Flow-Through NAD Sensor INTRODUCTION Nicotinamide adenine nucleotides are the coenzymes of more than 150 enzymes and they play a central role in cellular metabolism. The development of new methods for regenerating these coenzymes enables one to apply NAD-dependent enzymes for analytical purposes as well as in enzyme reactors.*The development of new methods for determining these coenzymes attracts a great deal of attention today. The commonly employed methods for the determination of nicotinamide coenzymes are based on the direct spectrophotometric registration of the changes in concentration of the reduced forms of the coenzyme^.^ However, the sensitivity of this method is low. To increase sensitivity, chemical or enzymatic enhancement methods were proposed, i.e., using the fact that the coenzyme present in the system participates in a multiple reduction-oxidation process. During this process one of the products of the system can be determined spectrophotometrically.4-11 In this case the sensitivity increases with the number of cycles made by the coenzyme. The limitations and difficulties of spectrophotometric methods occur when the coenzyme determination is made in strongly absorbing media. Some methods are deprived of such a shortcoming, namely, the one which is based on the estimation of radioactive carbon dioxide formed in the course of an enzymatic reactionlz and the other based on the measurement of oxygen concentration, which oxidizes NADH under the influence ~ l~minescent'~ of phenazine methosulfate (PMS).l 3 Recently, a m p e r ~ m e t r i c 'and methods have been proposed for the estimation of NAD in opaque solutions. The major drawback of these methods. with the exception of the luminescent method, is the need of large amounts of soluble enzymes and relatively complex electronic techniques. The development of the methods for the immobilization of the enzymes contributes greatly to the construction of new analytical systems without the defects characteristic of the homogeneous enzyme systems.I6," In this paper we are concerned with the investigation of a NAD sensor constructed on the basis of immobilized alcohol dehydrogenase and a flow-through voltametric electrode.
MATERIALS AND METHODS The following materials were used: horse liver alcohol dehydrogenase (EC 1.1.1. I , ADH), NAD, NADH, a 25% glutaraldehyde solution (Reanal, Hungary), Sepharose 6B (Pharmacia, Sweden), phenazine methosulfate (PMS, Gee Lawson, Great Britain), beef serum albumin (Olaine biochemical reagents plant, USSR), and cyanogen bromide (Koch Light, Great Britain). The activation of Sepharose by cyanogen bromide was carried out following the procedure given in Refs. 18 and 19, at pH 11.0 with 0.3 g cyanogen bromide/l mi Sepharose. On the immobilization of ADH, 5 ml activated Sepharose were washed on a glass filter with 50 ml 0.2M carbonate buffer, pH 9.0, then 5 ml of the same buffer and 1 ml ADH suspension (containing 28.6 units of enzyme) were added. The mixture was stirred for 20 hr at 4"C, then washed with 100 ml of the same buffer and 300 ml of a standard buffer solution (0.01M phosphate buffer, pH 8.0, containing 0.1M potassium chloride). The enzymatic activity was measured using an oxygen electrodez0by a modified Biotechnology and Bioengineering, Vol. XXI, Pp. 513-518 (1979) 0006-3592/79/0021-0513$01.OO
@ 1979 John Wiley & Sons, lnc.
