function of time. When it is desirable to maintain a known steady pO~, such electrodes are readily adapted al to perform this function without the need for special gas mixtures. Similar arrangements can be made to monitor or monitor and control substrates for isolated perfused organs, tissue slices, or homogenates, or enzymes. Glucose electrodes are proving useful in the measurement of liver metabolism by recording changes in glucose concentrations. Such monitoring is still best performed in small sample streams which are continuously diluted (about 20-fold) with buffer before being exposed to the thermostatted electrode. In measurements where simpler perfusion media can be used, enzyme electrodes can be mounted in an appropriate cuvet, such as in the YSI Model 53 oxygen monitor. Over the years, drift in pO2 electrodes has been reduced. Only about 1.8%/hr is the average drift for Clark-type transcutaneous pO2 electrodes. 58 Because an excess of substrate is desirable when measuring enzyme activity, methods based upon product formation rather than substrate depletion are being studied. Rapid methods using enzyme electrodes for the commonly measured blood enzymes such as CPK (creatine phosphokinase), SGOT (serum glutamic oxalacetic transaminase), L D H (lactic dehydrogenase), and the phosphatases are being developed. Acknowledgments The author is grateful for support by the Children's Hospital Research Foundation, to the Yellow Springs Instrument Co. for gifts of electrodes and instruments, and to Eleanor Clark for assisting in the preparation of this manuscript. 5a R. Huch, A. Huch, M. Albani, M. Gabriel, F. J. Schulte, H. Wolf, G. Rupprath, E Emmrich, U. Stechele, G. Duc, and H. Bucher, Pediatrics 57, 681 (1976).

[42] E l e c t r o c h e m i c a l A p p l i c a t i o n s o f O x i r e d u c t a s e s

By GEORGE BAUM and HOWARD H. WEETALL Introduction The occurrence and bioanalytical significance of oxireductases are discussed by Clark [41], this volume. As a consequence of the formation or disappearance of an electroactive species by enzymic action of oxidases, this class of enzymes readily lends itself to electroanalytical methods. The first report of an analytical device consisting of an oxidase combined with an electrochemical detector is attributed to Clark and METHODS IN ENZYMOLOGY, VOL. LVI

Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181956-6




© ~.

Cathode Anode

Immobilized enzyme

0 Oz permeable memorane

FIG. l. An enzyme electrode based on the oxygen sensor.

Lyons. 1 These investigators described a system to measure serum glucose oxidase, wherein oxygen is consumed and H202 is produced. The glucose oxidase was entrapped between two Cuprophane membranes and placed over the tip of the polarographic electrode. The oxygen uptake is determined with the platinum polarographic electrode set at the reduction potential of oxygen (Fig. 1). Glucose + Oz + H~O

glucoseoxidase ~- H202 + gluconic acid

Subsequently, the interest in the analytical uses of oxidases has steadily increased. A major difficulty in the analytical schemes involving enzymes in general is the inherent instability of biological materials. Many processes, such as oxidation, thermal unfolding, and dissociation of cofactor, result in gradual (or catastrophic !) loss of enzymic activity. Significant stability can be confirmed on an enzyme by immobilizing it on or in a stable matrix. This also eliminates the necessity of adding the enzyme to the solution to be measured. The many ways of immobilizing enzymes which have been reviewed in detail elsewhere (see this series, Vol. 34). Three recent texts give detailed descriptions of immobilization methods. 2-4 For this chapter we will consider only those methods directly applicable to enzyme electrodes. When choosing a method of immobilization one must consider several factors. These factors should include cost of enzyme, coupling efficiency, and compatibility of the immobilization method with the sensitivity of the enzyme to pH, temperature, or other factors capable of destroying enzyme activity. The physical nature of the application may also influence 1L. C. Clark and C. Lyons, Ann N. Y. Acad. Sci. 102, 29 (1962). 2 R. Goldman, L. Goldstein, and E. Katchalski, in "Biochemical Aspects of Reactions on Solid Supports" (G. R. Stark, ed.), p. 1-72. Academic Press, New York, 1971. 3 0 . R. Zaborsky "Immobilized Enzymes." Chem. Rubber Publ. Co., Cleveland, Ohio 1973. 4 H. H. Weetall, ed., "Immobilized Enzymes, Antigens, Antibodies and Peptides." Dekker, New York, 1975.




