BIOTECHNOLOGY AND BIOENGINEERING
VOL. XVIII (1976)
COMMUNICATIONS TO THE EDITOR Continuous Monitoring in Enzyme Immobilization INTRODUCTION I n connection with an investigation of the coupling of hormones to cyanogenhalide-activated carriers, we developed a simple and rapid methodology suitable for ascertaining the best reaction conditions. Coupling of enzymes to CNBr-activated gels is generally performed a t relatively low temperatures (4"C), with pH values between 5 and 8 over periods of 2 4 4 8 hr . Other modifications of the attachment procedure have been reported and sometimes higher coupling efficiency and immobilized activity have been achieved using varied conditions of pH,' temperature,z and pressure3 for shorter reaction times. We now describe a method lor continuous monitoring of the reaction between the protein (enzymes, hormones) and the activated gel. The technique is based on continuous spectrophotometric monitoring of the unbound protein recycled through a flow reactor containing the activated gel. MATERIALS AND METHODS Sepharose 4B, Pharmacia Fine Chemicals, Uppsala, Sweden, was utilized as a carrier. Electrophoretically pure human serum albumin (HSA) was purchased from Behringwerke, Marburg-Lahn, Germany. Horseradish-peroxidase (HRPO, E.C. 1.11.1.7), glucose oxidase (GDO, E.C. 1.1.3.4), and lactate dehydrogenase (LDH, E.C. 1,1.1.27) were Boehringer, Mannheim, Germany, products. Insulin (99% pure) and CNBr were purchased from Fluka, Buchs, Switzerland. Other chemicals used were reagent grade. The sepharose was activated according to the method reported by Axen and Ernbach.' Chemical coupling was carried out in a cycle reactor. In a typical example, 3.5 g (90 mg dry material) of water-swollen sepharose were suspended in 15 ml of 0.3M Na2C03pH 11.4 and treated with 500 mg of solid CNBr a t 8°C. After the reagent was completely dissolved the activated sepharose was filtered and washed with Na2C03solution, with the NaZCO3-NsC1 ( 0 . 3 M ) solutions at the desired p H and immediately transferred into a small chromatographic column which contained the protein solution. The effluent was continuously recycled into the column at a constant flow rate and the variation of the concentration of the protein in the effluent was monitored a t 280 nm in an 80 fil spectrophotometric cell. The total activation time was 28 min in all experiments. The amount of absorbed and coupled protein was given by the variation in absorbance with time. In addition, bound protein was determined by amino acid analysis carried out on the solid material after extensive washing with 0.3M NazCO3-NaCl solution a t p H 9, 0.3M NaCl, water, and complete acid hydrolisis with 6N HCI. The activities of immobilized enzymes were determined following the methodology previously reported5 and using the specific spectrophotometric reactions. 1017
@ 1976 by John Wiley & Sons, Inc.
1018 BIOTECHNOLOGY AND BIOENGINEERING, VOL. XVIII (1976)
RESULTS AND DISCUSSION The stability of activated sepharose with time and a t high p H values was first examined, for this purpose the gel treated with CNBr was kept at p H 10, 10.5, or 11 for 2 hrs a t 8°C before reaction with the protein. It was verified that the activated gel decays rapidly at high pH values even a t 8°C. Adsorption of the protein on the carrier was measured in gels treated with CNBr and then deactivated. With the substrates tested, adsorption values lower than 10% were found and washing with buffer and NaCl solution desorbed the protein completely. Dependence of the reaction rate of immobilization on some variables was then studied using HSA as the model protein. Figure 1 illustrates the way in which the coupling velocity between sepharose and HSA depends on pH at 8°C. Since the reaction rate increased with increasing pH, the optimum reaction p H can be easily and rapidly determined. At p H
.?
.6 E c 0 (D
.5
(u L
a
2
c a n
4
L
Y, 0
n
a
.3
.2
1
I
10
20
30
40
50
time rnin Fig. 1. Dependence of coupling reaction velocity on pH. Conditions: activated sepharose 3.5 g (90 mg dry); HSA 10 mg in 5 ml buffer solutions. pH 7.8 ( 0 ) 9; (0); 10 11 (A). Temperature, 8°C. Values: 5.1 (0);
(m);
=
10
20
30
40
50
(m);
t i m e min Fig. 2. Dependence of coupling reaction velocity on HSA concentration. Conditions: activated sepharose: 3.5 g (90 mg dry); HSA 5 mg 10 mg (0); 20 mg ( 0 ); 25 mg (0)in 5 ml buffer solution, pH 10. Temperature, 8°C.
m
: .4
F
0
aJ
+
~
.