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procedure.I3 A certain amount of ADH was introduced into the sealed cell thermostated at 25°C with Clark-type oxygen electrode, containing 10 ml O.IM ethanol, ImM NAD, ImM PMS, and 0.25mM oxygen,z0in a standard solution. The enzyme activity was measured by the initial rate of oxygen consumption. The enzymatic unit ( E ) corresponds to that amount of ADH necessary to catalyze the conversion of I pmol oxygen in 1 min in the conjugated reaction between ethanol, NAD, PMS, and oxygen. To prepare an enzyme membrane, 0.2 ml ADH suspension containing 5.7 E ADH and 50 pl glutaraldehyde solution were added to a solution of 20 mg albumin in 0.4 ml 0.05M phosphate buffer, pH 7.0. An additional amount of NADH (2.5 mg) was used when the membrane preparation involved the coenzyme. The mixture was poured onto a Nylon-6 net (0.1 mm thick and 3 x 3 cm in size) placed between two glassy sheets and stored for three days at 4°C. The construction of a flow-through voltametric sensor is shown in our previous work.21 The semipermeable membrane used was either a cellophane sheet or an ultrafiltration membrane MV 1-20 (U.S.S.R.)with an average pore size of 10-15 nm. The solution pumped through the sensor contained 0.5mM PMS in a standard solution. The electrode current was measured with a polarograph PIT-I connected with a recorder KSP-4 (both produced in the U.S.S.R.). RESULTS AND DISCUSSION The enzyme NAD sensor was constructed on the basis of a flow-through voltametric electrode.21The immobilized alcohol dehydrogenase was entrapped in a region near the surface of the electrode using the semipermeable membrane. The thickness of the enzyme layer was 0.1-0.5 mm. The immobilization of ADH was canied out by covalent attachment to Sepharose and by crosslinking with albumin using glutaraldehyde. Following the first method the enzyme preparation was obtained with 18% yield and showed an activity of 1.07 E M . Using the entrapment of the suspension of immobilized ADH or albumin-ADH under the ultrafiltration membrane, sensors 1, 2, and 3 were prepared (Table I). When a solution containing IM ethanol, 0.5mM PMS, and NAD, was pumped through the microcell of a flow-through sensor, the electrode current increases, as is shown in Figure 1. It can be seen that the response time exceeds 5 min and depends on the type of membrane used, the nature of the immobilized enzyme, and the thickness of the enzyme layer. The longest response time was obtained with a dialysis membrane. The shortest response time was observed in the case of an albumin enzyme membrane. The increase of the thickness of the enzyme layer leads
TABLE I Parameters of NAD Sensors
No. Means of immobilization 1 on Sepharose 2 on Sepharose 3 crosslinking with albumin
Enzyme layer thickness (mm) 0.2 0.5 0. I
Response Recovery Sensor velocity velocity sensitivity constant constant (nAipM NAD) (min-') (min-I) 47.6 142 5.4
0.12 0.073 0.26
0.10 0.053 0.23
":p
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Fig. 1. Dependence of current on the pumping time of a solution containing 1M ethanol, 0.5mM PMS, and lOpM NAD for the sensors based on ADH immobilized on Sepharose with the enzyme layer of 0.2 mm (1) and 0.5 mm (2) and for the sensor based on ADH crosslinked with albumin (3). Solution was pumped at a speed 0.53 mlimin, pH 8.0, and 30°C temperature. to an increase in response time. The increase of a sensor current is close to exponential. One-half period of steady-state current is equal to 2.7-9.5 min (Table I). The current decrease with the elution of coenzyme, when blank solution is pumped after the coenzyme-containing solution, proceeds at the same speed (Table I). The sensor's sensitivity depends on the immobilization technique as well as on the thickness of the enzyme layer (Table I). With ADH immobilized on Sepharose the sensitivity of the sensor reaches 142 nAipl4 NAD. In the case of the sensors based on the albumin-enzyme membrane the sensitivity was 5.4 and 1.6 nAIpl4 NAD when ADH was immobilized in the presence of NADH or without it, respectively. To increase the number of analyses made during the same time period, the NADcontaining solution can be pumped for a definite time period, not necessarily until the maximum of a steady-state current. The sensor readings show the initial rate of the process when the pumping time does not exceed one-half the period of the steady-state current. Figure 2 indicates that under such an operating mode a linear
Fig. 2. Calibration curves for the sensor based on ADH immobilized on Sephar( I ) and for the sensor with an albuminose with the layer thickness of 0.2 mm (0) ADH membrane ( 0 )(2). In the first case the solutions were pumped for 5 min, in the latter one, until the current reaches its steady-state value.
