. Ernback, Eur. J. Biochem. 18, 351 (1971). " R . Goldman, 0. Kedem, I. H. Silman, S. R. Caplan, and E. Katchalski, Biochemistry 7, 486 (1968). S. Gestrelius, B. Mattiasson, and K. Mosbach, Eur. J. Biochem. 36, 89 (1973).