L
.2 .
.4
.6
.8
.’ -
1.2 -
5
(m),
10
15
20
t i m e min Fig. 3. Coupling of LDH GDO ( O ) ,HRPO ( O ) , and insulin ( 0 )to sepharose. Conditions: activated sepharose: 3.5 g (90 mg dry) ; LDH 10 mg (AZ801.71) ; GDO 10 mg (A280 1.11); HRPO 10 mg ( A p 8 0 2.15); insulin 10 mg (AZs02.0) in 5 ml of buffer solution, pH 9. Reaction temperature, 8°C.
a
n
.A 0
m
0
aJ
m
L
(D (u
m
I
C
0
L
0 E
,6-\
E
5,
;;
1020 BIOTECHNOLOGY AND BIOENGINEERING, VOL. XVIII (1976) 9 the reaction velocity w&s sufficiently high so that maximum binding could be achieved within 50 min. Figure 2 illustrates the effect of protein concentration on reaction rate. As expected, the increase in protein concentration had only the effect of increasing the initial reaction rate. The initial velocity increases with HSA concentration, while equilibrium is reached after 130 min. The effect of temperature on the immobilization was tested using HRPO, GDO, LDH, and insulin. At p H 9, the initial velocity tripled by increasing the coupling reaction temperature, as shown by the results illustrated in Figures 3 and 4. The technique described allows the reaction to be stopped when the equilibrium position is reached and to avoid treatment of the enzyme in severe conditions for long periods of time, thus reaching higher activity. As an example an equal amount of HRPO was bound to identical quantities of activated sepharose (0.8 mg/ml sepharose, determined by amino acid analysis) at 8 and 25°C with differing
1.2
.1
.B
i
a
.6
4
.2
5
10
15
20 time
25
min.
(u),
Fig. 4. Effect of temperature on coupling of LDH GDO (El),HRPO (O), Insulin ( 0 )to sepharose. (a) Conditions: activated sepharose: 3.5 g (90 mg dry); LDH, GDO, HRPO, insulin, 10 mg in 5 ml of buffer solution, p H 9. Reaction temperature, 25°C. (b) Sequential addition of LDH (4 mg X 5 ml) to LDH-sepharose.
COMMUNICATIONS TO T H E EDITOR
1021
reaction velocities; correspondingly equal apparent activity was found for both samples of the immobilized enzymes. Continuous protein monitoring may allow CNBr-activated gels to be used more efficiently. By sequential addition of the protein to the gel it was possible to immobilize a desired amount of enzyme. An example is illustrated in Figure 4b; by using LDH a t 25°C in a two-step process, an increasing amount (see curve A) of the enzyme was progressively immobilized within 25 min. Using properly swollen gels, the results obtained show that in a binding protein the equilibrium position is reached within a shorter time than those generally used. This observation is particularly useful when enzymes are poorly stable3 in the alcaline medium, immobilization being achieved completely in a few minutes. When a known amount of two or more enzymes i s to be immobilized on to an equal amount of carrier6 it should be easy by continuous monitoring to stop the reaction a t the desired value.
References 1. P. Cuatrecasas, Biochemistry, 63, 450 (1969). 2. Afinity Chromatography: Principles and Methods, Pharmacia Fine Chemicals AB, Uppsala, Rahms & Lund, 1974. 3. H. P. Gregor and P. W. Rauf, Biotechnol. Bioeng., 17,445 (1975). 4 . R. Axen and S. Ernback, Eur. J. Biochem., 18, 351 (1971). 5. P. Cremonesi, G. Mazzola, B. Focher, and G. Vecchio, Die Angew. Makromol. Chemie, 48, 17 (1975). 6. P. Cremonesi, unpublished results.