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dependence of the electrode current on the coenzyme concentration up to 2 0 - 3 0 N is observed. As in the case of a steady-state current (Table I), the slope of the standard curves is higher for the sensor based on Sepharose than that on an albumin membrane. The sequence of the processes occurring in the sensor consists of the following steps. On pumping through the microcell of a buffer solution containing ethanol, PMS, and NAD, the components of the solution diffuse across the semipermeable membrane to the region containing the immobilized enzyme, where the enzymatic reaction of oxidation of ethanol via NAD occurs. Furthermore, the oxidation of NADH formed proceeds rapidly in the presence of PMS.I3 The reduced form of PMS is oxidized on the platinum anode. The reduction of silver ions and oxygen takes place on the reference electrode. In the range of low coenzyme concentrations the calibration curves are linear. If the overall rate of the process is determined by the rate of the enzymatic reaction, then the kinetic parameters can be derived on the basis of the linearization of the data obtained from the calibration curves in Lineweaver-Burk coordinates.z2The calculated values of apparent Michaelis constant are equal to 125 and 8 3 N for electrodes I and 3, respectively. Within the limits of accuracy these values are close to the Michaelis constant of a native enzyme. Digital simulation of an amperometric enzyme-sensor’s action indicated that the steady-state current half-period is equal to 1/10of an enzyme layer thickness squared over the diffusion coefficient. 22 Consequently, the calculated diffusion coefficient of NAD is equal to 6.3 x 10-’-2.6 x lo-’ cm*/sec, taking into account the thickness of an ultrafiltration membrane (0.1 mm). The values obtained two to three times lower than the diffusion coefficient of water, calculated by using the molecular weight of an coenzyme. It follows that the diffusive difficulties appear to be of no significance to the coenzyme in the case of sensors based on an ultrafiltration membrane and Sepharose (electrodes I and 2 in Table I). However, in applying a dialysis membrane, those difficulties become apparent. This results in a long sensor response time. For this reason dialysis membranes are practically of no use in the construction of such sensors. At high substrate concentrations the value of the sensor’s steady-state current is directly proportional to the enzyme concentration and its layer thickness. z2 Therefore, the comparison of the sensor’s steady-state currents made on the basis of Sepharose and those on an albumin enzyme membrane indicates that enzyme activity is significantly lower in the case of an albumin membrane. ?he loss in the sensitivity of the sensor with an albumin membrane produced in the absence of NADH is also related to the decrease of the enzyme activity. One of the most significant characteristics of the sensors is their long-term stability. The changes in readings of the sensors based on ADH (the immobilization of which was camed out on Sepharose or in a membrane) are represented by curves 1 and 2, respectively, in Figure 3. The stability of the sensor based on the enzyme immobilization on Sepharose is higher than that involving an albumin membrane. The initial increase in electrode sensitivity is due to the formation of an enzyme layer and the change in diffusion parameters. Such current increase has also been observed for other amperometric electrode^.^^ The decrease is due to the deactivation of the enzyme that proceeds at a greater speed in the presence of ADH entrapped in albumin membrane. However, the sensor retains its sensitivity on the level of 8040% during eight days. The flow-through sensors can also be used for the determination of a reduced form of NAD because of the rapid oxidation of NADH by PMS. The substitution of ADH