the choice of the immobilization method. The most common immobilization techniques are adsorption, covalent attachment, covalent crosslinking, physical entrapment, and microencapsulation. Many reports of electrochemical determinations with immobilized enzymes have appeared. Several reviews are now available. 5-9 Although very few of the systems have been accepted for routine clinical use, we believe that there is considerable potential for this technology. In this chapter, we will describe some selected significant developments and provide experimental methods which may enable the reader to further exploit the use of immobilized oxidases for analytical clinical chemistry. There are two basic approaches to the construction of a system for electrochemical measurements with immobilized oxidoreductases. The enzyme may be brought into intimate contact with the sensor, an arrangement we refer to as an enzyme electrode, or the enzymic reaction may be conducted at a separate site and the reaction products are then transported to the sensor, an arrangement we refer to as a microflow immobilized enzyme reactor (IMER). Enzyme Electrode Applications Kinetic Considerations. A simple examination of the MichaelisMenton equation reveals that only at substrate concentrations significantly less than the Michaelis constant will the rate of the enzymic reaction be proportional to substrate concentration. V --

KaE[S] IS] + K m

A + [S] -> K m v = K3E A + [S] .~ K m K3E[S] V --


The rate of enzymic conversion is then determined by the rate of substrate depletion or product formation at the electrode surface. Several rigorous analyses of the mass transport process have been described. At 5G. G. Guilbault, Crit. Rev. Anal. Chem. 1, 391 (1970). 6 G. D. Christian, Adv. Biomed. Eng. Med. Phys. 4, 95 (1971). 7 D. A. Gough and J. D. Andrade, Science 1811, 380 (1973). 8 G. G. Guilbault, in "Immobilized Enzymes, Antigens, Antibodies, and Peptides" (H. H. Weetall, ed.), p. 293. Dekker, New York, 1975. 9 N. Lakshiminarayanaiah, " M e m b r a n e Electrodes," p. 335. Academic Press, New York, 1976.




high enzymic activity and [S] ~ Km, the relationships derived by Blaedel et al. lo reduce to




where [P'] is the concentration of the product of the sensor, n is the stoichiometry coefficient for the substrate to product conversion, Ds and Dp are diffusion coefficients of the substrate and product, respectively, and [S]b is the substrate concentration in the bulk phase. At conditions of high enzymic activity, low substrate concentration and high membrane thickness, the observed reaction rate is diffusion limited. Selected Electrodes. GLUCOSE OXIDASE. A very convenient and compact arrangement results when the enzyme is immobilized in the proximity of the sensor. Many systems have been described for the determination of glucose using an entrapped glucose oxidase layer over the sensor tip. Subsequent to the report by Clark and Lyons, 1 Updike and Hicks 11 entrapped glucose oxidase in a polyacrylamide gel which was then set over the end of a platinum electrode. Physical entrapment methods are suitable when the substrate is of sufficiently low molecular weight to diffuse through the barrier to the enzyme. In general, the stability as well as the availability of the enzyme can be increased by covalent immobilization of the enzyme in a thin layer at the electrode surface. The increase in stability of glucose oxidase by immobilization was shown by Guilbault and Lubrano. n These authors compared the stability of three glucose oxidase preparations: covalently bound, physically entrapped, and solubilized. Glucose oxidase chemically coupled to a hydrazyl derivative of polyacrylic acid was clearly the most stable preparation. The electrode configuration used by Guilbault and Lubrano are shown in Fig. 1. A thin film of the immobilized glucose oxidase is held in place by a cellophane membrane. The membrane protects the structural integrity of the enzyme composite, prevents penetration of macromolecular components, and will prevent loss of any uncoupled glucose oxidase. An instrument that uses an immobilized glucose oxidase membrane over a platinum electrode for the determination of serum glucose is presently manufactured by Yellow Springs Instrument Co. The electrode is operated at the reduction potential of HzO2. In these "glucose electrodes" the immobilized glucose oxidase preparation will normally contain some catalase as a contaminant. Some catalase activity is beneficial as a protective agent to prevent deactivation 10W. J. Biaedel, T. R. Kissel, and R. C. Boguslaski, Anal. Chem. 44, 2030 (1972). 11S. J. Updike and G. P. Hicks, Nature (London) 214, 986 (1974). 12G. G. Guilbault and G. J. Lubrano, Anal. Chim. Acta 64, 439 (1973).