ROBERTO BOVARA PIERO PASTA PIETRO CREMONESI Laboratorio di Chimica degli Ormoni, C.N.R. Via Mario Bianco 9 20131-Milano Italy Accepted for Publication January 27, 1976
VOL. XVIII (1976)
COMMUNICATIONS TO THE EDITOR Continuous Monitoring in Enzyme Immobilization INTRODUCTION I n connection with an investigation of the coupling of hormones to cyanogenhalide-activated carriers, we developed a simple and rapid methodology suitable for ascertaining the best reaction conditions. Coupling of enzymes to CNBr-activated gels is generally performed a t relatively low temperatures (4"C), with pH values between 5 and 8 over periods of 2 4 4 8 hr . Other modifications of the attachment procedure have been reported and sometimes higher coupling efficiency and immobilized activity have been achieved using varied conditions of pH,' temperature,z and pressure3 for shorter reaction times. We now describe a method lor continuous monitoring of the reaction between the protein (enzymes, hormones) and the activated gel. The technique is based on continuous spectrophotometric monitoring of the unbound protein recycled through a flow reactor containing the activated gel. MATERIALS AND METHODS Sepharose 4B, Pharmacia Fine Chemicals, Uppsala, Sweden, was utilized as a carrier. Electrophoretically pure human serum albumin (HSA) was purchased from Behringwerke, Marburg-Lahn, Germany. Horseradish-peroxidase (HRPO, E.C. 1.11.1.7), glucose oxidase (GDO, E.C. 1.1.3.4), and lactate dehydrogenase (LDH, E.C. 1,1.1.27) were Boehringer, Mannheim, Germany, products. Insulin (99% pure) and CNBr were purchased from Fluka, Buchs, Switzerland. Other chemicals used were reagent grade. The sepharose was activated according to the method reported by Axen and Ernbach.' Chemical coupling was carried out in a cycle reactor. In a typical example, 3.5 g (90 mg dry material) of water-swollen sepharose were suspended in 15 ml of 0.3M Na2C03pH 11.4 and treated with 500 mg of solid CNBr a t 8°C. After the reagent was completely dissolved the activated sepharose was filtered and washed with Na2C03solution, with the NaZCO3-NsC1 ( 0 . 3 M ) solutions at the desired p H and immediately transferred into a small chromatographic column which contained the protein solution. The effluent was continuously recycled into the column at a constant flow rate and the variation of the concentration of the protein in the effluent was monitored a t 280 nm in an 80 fil spectrophotometric cell. The total activation time was 28 min in all experiments. The amount of absorbed and coupled protein was given by the variation in absorbance with time. In addition, bound protein was determined by amino acid analysis carried out on the solid material after extensive washing with 0.3M NazCO3-NaCl solution a t p H 9, 0.3M NaCl, water, and complete acid hydrolisis with 6N HCI. The activities of immobilized enzymes were determined following the methodology previously reported5 and using the specific spectrophotometric reactions. 1017
@ 1976 by John Wiley & Sons, Inc.
1018 BIOTECHNOLOGY AND BIOENGINEERING, VOL. XVIII (1976)
RESULTS AND DISCUSSION The stability of activated sepharose with time and a t high p H values was first examined, for this purpose the gel treated with CNBr was kept at p H 10, 10.5, or 11 for 2 hrs a t 8°C before reaction with the protein. It was verified that the activated gel decays rapidly at high pH values even a t 8°C. Adsorption of the protein on the carrier was measured in gels treated with CNBr and then deactivated. With the substrates tested, adsorption values lower than 10% were found and washing with buffer and NaCl solution desorbed the protein completely. Dependence of the reaction rate of immobilization on some variables was then studied using HSA as the model protein. Figure 1 illustrates the way in which the coupling velocity between sepharose and HSA depends on pH at 8°C. Since the reaction rate increased with increasing pH, the optimum reaction p H can be easily and rapidly determined. At p H
.?
.6 E c 0 (D
.5
(u L
a
2
c a n
4
L
Y, 0
n
a
.3
.2
1
I
10
20
30
40
50
time rnin Fig. 1. Dependence of coupling reaction velocity on pH. Conditions: activated sepharose 3.5 g (90 mg dry); HSA 10 mg in 5 ml buffer solutions. pH 7.8 ( 0 ) 9; (0); 10 11 (A). Temperature, 8°C. Values: 5.1 (0);
(m);
=
10
20
30
40
50
(m);
t i m e min Fig. 2. Dependence of coupling reaction velocity on HSA concentration. Conditions: activated sepharose: 3.5 g (90 mg dry); HSA 5 mg 10 mg (0); 20 mg ( 0 ); 25 mg (0)in 5 ml buffer solution, pH 10. Temperature, 8°C.
m
: .4
F
0
aJ
+
~
.