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Fig. 3. Changes in the sensors response, constructed on the basis of ADH im(1)and the sensor mobilized on Sepharose with the layer thickness of 0.2 mm (0) based on an albumin-ADH membrane (0)(2),stored at 4°C and tested periodically within 1 hr. Solution containing 1OpM NAD was pumped until the current reaches its steady-state value. by NADP-dependent dehydrogenases makes it possible to apply those electrodes for the determination of NADP(H). References 1 . A. L. Lehninger, Biochemistry (Worth, New York, 1972). 2. P. Davies and K. Mosbach, Biochim. Biophys. Acta, 370, 329 (1974). 3. M. Klingenberg. in Methods of Enzymatic Analysis, H. U. Bergmeyer, Ed. (Academic, New York, 1965),p. 528. 4. T.F. Slater, B. Sawyer, and U. Strauli, Arch. Int. Physiol. Biochem., 72,427 (1964). 5 . C. L. Woodley and N. K . Gupta, Anal. Biochem., 43, 341 (1971). 6. M. P. Schulman, N. K. Gupta, A. Omachi, G. Hoffman, and W. E. Marshall, Anal. Biochem., 60,302 (1974). 7. H. H. Rasmussen, J. R. Nielsen, and P. Schack, Anal. Biochem., 50, 642 ( 1972). 8. T . Kato, S . J . Berger, J . A. Carter, and 0. H . Lowry, Anal. Biochem., 53, 86 (1973). 9. S. Bitny-Szlachto and A. Sollich, Acta Biochim. Pol., 17, 175 (1970). 10. C. Bernofsky and K. M. Royal, Biochim. Biophys. Acta, 215, 210 (1970). 1 1 . G. E. Glock and P. McLean, Biochem. J . , 61,381 (1955). 12. I. Pastan, V. Wills, B. Herring, and J . B. Field, J . Biol. Chem., 238, 3362 (1963). 13. A. L.Greenbaum, J. B. Clark, and P. McLean, Biochem. J . , 95, 161 (1965). 14. T.C. Wallace, M. B. Leh, and R. W. Coughlin, Biotechnol. Bioeng., 19, 901 ( 1977). 15. E. Jablonski and M. DeLuca, Proc. Nar. Acad. Sci. USA, 73, 3848 (1976). 16. D. N. Gray, M. H. Keyes, and B. Watson, Anal. Chem. A , 49, 1067 (1977). 17. I. V. Berezin, V. K. Antonov, K. Martinek, Eds., Immobilized Enzymes (Moscow State University Publishing House, Moscow, 1976),Vols. I and 11. 18. R. Axen, J. Porath, and S. Ernback, Nature, 214, 1302 (l%7). 19. P. Cuatrecasas, J . B i d . Chem., 245, 3059 (1970). 20. B. S. Panavo and J. J . Kulys, in Methods in Biochemistry (Nauka, Vilnius, 1976),p. 130.
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21. A. A. Malinauskas and J. J. Kulys, Anal. Chirn. Acta, 98, 31 (1978). 22. L. D. Me11 and J. T. Maloy, Anal. Chern., 47, 299 (1975). 23. G. G. Guilbault and G. J . Lubrano, Anal. Chirn. Acra, 64, 439 (1973).
A. A. MALINAUSKAS J. J. KULYS Institute of Biochemistry Academy of Sciences Lithuanian SSR Vilnius, U.S .S.R. Accepted for Publication July 14, 1978
MATERIALS AND METHODS The following materials were used: horse liver alcohol dehydrogenase (EC 1.1.1. I , ADH), NAD, NADH, a 25% glutaraldehyde solution (Reanal, Hungary), Sepharose 6B (Pharmacia, Sweden), phenazine methosulfate (PMS, Gee Lawson, Great Britain), beef serum albumin (Olaine biochemical reagents plant, USSR), and cyanogen bromide (Koch Light, Great Britain). The activation of Sepharose by cyanogen bromide was carried out following the procedure given in Refs. 18 and 19, at pH 11.0 with 0.3 g cyanogen bromide/l mi Sepharose. On the immobilization of ADH, 5 ml activated Sepharose were washed on a glass filter with 50 ml 0.2M carbonate buffer, pH 9.0, then 5 ml of the same buffer and 1 ml ADH suspension (containing 28.6 units of enzyme) were added. The mixture was stirred for 20 hr at 4"C, then washed with 100 ml of the same buffer and 300 ml of a standard buffer solution (0.01M phosphate buffer, pH 8.0, containing 0.1M potassium chloride). The enzymatic activity was measured using an oxygen electrodez0by a modified Biotechnology and Bioengineering, Vol. XXI, Pp. 513-518 (1979) 0006-3592/79/0021-0513$01.OO
@ 1979 John Wiley & Sons, lnc.