of glucose oxidase by hydrogen peroxide. The overall stoichiometry of the reaction is glucose oxidase , gluconic acid + H202 catalase H202 ' HzO + ½Oz


The glucose oxidation reaction can also be monitored by an iodide selective electrode. In the presence of peroxidase, hydrogen peroxide oxidizes added iodide to iodine. The rate of loss of iodine, which can be detected with the solid-state iodide selective electrode, 13,a4 is related to the initial amount of glucose in the sample. H~O2 + 2I

+ 2H ÷

peroxidase ~I2 + 2HzO

Another reaction to which the glucose oxidation reaction can be coupled is the reduction of quinone to hydroquinone.lS The hydroquinone produced is reduced at a platinum polarographic electrode to regenerate quinone. The current generated between the electrodes is related to the amount of hydroquinone produced. With the iodide and the quinone addition methods, special procedures are required to eliminate interference from electro-oxidizable species in serum such as ascorbic acid, tyrosine, and uric acid. Pretreatment of the serum sample and differential electrode arrangements have been used to eliminate the effects of endogenous interfering substances. Glucose + quinone + H 2 0

glucose oxidase , gluconic acid + hydroquinone

ALCOHOL OXlDASE. Alcohol oxidase (AO) cross-linked to plasma albumin by glutaraldehyde may be used in amperometric determinations of serum ethanol. TM Oz + CH3CHzOH

alcohol oxidase ~ CH.~CHO + H20~

The alcohol oxidase enzyme electrode responds to alcohols, aldehydes, and carboxylic acids. The reaction conditions can be optimized for an ethanol over methanol response. At a polarizing voltage of - 0 . 6 V, pH 8, and low ionic strength, ethanol consumes oxygen 12,000 times more rapidly than methanol. The rate of formaldehyde oxidation under these conditions is 17,600 times faster than ethanol oxidation! GLUTAMATE DEHYDROGENASE. A novel scheme to use an ammonium selective electrode to measure glutamate concentration was re13 R. 14G. 16 D. 16 M.

A. Llenado and G. A. Rechnitz, Anal. Chem. 45, 826 and 2165 (1973). Nagy, L. H. yon Strop, and G. G. Guilbault, Anal. Chim. Acta 66, 443 (1973). L. Williams, A. Doirg, and A. Korosi, Anal. Chem. 42, 118 (1970). N a n j o and G. G. Guilbault, Anal. Chim. Acta 75, 169 (1975).




ported by Davies and Mosbach. 17 Cofactor NAD ÷ is covalently bound to dextran and is placed together with glutamate dehydrogenase (GDH) at the tip of an ammonium selective glass electrode. The enzyme couple is GDH

Glutamate ~


NAD÷ Lactate -

~. Ketoglutamate + NH4+

~ ' ~ - / LDH



The response range for this system is rather narrow, 10-3 to 10-4 M. Possibly a more selective electrode, such as the airgap ammonium electrode developed by Ru2i~ka and Hansen, is would increase the response range of the system. Immobilization of glutamate dehydrogenase does not present any special difficulties. TM However, it be perceived that either the enzyme or its cofactor must remain mobile in order to maintain activity. Broun e t a l . 2° have claimed that enzyme systems having mobile cofactors can be efficiently immobilized in a cellophane matrix by cross-linking with glutaraldehyde. The procedure for preparing a glucose oxidase membrane by cross-linking the enzyme in a cellophane matrix is given in the experimental section. AMINO ACID OXIDASE (AAO). Several enzyme electrodes for L-amino acids have been prepared using the enzyme L-amino acid oxidase. AAO

RCHNH.~CO2- + H 2 0 + O~

, RCOCO2- + NH4 ÷ + HzO2

The H~Oz product decarboxylates the a-keto acid RCOCO2- + H~O2 ~ RCO2

+ COs + H~O

Guilbault and Hrabankova 2~ immobilized the enzyme in a polyacrylamide gel and placed the membrane over a cation selective glass electrode. The presence of catalase is also beneficial in this system particularly in minimizing the nonenzymic decarboxylation reaction. The overall reaction is then AAO

2RCHNH3+COz - + HzO + 02

' 2 R C O C O 2 - + NH4 +

17 p. Davies and K. M o s b a c h , Biochim. Biophys. Acta 370, 329 (1974). 18 j. Ru~,i~ka and E. H. H a n s e n , Anal. Chirn. Acta 69, 129 (1974). 19 L. H a v e k e s , F. B u c h m a n n , and J. Visser, Biochim. Biophys. Acta 334, 272 (1974). 20G. Broun, D. T h o m a s , G. Gelf, D. D o m u r a d o , A. M. Berjonneau, and C. Guillon, Biotechnol. Bioeng. 15, 359 (1973). zl G. G. Guilbault and E. H r a b a n k o v a , Anal, Left. 3, 53 (1970).




