ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 7, No. 6Copyright © 1977, Institute for Clinical Science

Analytical Applications of Immobilized Enzymes

ALFRED H. FREE, Ph .D.

Ames Company, Div. Miles Laboratories, Inc.Elkart, IN 46514

ABSTRACT

Several clinical laboratory methods using enzymes as reagents now may utilize immobilized enzymes. These are enzymes attached to solid surfaces by adsorption, covalent binding, cross linking or similar means. Im­mobilized enzymes are widely used presently in convenient tests for urine glucose and galactose or blood glucose and urea, and in serum glucose or urea determinations by automated methods. Advantages include enhanced stability, enzyme conservation, reuse and economy.

Limitations in clinical analysis include the requirement for proper handl­ing of both the immobilized enzyme and the specimen with which it is used. Immobilized enzymes of the future should aid in new discoveries regarding sequential enzyme-catalyzed reactions in living cells and expand the utility of enzymes as reagents in analytical laboratory science.

Introduction

Enzymes have a critical role in the metabolic processes within all cells. The term enzyme was first suggested by Willy Kuhne about one hundred years ago (1876) as an alternative for the noun “ferment,” and derives from the Greek “in yeast.” In only a relatively few in­stances in biological systems does an en­zyme ever function other than in the specific cell where it was created. It is not surprising that the first understanding of enzymes in human physiology related to situations where the enzymes do leave the cells where they are formed. Over the centuries, studies of the enzymes of saliva, gastric juice and pancreatic juice have elucidated the role that these ex­

tracellular enzymes play in digestion. Their study has also been of utility in clinical laboratory science. The author has had an interest and involvement in this subject for many years.4,5 However, the natural habitat of the multitude of en­zymes that are known to function in biological systems is within cells where they are immobilized not only within the cell but are closely attached to specific subcellular structures.

Enzymes have been used for analytical purposes for approximately one century but it is only within the past two decades that significant attention has been given to the employment of immobilized en­zymes in analytical laboratory prac­tice.7,11,12

479

480 FREE

Nature of Enzyme Immobilization

Enzymes as proteins or as functional groups within the proteins are readily linked or attached to solid surfaces by a variety of means. The physical adsorption of an enzyme on an insoluble matrix is one of the most common forms of im­mobilization. Cellulose mats have been employed by us quite extensively but many types of insoluble surfaces or mat­rices have been used, including controlled-pore glass beads, porous titania bodies and charcoal. Additionally, enzymes may be attached to synthetic ion exchange columns.

A second type of immobilization of en­zymes is to establish the protein mole­cule inside the lattice of a gel which has pores too small to allow the entrapped protein to escape. A number of enzymes immobilized by this method have been prepared and studied. Acrylamide gels have been extensively used for this pur­pose.

A third means of immobilization of en­zymes is to create a covalent binding of the protein to a water insoluble carrier. This can be established by binding func­tional groups of the enzyme which are not essential for its activity. Car- boxymethyl cellulose azide, sepharose cyanogen bromide activated, and poly­styrene tubing converted to poly-p- aminostyrene and diazotized are some examples.

A fourth approach is to create cross- linking of the protein using a suitable bifunctional reagent. The bifunctional reagents used quite commonly are those which have two identical functional

T A B L E I

Methods for Immobilization of Enzymes

Physical adsorptionOcclusion in cross-linked polymeric matrices Covalent binding to water insoluble carrier Intermolecular cross-linking using bifunctional

reagents Microencapsulation

groups such as glutaraldehyde and bisdiazobenzidine-2-2-disulfonic acid.

A fifth method involves microencapsu­lation within semipermeable membranes which are either permanent or non­permanent. All of these methods have been discussed in detail by Zaborsky.12 These methods are listed in table I.

Examples of Analytical Applications

During the past two decades, solid state chemistry has been used with im­mobilized enzymes to establish several convenience test systems. The first of these was a test for glucose in urine which responds with a color change within 10 seconds when a dip-and-read test is moistened with a urine containing glucose.2 This test, which is called Clini- stix* contains two immobilized enzymes (glucose oxidase and peroxidase) which are adsorbed onto a cellulose mat. In fig­ure 1 are shown the chemical reactions involved. A somewhat comparable sys­tem which utilizes immobilized galactose oxidase provides a specific test for galac­tose in urine. In figure 2 are shown the equations for these reactions, and in fig­ure 3 is shown a photograph of the urine galactose test called Galactostix.* Im­mobilized enzymes have also been used in creating rapid test systems using whole blood for the evaluation of blood sugar and blood urea.3

After the enzymes and chromogens have been absorbed on the cellulose fi­bers with each of these systems, the fi­bers are coated with ethyl cellulose. Glu­cose or urea readily and rapidly diffuse through the ethyl cellulose membrane, but the presence of the coating prevents the cells from adsorbing on the cellulose and allows them to be washed away. In figure 4 are shown the reaction equations of Azostix,* the rapid blood urea test. All

*Registered trademark of Miles Laboratories, Inc., Elkhart, IN.