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BIOTECHNOLOGY AND BIOENGINEERING VOL. XXI (1979)
procedure.I3 A certain amount of ADH was introduced into the sealed cell thermostated at 25°C with Clark-type oxygen electrode, containing 10 ml O.IM ethanol, ImM NAD, ImM PMS, and 0.25mM oxygen,z0in a standard solution. The enzyme activity was measured by the initial rate of oxygen consumption. The enzymatic unit ( E ) corresponds to that amount of ADH necessary to catalyze the conversion of I pmol oxygen in 1 min in the conjugated reaction between ethanol, NAD, PMS, and oxygen. To prepare an enzyme membrane, 0.2 ml ADH suspension containing 5.7 E ADH and 50 pl glutaraldehyde solution were added to a solution of 20 mg albumin in 0.4 ml 0.05M phosphate buffer, pH 7.0. An additional amount of NADH (2.5 mg) was used when the membrane preparation involved the coenzyme. The mixture was poured onto a Nylon-6 net (0.1 mm thick and 3 x 3 cm in size) placed between two glassy sheets and stored for three days at 4°C. The construction of a flow-through voltametric sensor is shown in our previous work.21 The semipermeable membrane used was either a cellophane sheet or an ultrafiltration membrane MV 1-20 (U.S.S.R.)with an average pore size of 10-15 nm. The solution pumped through the sensor contained 0.5mM PMS in a standard solution. The electrode current was measured with a polarograph PIT-I connected with a recorder KSP-4 (both produced in the U.S.S.R.). RESULTS AND DISCUSSION The enzyme NAD sensor was constructed on the basis of a flow-through voltametric electrode.21The immobilized alcohol dehydrogenase was entrapped in a region near the surface of the electrode using the semipermeable membrane. The thickness of the enzyme layer was 0.1-0.5 mm. The immobilization of ADH was canied out by covalent attachment to Sepharose and by crosslinking with albumin using glutaraldehyde. Following the first method the enzyme preparation was obtained with 18% yield and showed an activity of 1.07 E M . Using the entrapment of the suspension of immobilized ADH or albumin-ADH under the ultrafiltration membrane, sensors 1, 2, and 3 were prepared (Table I). When a solution containing IM ethanol, 0.5mM PMS, and NAD, was pumped through the microcell of a flow-through sensor, the electrode current increases, as is shown in Figure 1. It can be seen that the response time exceeds 5 min and depends on the type of membrane used, the nature of the immobilized enzyme, and the thickness of the enzyme layer. The longest response time was obtained with a dialysis membrane. The shortest response time was observed in the case of an albumin enzyme membrane. The increase of the thickness of the enzyme layer leads
TABLE I Parameters of NAD Sensors
No. Means of immobilization 1 on Sepharose 2 on Sepharose 3 crosslinking with albumin
Enzyme layer thickness (mm) 0.2 0.5 0. I
Response Recovery Sensor velocity velocity sensitivity constant constant (nAipM NAD) (min-') (min-I) 47.6 142 5.4
0.12 0.073 0.26
0.10 0.053 0.23
":p
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Fig. 1. Dependence of current on the pumping time of a solution containing 1M ethanol, 0.5mM PMS, and lOpM NAD for the sensors based on ADH immobilized on Sepharose with the enzyme layer of 0.2 mm (1) and 0.5 mm (2) and for the sensor based on ADH crosslinked with albumin (3). Solution was pumped at a speed 0.53 mlimin, pH 8.0, and 30°C temperature. to an increase in response time. The increase of a sensor current is close to exponential. One-half period of steady-state current is equal to 2.7-9.5 min (Table I). The current decrease with the elution of coenzyme, when blank solution is pumped after the coenzyme-containing solution, proceeds at the same speed (Table I). The sensor's sensitivity depends on the immobilization technique as well as on the thickness of the enzyme layer (Table I). With ADH immobilized on Sepharose the sensitivity of the sensor reaches 142 nAipl4 NAD. In the case of the sensors based on the albumin-enzyme membrane the sensitivity was 5.4 and 1.6 nAIpl4 NAD when ADH was immobilized in the presence of NADH or without it, respectively. To increase the number of analyses made during the same time period, the NADcontaining solution can be pumped for a definite time period, not necessarily until the maximum of a steady-state current. The sensor readings show the initial rate of the process when the pumping time does not exceed one-half the period of the steady-state current. Figure 2 indicates that under such an operating mode a linear
Fig. 2. Calibration curves for the sensor based on ADH immobilized on Sephar( I ) and for the sensor with an albuminose with the layer thickness of 0.2 mm (0) ADH membrane ( 0 )(2). In the first case the solutions were pumped for 5 min, in the latter one, until the current reaches its steady-state value.