The cation selective glass electrode also responds to H +, Na +, and K ÷. The use of a nonacton impregnated silicon membrane electrode significantly reduced interferences from the other cations.22 The use of the iodide selective electrode with a polyacrylamide membrane containing both AAO and horseradish peroxidase to monitor H202 in the above reaction has also been reported, za Broun co-cross-linked catalase and L-amino acid oxidase with glutaraldehyde, z4 The membrane was then placed over the oxygen-permeable membrane of the amperometric oxygen electrode. 2a There is very little selectivity to this electrode since most of the L-amino acids are deaminated by L-amino acid oxidase. The Km varies considerably among the amino acids. Tyrosine and leucine are rapidly oxidized, while histidine and methionine are oxidized slowly. CHOLESTEROL OXIDASE. Clark and Emory 2~ described an electrode system for cholesterol based on the polarographic measurement of the hydrogen peroxide that is formed by the action of soluble cholesterol oxidase on cholesterol. Both egterified and free cholesterol can be measured by the stepwise addition of cholesterol oxidase followed by cholesterol ester hydrolase (see Clark [41], this volume, for further details). Ascorbic acid is reported to interfere z~with the assay when the reaction is coupled to a peroxidase colorimetric assay. An alternative detection system using soluble enzymes was devised by Papastathopoulas and Rechnitz. 26 Molybdate salts can be used to catalyze the oxidation of iodide by hydrogen peroxide. HzO~ + 21- +

2H +

Mow 'Is + 2H20

The iodine can now be measured by the iodide selective electrode. As these enzymes become available at reduced cost and higher specific activity, it can be anticipated that immobilization of these enzymes in a format suitable for electroanalytical measurements will be accomplished.

Preparation of Enzyme Electrodes

Soluble Immobilized Systems. Prepare a thick paste of the enzyme and spread over the electrode surface. Cover the paste with a 20-25/xm thick layer of dialysis tubing. If maintained at 10°-15 ° between uses such systems may be stable for weeks. 22 G. G. Guilbault and E. Hrabankova, Anal. Chim. Acta 56, 285 (1971). 23 G. G. Guilbault and G. Nagy, Anal. Lett. 6, 301 (1975). 24 C. Tran-Mink and G. Broun, Anal. Chem. 47, 1359 (1975). 25 L. C. Clark and C. R. Emory, in "Ion and Enzyme Electrodes in Biology and Medicine" (M. Kessler et al., eds.), p. 161. Urban & Schwarzenberg, Berlin, 1976. zn D. S. Papastathopoulas and G. A. Rechnitz, Anal. Chem. 47, 1792 (1975).




Entrapped Systems. Place 10-15 IU of enzyme activity, physically or chemically trapped in a gel, on a piece of dialysis tubing and wrap the tubing around the electrode, spreading the enzyme evenly around the electrode surface. Place a rubber O ring around the electrode to hold the cellophane in place. Place the electrode overnight in buffer for a few hours before use. Store in buffer at 4° between use. Gel System. Holding the electrode upside down, cover it with a thin nylon net (about 90/xm thick, sheer nylon stocking), and secure it with a rubber O ring as previously described. 8Prepare the enzyme gel solution by mixing 0.1 g of enzyme (purity about 10-15 U/mg) with 1.0 ml of acrylamide gel solution prepared as previously described. Gently pour the enzyme gel solution onto the nylon net in a thin film, making sure all the pores of the net are saturated. One milliliter of this solution should be enough for several electrodes. Place the electrode in a water-jacketed cell at 0.5 ° and remove oxygen by purging with N2 before and during polymerization. Complete the polymerization by irradiating with a 150 W projector spotlight for 1 hr. Place a piece of dialysis membrane over the outside of the nylon net for further protection, and secure with a second rubber O ring. Soak the electrode in buffer solution overnight and store in buffer between use. Direct Polymerization onto the Electrode Membrane. This can be carried out by direct attachment of the enzyme to the surface of the electrode by the silanization technique, if the electrode is glass, oF by direct chemical attachment on the electrode surface, in the case of a plastic electrode. In the latter situation, membranes can be prepared by dropping 0.1 ml of soluble enzyme solution onto the surface of a gas diffusion membrane. The membrane is set aside for 12 hr at 4* to allow evaporation of the solvent. Glutaraldehyde solution is then added dropwise (2.5% in phosphate buffer, pH 6.5). The membrane is set aside for another 1.5 hr at 4 °, and is then rinsed carefully with water to remove free enzyme and buffer. Glucose Oxidase--Polyacrylamide Adduct. Acrylic acid (Aldrich) was polymerized with a few milligrams of ammonium persulfate at 80°---110 ° for several hours. The viscous polyacrylic acid was then saturated with excess p-nitroaniline by stirring overnight. The gel was diluted with an equal volume of water and the nitro group was reduced by the dropwise addition of titanium trichloride (26%) with virorous stirring. The reduction is complete when viscous reaction mixture changes from yelloworange to blue-black precipitate. The precipitate was washed several times with water and cold (0°) 2 M nitrous acid was added slowly with vigorous stirring until the precipitate turned white. The diazonium derivative was quickly washed with cold 0.1 M phosphate buffer pH 6.0. A cold solution of glucose oxidase was added and the mixture stirred to 1-8 hr in an ice bath. The precipitate was then washed with cold buffer.