ANALYTICAL APPLICATIONS O F IM M OBILIZED ENZYMES 481

F i g u r e 1. Chemical equations of reaction of “ Dip-and-read” test for urine glucose employing immobilized enzymes.

H —C = 0I( C H O H ) 4

c h 2o h

d -G LU C O SE

H2O + O2

/0HIMMOBILIZED ^ 0

------------------- ► IC H O H ) ,GLUCOSE OXIDASE I

C H ,O H

GLUCONIC

ACID

H ,02U2

HYDROGEN

PEROXIDE

HoO2U2

HYDROGEN

PEROXIDE

(c 6h 3 • c h 3 • n h 2)2

ORTHOTOLIDINE

IMMOBILIZED

PEROXIDASE

- ( C 6 h 3 c h 3 - NH)2

OXIDATION PRODUCT

OF ORTHOTOLIDINE (Blue)

of these systems are employed as discrete testing methods and are completely dis­posable.

Research Products, Miles Laboratories, Inc., has developed analytical systems for automated instruments utilizing im­mobilized enzymes. These products, which are based on the work of Hornby

and his associates,1,6 are called Catalinks. Catalinks Glucose Oxidase is a nylon tube with a 1 mm internal diameter. Glu­cose oxidase is covalently bound to the inner surface of the tube. The Catalinks tube is established in the flow pattern of a continuous analyzer and as the fluid passes through the tube, the immobilized

enzyme catalyzes the oxidation of glucose to d-gluconic acid and hydrogen peroxide. The equations in the measurement of glucose are shown in figure 5. In figure 6 is shown a photograph of a Catalinks unit.

A second form of Catalinks utilizes immobilized urease which is covalently bound to the inner surface of a 1 mm inner diameter nylon tube. In this case, the immobilized enzyme urease cat­alyzes the hydrolysis of urea to am­monia and carbon dioxide. The ammonia in turn reacts with sodium hypochlorite to form chloramine, which then reacts at the para-position of salicylic acid yield-

F i g u r e 2. Chemical equations of reactions of “ Dip-and-read” test for urine galactose employing immobilized enzymes.

H—C=0 I

H-C-OHI

HO-C-HI

HO-C-HI

H-C-OHI

H-C-OHIH

d-GALACTOSE

H oO + OoIMMOBILIZED

GALACTOSEOXIDASE

HoO-2U2

H—C=0 I

H-C-OHI

HO-C-Ht

h o - c - hI

H-C-OHI

H—C=0

d-GALACTO HYDROGEN

HEXODIALDOSE PEROXIDE

IMMOBILIZEDHoOo + (CcHo ■ CHq ' NHo)o ------------------ ► (Cr Ho CHo MH)o

PEROXIDASE

HYDROGEN ORTHOTOLIDINE OXIDATION PRODUCTPEROXIDE OF ORTHOTOLIDINE

(Blue)

482 FREE

F ig u r e 3. Photograph of urine galactose test.

ing 2-hydroxy-5-aminobenzoic acid. Sub­sequently, the latter product reacts with another molecule of salicylate to produce an indophenol derivative which, when oxidized by excess hypochlorite, finally produces a greenish-blue indophenol of salicylic acid. The overall rate and repro­ducibility of the reaction is enhanced by nitroprusside.

Kunz and Stastny9 have described a procedure for measuring glucose in serum utilizing immobilized glucose oxidase in which the enzyme is attached to controlled-pore glass beads. Good sta­bility and performance were reported.

Leon et al10 have described a continu­ous flow tubular reactor containing im­mobilized glucose oxidase. This was used with an automated system which performed as many as 25,000 measure­ments with a single enzyme tube.

Kiang et al8 have employed im­mobilized glucose oxidase and per­oxidase in an analytical system in

which the enzymes are attached to a cylindrical magnetic stirrer so that the enzymes are exposed to the reaction mix­ture quite rapidly. This approach is in contrast to most methods in which the immobilized enzymes have been statio­nary. In this procedure, relatively high concentrations of ascorbic acid, uric acid and creatinine did not interfere.