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dependence of the electrode current on the coenzyme concentration up to 2 0 - 3 0 N is observed. As in the case of a steady-state current (Table I), the slope of the standard curves is higher for the sensor based on Sepharose than that on an albumin membrane. The sequence of the processes occurring in the sensor consists of the following steps. On pumping through the microcell of a buffer solution containing ethanol, PMS, and NAD, the components of the solution diffuse across the semipermeable membrane to the region containing the immobilized enzyme, where the enzymatic reaction of oxidation of ethanol via NAD occurs. Furthermore, the oxidation of NADH formed proceeds rapidly in the presence of PMS.I3 The reduced form of PMS is oxidized on the platinum anode. The reduction of silver ions and oxygen takes place on the reference electrode. In the range of low coenzyme concentrations the calibration curves are linear. If the overall rate of the process is determined by the rate of the enzymatic reaction, then the kinetic parameters can be derived on the basis of the linearization of the data obtained from the calibration curves in Lineweaver-Burk coordinates.z2The calculated values of apparent Michaelis constant are equal to 125 and 8 3 N for electrodes I and 3, respectively. Within the limits of accuracy these values are close to the Michaelis constant of a native enzyme. Digital simulation of an amperometric enzyme-sensor’s action indicated that the steady-state current half-period is equal to 1/10of an enzyme layer thickness squared over the diffusion coefficient. 22 Consequently, the calculated diffusion coefficient of NAD is equal to 6.3 x 10-’-2.6 x lo-’ cm*/sec, taking into account the thickness of an ultrafiltration membrane (0.1 mm). The values obtained two to three times lower than the diffusion coefficient of water, calculated by using the molecular weight of an coenzyme. It follows that the diffusive difficulties appear to be of no significance to the coenzyme in the case of sensors based on an ultrafiltration membrane and Sepharose (electrodes I and 2 in Table I). However, in applying a dialysis membrane, those difficulties become apparent. This results in a long sensor response time. For this reason dialysis membranes are practically of no use in the construction of such sensors. At high substrate concentrations the value of the sensor’s steady-state current is directly proportional to the enzyme concentration and its layer thickness. z2 Therefore, the comparison of the sensor’s steady-state currents made on the basis of Sepharose and those on an albumin enzyme membrane indicates that enzyme activity is significantly lower in the case of an albumin membrane. ?he loss in the sensitivity of the sensor with an albumin membrane produced in the absence of NADH is also related to the decrease of the enzyme activity. One of the most significant characteristics of the sensors is their long-term stability. The changes in readings of the sensors based on ADH (the immobilization of which was camed out on Sepharose or in a membrane) are represented by curves 1 and 2, respectively, in Figure 3. The stability of the sensor based on the enzyme immobilization on Sepharose is higher than that involving an albumin membrane. The initial increase in electrode sensitivity is due to the formation of an enzyme layer and the change in diffusion parameters. Such current increase has also been observed for other amperometric electrode^.