Glucose Oxidase Immobilized in a Cellophane Matrix. A 100-cm z cellophane sheet 30/xm thick is impregnated with 3 ml of glucose oxidase (6 mg/ml, 781 U/mg) in 0.02 M phosphate buffer, pH 6.8, by alternate evacuation and air equilibration at 4° in a dessicator. The sheets are then impregnated with a solution of 2.5% glutaraldehyde in phosphate buffer in the same manner. Exhaustive washing is required to remove non-cross-linked material. Unreactive aldehyde functions are blocked by glycine. Up to 0.1 mg/cm 2 of protein can be bound by this method. The specific activity of the bound glucose oxidase is 10% of the specific activity of the free enzyme.

Microflow I M E R

When the enzyme is constrained at the sensor tip, enzyme loading is severely restricted. In order to obtain sufficient substrate conversion for detection, extended reaction times may be required. One way to increase the quantity of enzyme is to immobilize the enzyme in a packed or hollow tube reactor. The reactor may consist of packed porous or solid particles or the reactor may be a hollow tube with the enzyme immobilized on the reactor walls. All of these reactor designs have been investigated. Kinetic Considerations. The kinetic behavior of enzymic column reactors have been analyzed by several groups. 27-29 Starting from the integrated form of the Michaelis-Menton equation, Bar-Eli and Katchalski 27 derived the following equation for conversion of substrate at [S] >> Km. AS = K3Eo(h/v)

Where h and v are the height and volume of the column, respectively. A similar relationship was derived by Lilly et al.28 PS0 = Kmhv(1 - P) + K~EB/Q Where P = S/So, Q is the flow rate, andB is the fractional void volume of the column. The essential difference between the enzyme electrodes and the IMER is that the enzyme electrode functions effectively at [S] ~ K m, whereas the column reactors are more effective at [S] -> Kin. A detailed analysis of the engineering aspects of IMER systems is given by Pitcher and Havewala. 29 Selected Reactors PACKED CPG IMER. A packed column of glucose oxidase on controlled-pore glass (CPG) was used by Weibel et al. 3° 27 A. Bar-Eli and E. Katchalski, J. Biol. Chem. 238, 1690 (1963). 2a M. D. Lilly, W. E. Hornby, and E. M. Crook, Biochem. J. 100, 718 (1966). 29 W. Pitcher and N. Havewala, in "Immobilized Enzymes, Antigens, Antibodies, and Peptides" (H. H. Weetall, ed.), p. 93. Dekker, New York, 1975. 30 M. K. Weibel, W. Dritschilo, H. J. Bright, and A. E. Humphry, Anal. Biochem. 52, 402 (1973).