L
.2 .
.4
.6
.8
.’ -
1.2 -
5
(m),
10
15
20
t i m e min Fig. 3. Coupling of LDH GDO ( O ) ,HRPO ( O ) , and insulin ( 0 )to sepharose. Conditions: activated sepharose: 3.5 g (90 mg dry) ; LDH 10 mg (AZ801.71) ; GDO 10 mg (A280 1.11); HRPO 10 mg ( A p 8 0 2.15); insulin 10 mg (AZs02.0) in 5 ml of buffer solution, pH 9. Reaction temperature, 8°C.
a
n
.A 0
m
0
aJ
m
L
(D (u
m
I
C
0
L
0 E
,6-\
E
5,
;;
1020 BIOTECHNOLOGY AND BIOENGINEERING, VOL. XVIII (1976) 9 the reaction velocity w&s sufficiently high so that maximum binding could be achieved within 50 min. Figure 2 illustrates the effect of protein concentration on reaction rate. As expected, the increase in protein concentration had only the effect of increasing the initial reaction rate. The initial velocity increases with HSA concentration, while equilibrium is reached after 130 min. The effect of temperature on the immobilization was tested using HRPO, GDO, LDH, and insulin. At p H 9, the initial velocity tripled by increasing the coupling reaction temperature, as shown by the results illustrated in Figures 3 and 4. The technique described allows the reaction to be stopped when the equilibrium position is reached and to avoid treatment of the enzyme in severe conditions for long periods of time, thus reaching higher activity. As an example an equal amount of HRPO was bound to identical quantities of activated sepharose (0.8 mg/ml sepharose, determined by amino acid analysis) at 8 and 25°C with differing
1.2
.1
.B
i
a
.6
4
.2
5
10
15
20 time
25
min.
(u),
Fig. 4. Effect of temperature on coupling of LDH GDO (El),HRPO (O), Insulin ( 0 )to sepharose. (a) Conditions: activated sepharose: 3.5 g (90 mg dry); LDH, GDO, HRPO, insulin, 10 mg in 5 ml of buffer solution, p H 9. Reaction temperature, 25°C. (b) Sequential addition of LDH (4 mg X 5 ml) to LDH-sepharose.
COMMUNICATIONS TO T H E EDITOR
1021
reaction velocities; correspondingly equal apparent activity was found for both samples of the immobilized enzymes. Continuous protein monitoring may allow CNBr-activated gels to be used more efficiently. By sequential addition of the protein to the gel it was possible to immobilize a desired amount of enzyme. An example is illustrated in Figure 4b; by using LDH a t 25°C in a two-step process, an increasing amount (see curve A) of the enzyme was progressively immobilized within 25 min. Using properly swollen gels, the results obtained show that in a binding protein the equilibrium position is reached within a shorter time than those generally used. This observation is particularly useful when enzymes are poorly stable3 in the alcaline medium, immobilization being achieved completely in a few minutes. When a known amount of two or more enzymes i s to be immobilized on to an equal amount of carrier6 it should be easy by continuous monitoring to stop the reaction a t the desired value.
References 1. P. Cuatrecasas, Biochemistry, 63, 450 (1969). 2. Afinity Chromatography: Principles and Methods, Pharmacia Fine Chemicals AB, Uppsala, Rahms & Lund, 1974. 3. H. P. Gregor and P. W. Rauf, Biotechnol. Bioeng., 17,445 (1975). 4 . R. Axen and S. Ernback, Eur. J. Biochem., 18, 351 (1971). 5. P. Cremonesi, G. Mazzola, B. Focher, and G. Vecchio, Die Angew. Makromol. Chemie, 48, 17 (1975). 6. P. Cremonesi, unpublished results.
ROBERTO BOVARA PIERO PASTA PIETRO CREMONESI Laboratorio di Chimica degli Ormoni, C.N.R. Via Mario Bianco 9 20131-Milano Italy Accepted for Publication January 27, 1976