A review of current clinical laboratory practice indicates some 15 enzymes which receive significant usage as analyt­ical reagents. These enzymes are listed in table II. Nine of these are commercially available in one or more immobilized forms, and these are identified in the ta­ble. Additionally, research studies have described immobilized models of several of the other enzymes listed in table II.

Advantages of Immobilized Enzymes as Reagents

A number of advantages which im­mobilized enzymes may provide over

NH„

l\!H,

C=0 + 3H20 + BROMTHYM OL B LU E ■IM M OBILIZED

U REASEYELLO W COLOR

UREA

C 0 2 + 2 NH4 OH + BROMTHYM OL BLU E

G REEN to BLU E COLOR (due to higher pH)

F ig u r e 4. Chem ical equation o f reaction o f b lood urea test em ploy ing im m ob ilized enzyme urease.

ANALYTICAL APPLICATIONS OF IMMOBILIZED ENZYMES 483

H—C O HO—C O,1 C A TA L IN K S I(CHOH)4 + H20 + 0 ? --------------------------------► (CHOH)d + H? 0 ,J, „ GLUCOSE O XID ASE Ic h 2o h c h 2o h

d-GLUCOSE GLUCONIC HYDROGENACID PER O XID E

2H20 2 + 4 - AMINOPHENAZONE ----------PERO XID ASE-------- ► QUINONEIM INE D YE

+ 3,5 D ICHLO RO -2-HYD RO XYBEN ZEN E SU LFO N ATEHYDROGENPER O XID E

Figure 5. Chemical equations of reactions of serum glucose with immobilized glucose oxidase of Catalinks.

soluble enzymes includes enzyme con­servation, enzyme reuse, enhanced sta­bility, economy, convenience, reproduc­ible results, ready-to-use, space-saving, adaptability to sequential reactions and adaptability to various systems. Every application of an immobilized enzyme does not necessarily result in the achievement of each advantage; how­ever, in the majority of the applications which have been established, most of the advantages are manifested.

Hornby and Inman6 described the use of immobilized enzymes in continuous

flow analyzers. In one example, during the course of approximately one month, some 3,500 glucose measurements were made on serum with one coil of tubing containing immobilized glucose oxidase. In another experiment of about the same duration, approximately 5,000 serum urea measurements were made. In either in­stance, no changes in the sensitivity ad­justments of the instrument were made. With sensitivity adjustments, several times as many measurements can be made with a single tube. The amount of enzyme which is required is very much

Figure 6. Immobilized enzyme tube for glucose analysis.

:

484 FREE

TABLE IIEnzymes Used in Clinical Analyses

Name Measurement

Alcohol dehydrogenase* ethyl alcoholCatalase* in coupled systemsCatechol methyltransferase epinephrine,

norepinephrineCholesterol esterase coupled system for

cholesterolCholesterol oxidase cholesterolCreatine kinase creatineGalactose oxidase* galactoseGlucose oxidase* glucose3-Glucuronidase coupled system-

urinary steroidsHexokinase* glucoseLactic dehydrogenase* lactic acidLipase triglyceridesPeroxidase* coupled systemUrease* ureaUricase* uric acid

♦Commercially available in immbilized form.

less when it is immobilized with the quantity being from 1/3 to 1/10 or 1/20 as great.

In general, immobilized enzymes are more stable than their counterparts which are free in solution. The reason for this is not understood, although it may be due to the fact that the insoluble enzyme has its stability enhanced by its physical or chemical bonds to the insoluble mat­rix. Increased stability of the enzyme in a system provides a strengthening of what, in many cases, is the weakest link.

Limitations of Immobilized Enzymes as Reagents

Immobilized enzymes present a form of analytical tool which requires care and consideration on the part of the user if good results are to be obtained. Proper storage is of great importance with all en­zymes but is of particular significance with immobilized enzymes.

The reagent dip-and-read systems for glucose, galactose and urea, which in­volve enzymes absorbed onto cellulous mats, require storage in an atmosphere with a very low humidity. This is readily

achieved by the use of a closed, glass bot­tle and desiccant packets. However, if the bottle top is not replaced promptly after removing a reagent strip, moisture will enter and cause deterioration of the prod­uct.

In contrast, the immobilized enzymes in Catalinks need to be kept moist to pro­vide for optimum storage. Enzyme poisons and inhibitors are limiting fac­tors, although in some immobilized en­zyme systems there is much less influ­ence of inhibitors.8

In systems involving immobilized en­zymes within tubes, it is necessary to use small bore tubing in order to have the so­lution being analyzed come in contact with the enzyme. However, such tubes are prone to becoming obstructed if care is not taken to eliminate any particulate matter or fibrin clots.