Gilson Minipuls pump




r-< ~

Buffer air-saturated

Glucosebuffersolution air-saturated

Amplifiebi r as



Miniature column




Flow meter


FIG. 2. Apparatusfor measuringenzyme activity. From Kunz and Stastny21 and by Kunz and Stastny 31 in an analytical system for serum glucose. The sample is swept through the column with oxygen saturated buffer past a polarographic oxygen electrode (Fig. 2). Kunz and Stastny examined three modes of operation: (1) an endpoint method for prediluted sample, (2) an endpoint method for undiluted sample, and (3) a maximum reaction rate measurement for undiluted sample. The endpoint method requires the integration of the area of the oxygen consumption curve. All three modes of operation gave excellent correlation with the concentration of added glucose. A 1-ml column was sufficient to quantitatively oxidize all of the glucose in a 9/xl sample at a flow rate of 5 ml/min. PACKED PELLICULARIMER. A porous support, such as controlledpore glass, can retain as much as 100 mg of enzyme per gram of support. Under conditions of high substrate concentration, the rate of enzymic conversion can become diffusion controlled. An approach to reduce the diffusion pathway is to immobilize the enzyme in a thin film on a solid

~1H. J. Kunz and M. Stastny, Clin.


20, 1018(1974).




nonporous support. The mass transfer characteristics of these pellicular immobilized enzyme supports has been analyzed by Horvath. a2 HOLLOW COLUMN IMER. When enzyme activity per unit weight of support is low, column length or diameter must be increased in order to obtain the desired degree of conversion. Long packed columns can present problems of restricted flow rates. The enzyme may also be immobilized on the inner surface of a hollow tube reactor. 33 In this way, long columns with minimal pressure drops can be constructed. Although nylon tubing appears to be the preferred column material, there is no inherent reason why a wide variety of organic or inorganic columns could not be used, since the chemistry for immobilization has now been well developed. Hornby has described the preparation of an immobilized glucose oxidase on nylon tubing for serum glucose determinations, a4 The nylon is first leached with a CaCIz solution to remove amorphous nylon. This is an etching process that increases the internal surface area of the tubing. An additional result is that amorphous nylon regions are physically less stable and their removal increases the stability of the enzyme column. Hornby and co-workers have also immobilized alcohol dehydrogenase (ADH) in a system for the assay of either pyruvate or lactate in the presence of NAD ÷ and alcohol. ADH

Ethanol ~ NAD+~ Lactate ~

~ Acetaldehyde tNADH + H+


~ Pyruvate

Preparation of Immobilized Enzymes for LMER

Polyacrylamide Entrapped Enzyme. Dissolve 1.15 g of N,N'-methylenebisacrylamide in 40 ml of 0.1 M phosphate buffer, pH 6.8, by heating until dissolved. Cool to 35° and add 6.06 g of acrylamide monomer. After mixing, filter into a flask containing 5.5 mg of riboflavin and 5.5 mg potassium persulfate. Bring to 50 ml. The gel solution may be stored in the dark for several months until required. The enzyme is immobilized by mixing 0.1 g of enzyme with 1.0 ml of the gel solution. Deoxygenate with N2 and polymerize with a 150 W 32 C. Horvath and J. M. Engasser, Ind. Eng. Chem., Fundam. 12, 229 (1973). '~3D. J. Inman and W. E. Hornby, Biochem. J. 129, (1972). 34 W. E. Hornby, D. J. Inman, and A. McDonald, FEBS Left. 23, 114 (1973).