^^ The decrease is due to the deactivation of the enzyme that proceeds at a greater speed in the presence of ADH entrapped in albumin membrane. However, the sensor retains its sensitivity on the level of 8040% during eight days. The flow-through sensors can also be used for the determination of a reduced form of NAD because of the rapid oxidation of NADH by PMS. The substitution of ADH
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Fig. 3. Changes in the sensors response, constructed on the basis of ADH im(1)and the sensor mobilized on Sepharose with the layer thickness of 0.2 mm (0) based on an albumin-ADH membrane (0)(2),stored at 4°C and tested periodically within 1 hr. Solution containing 1OpM NAD was pumped until the current reaches its steady-state value. by NADP-dependent dehydrogenases makes it possible to apply those electrodes for the determination of NADP(H). References 1 . A. L. Lehninger, Biochemistry (Worth, New York, 1972). 2. P. Davies and K. Mosbach, Biochim. Biophys. Acta, 370, 329 (1974). 3. M. Klingenberg. in Methods of Enzymatic Analysis, H. U. Bergmeyer, Ed. (Academic, New York, 1965),p. 528. 4. T.F. Slater, B. Sawyer, and U. Strauli, Arch. Int. Physiol. Biochem., 72,427 (1964). 5 . C. L. Woodley and N. K . Gupta, Anal. Biochem., 43, 341 (1971). 6. M. P. Schulman, N. K. Gupta, A. Omachi, G. Hoffman, and W. E. Marshall, Anal. Biochem., 60,302 (1974). 7. H. H. Rasmussen, J. R. Nielsen, and P. Schack, Anal. Biochem., 50, 642 ( 1972). 8. T . Kato, S . J . Berger, J . A. Carter, and 0. H . Lowry, Anal. Biochem., 53, 86 (1973). 9. S. Bitny-Szlachto and A. Sollich, Acta Biochim. Pol., 17, 175 (1970). 10. C. Bernofsky and K. M. Royal, Biochim. Biophys. Acta, 215, 210 (1970). 1 1 . G. E. Glock and P. McLean, Biochem. J . , 61,381 (1955). 12. I. Pastan, V. Wills, B. Herring, and J . B. Field, J . Biol. Chem., 238, 3362 (1963). 13. A. L.Greenbaum, J. B. Clark, and P. McLean, Biochem. J . , 95, 161 (1965). 14. T.C. Wallace, M. B. Leh, and R. W. Coughlin, Biotechnol. Bioeng., 19, 901 ( 1977). 15. E. Jablonski and M. DeLuca, Proc. Nar. Acad. Sci. USA, 73, 3848 (1976). 16. D. N. Gray, M. H. Keyes, and B. Watson, Anal. Chem. A , 49, 1067 (1977). 17. I. V. Berezin, V. K. Antonov, K. Martinek, Eds., Immobilized Enzymes (Moscow State University Publishing House, Moscow, 1976),Vols. I and 11. 18. R. Axen, J. Porath, and S. Ernback, Nature, 214, 1302 (l%7). 19. P. Cuatrecasas, J . B i d . Chem., 245, 3059 (1970). 20. B. S. Panavo and J. J . Kulys, in Methods in Biochemistry (Nauka, Vilnius, 1976),p. 130.
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21. A. A. Malinauskas and J. J. Kulys, Anal. Chirn. Acta, 98, 31 (1978). 22. L. D. Me11 and J. T. Maloy, Anal. Chern., 47, 299 (1975). 23. G. G. Guilbault and G. J . Lubrano, Anal. Chirn. Acra, 64, 439 (1973).
A. A. MALINAUSKAS J. J. KULYS Institute of Biochemistry Academy of Sciences Lithuanian SSR Vilnius, U.S .S.R. Accepted for Publication July 14, 1978