In some limited use situations, it may not be possible to find a commercial form of the immobilized enzyme which is adaptable to the usage desired. Corre­spondingly, in such cases it may not be practical to prepare the immobilized en­zyme in the laboratory.

In summary, immobilized enzymes have limitations. In many instances the limitations are related to the requirement for proper handling and appropriately fol­lowing directions. Immobilized enzymes may be subject to inhibitors and poisons. Fine bore tubes may become plugged. In some instances, the use of immobilized enzymes may not be practical.

Future of Immobilized Enzymes

Speculating about the future presents interesting opportunities to interrelate the past and the present. Enzymes are amazing catalysts. Within living cells, enzymes catalyze sequential chemical reactions which biochemists are just be­ginning to understand. Perhaps, in the fu­ture, sequences which occur within cells

ANALYTICAL APPLICATIONS OF IM M OBILIZED ENZYMES 485

can be achieved through the use of im­mobilized enzymes. Then we can antici­pate the discovery of new knowledge of a myriad of reaction systems which may reveal defects and inadequate function­ing within cells and tissues which have heretofore only been suspected. Perhaps, in the future, immobilized enzymes can be transplanted into those unfortunate individuals whose genetic backgrounds cause them to be deficient in a necessary enzyme.

The expanded use of enzymes in analytical clinical analysis is quite prob­able. Enzymes provide a high degree of specificity and sensitivity which should supersede the many non-specific empiri­cal methods which have been used in the past. It is quite likely that the relatively high cost of high purity enzymes will have a marked effect in encouraging the use of immobilized enzymes which are able to function effectively with mark­edly lower quantities of enzymes.

The capability of being able to locate an enzyme at a discrete, specific place provides an opportunity to place the en­zyme where it will not be exposed to in­hibitors or to enzyme poisons. Corre­spondingly, the enzyme can be located at a spot within the system where it will be possible to have it function and then cease to function because the reacting mixture has moved away from the im­mobilized enzyme.

Quite definitely, immobilized enzymes present interesting and exciting chal­lenges to the clinical laboratory scientist.

References

1. C a m p b e l l , J., H o r n b y , W. E., and M o r r is , D. L.: The preparation of several new nylon type-glueose oxidase derivatives and their in­corporation into the ‘reagentless’ automated analysis of glucose. Biochim. Biophys. Acta 284:307-316, 1975.

2. F r e e , A. H ., A d a m s , E . C., Ke r c h e r , M . L.,

F r e e , H . M ., and C o o k , M . H .: S im p le specific

test for urine glucose. C lin . Chem . 3:163-168, 1957.

3. F r e e , A. H. and L o w e r , G. D.: Diagnostic Composition for Blood Urea. U.S. Patent Of­fice No. 3; 145, 086, August 18, 1964.

4. F r e e , A. H . and M y e r s , V. C.: Clinical en­zyme studies. Factors involved, clinical use­fulness and a suggested method for expressing enzyme activities. Amer. J. Clin. Path. 13:42-56, 1943.

5. F r e e , A. H. and M y e r s , V. C.: The estimation of the enzymes, amylase, proteinase, and lipase in duodenal contents. J. Lab. Clin. Med. 28:1387-1398, 1943.

6 . Inman, D. J. and H o r n b y , W. E.: The im­mobilization of enzymes on nylon structures and their use in automated analysis. Biochem. J. 129:255-262, 1972.

7. Ka t c h a l s k i, E., S il m a n , I., and G o l d m a n ,

R.: Effect of the microenvironment on the mode of action of immobilized enzymes. Adv. Enzymol. 34:445-536, 1971.

8 . K ia n g , S. W., Ku a n , J. W., Ku a n , S. S., and G u il b a u l t , G. G.: Measurement of glucose in plasma, with use of immobilized glucose oxidase and peroxidase. C lin. Chem. 22:1378-1382, 1976.

9. Ku n z , H. J. and St a s t n y , M.: Immobilized glucose oxidase used to measure glucose in serum. Clin. Chem. 20:1018-1022, 1974.

10. L e o n , L . P., N a r a y a n a n , S., D e l l e n b a c h ,

R., and H o r v a t h , C.: Immobilized glucose oxidase used in the continuous-flow determi­nation of serum glucose. C lin . Chem. 22:1017-1023, 1976.

11. S i l m a n , I. and Ka t c h a l s k i , E.: Water- insoluble derivatives of enzymes, antigens and antibodies. Ann. Rev. Biochem. 35:873-908,1966.

12. Z a b ORSKY, O. R.: Immobilized Enzymes. Cleveland, CRC Press, 1973.