photoflood lamp for 1 hr. The preparation can be sliced or chopped to the desired particle size. Preparation of Immobilized Glucose Oxidase on Nylon Tubing. ACTIVATION OF NYLON TUBING. A 3-m length of nylon tubing (1 mm diameter) is leached with a solution of 18.6% CaCIz in 18.6% water in methanol for 20 min at 50° to remove amorphous nylon. The tubing is washed with 250 ml of water at 10 ml/min. The tubing is then partially hydrolyzed with 3.65 N HCI at 45 ° for 40 rain in a circulating system at 5 ml/min and then washed with 250 ml of water at 10 ml/min. COVALENT ATTACHMENT OF GLUCOSE O X I D A S E . The tubing is prefused with a 12.5% solution of glutaraldehyde in 0.1 borate buffer, pH 8.5, for 20 min at 0° at a flow rate of I0 ml/min. The tubing is then washed with 50 ml of borate buffer and 10 ml of triazole-treated glucose oxidase a5 (3.5 mg protein/per milliliter) is recirculated through the tubing for 4 hr at 0° at a flow rate of 2.5 ml/min. Unreacted enzyme is removed by washing with 500 ml of 0.2 M NaC1 at 10 ml/min. Inorganic Supports. The covalent attachment of most enzymes to an inorganic support can be easily achieved on functional organic derivatives of the support material. The protocols given below are primarily for silica related particles. PREPARATION OF A SULFHYDRYL-INORGANIC CARRIER. One gram of support is added to 5 ml of a 10% aqueous solution of ~/-aminopropyltriethoxysilane which has been adjusted to pH 4.0 with HCI. The reaction mixture is stirred and heated to 70°C for 4 hr. The solution is decanted and the support is washed several times with water. This silanized support is then heated for approximately 2 hr at 120°. The resulting product has an alkylamine functional group which can undergo a wide variety of coupling reactions or it can be further derivatized to an arylamine. PREPARATION OF A SULFHYDRYL--INORGANIC CARRIER. One gram of support is added to 5 ml of a 10% aqueous solution of y-mercaptopropyltrimethoxysilane which has been adjusted to pH 5 with 6 N HCI. The reaction mixture is stirred and heated to reflux for about 2 hr. The reaction mixture is decanted and the support is washed several times with pH 5 solution, followed by several washings with water. The product is then heated for 2 hr at 120°C. The resulting product is a sulf3~Glucose oxidase (200 mg) from Aspergillus niger (type II; Sigma Chemical Co.) was incubated for 3 hr at 37° in 40 ml of 0.1 M KH2PO4 buffer containing 4 mmole of 3amino-l,2,4-triazole and 160/xmole H202 previously titrated to pH 7.5 with 2 M NaOH. The solution was then dialyzed at 4 ° against water (four times, 2 liters) and 0.2 M KH~PO4, pH 7.5 (once, 2 liters). This procedure was considered necessary to minimize contaminating catalase activity.




hydryl derivative which can be used for the covalent attachment of sulfhydryl enzymes such as urease. D I A Z O T I Z A T I O N OF AN ARYLAM1NE SUPPORT. TO 1 g of an arylamine carrier add l0 ml of 2 N HC1. This is cooled in an ice bath to 0°. After cooling, add 0.25 g NaNO2. The reaction is continued in an ice bath for 30 min. The diazotized product is thoroughly washed on a Buchner funnel with cold water and 1% sulfamic acid if desired. The enzyme in a 1% solution dissolved in an appropriate buffer, usually 0.05 M NaHCO3, pH 8.5, is added to the carrier. The reaction is continued for at least 1 hr at 0° with continuous monitoring of the pH. Diluted NaOH is added as necessary to maintain pH. The reaction can be continued overnight if desired. The resulting product is filtered and washed with buffer. T1AZINE COUPLING. TO 1 g of an alkylamine support is added 0.2 ml of triethylamine, 0.3 g of 1,3-dichloro-5-methoxytriazine in l0 ml of benzene. The mixture is reacted at 50°C for 2 hr. The activated product is washed with toluene and dried at 100°C for 1 hr. The activated derivative is coupled with a 1% enzyme solution in an appropriate buffer such as 0.05 M borate, pH 8.5, for at least 1 hr. GLUTARALDEHYDE COUPLING. To 1 g of an alkylamine support add 25 ml of a 2.5% solution of glutaraldehyde in 0.05 M phosphate buffered saline solution. Allow the carrier to activate for at least 60 min. Wash the product exhaustively with distilled water. Coupling is achieved by addition of a 1% solution of enzyme to the carder at pH 6.5-7.5 in an appropriate buffer. PREPARATION OF URICASE IMMOBILIZED ON CONTROLLED-PORE GLASS

(CPG). Dextran-coated CPG 550 ,~ 40/80 mesh (Pierce Chemical Co.) is activated by the cyanogen bromide method. Cyanogen bromide (0.10g) was added to the CPG (0.20 g) in 2 ml of water. The pH was adjusted to 10.0 after 15 min at 23°, the support was washed several times with water followed by 0.1 M borate buffer, pH 8.5. Uricase (0.5 mg) (Sigma Chemical Corp.) was added to the buffer and the mixture was shaken for 1 hr at room temperature. The preparation was then washed exhaustively With water and 0.5 M NaC1. The activity of the preparation as measured by 02 consumption was l0 U/g at a uric acid concentration of 0.20 rag%.

Electrochemical applications of oxireductases.

[42] ELECTROCHEMICAL APPLICATIONS OF OXIREDUCTASES 479 function of time. When it is desirable to maintain a known steady pO~, such electrodes are r...
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