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Bioluminescent determination of reduced nicotinamide adenine dinucleotide with immobilized bacterial luciferase and flavin mononucleotide oxidoreductase on collagen film
Analytica Chimica Acta, 161(1984) 355-358 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
Short Communication
BIOLUMINESCENT DETERMINATION OF REDUCED NICOTINAMIDE ADENINE DINUCLEOTIDE WITH IMMOBILIZED BACTERIAL LUCIFERASE AND FLAVIN MONONUCLEOTIDE OXIDOREDUCTASE ON COLLAGEN FILM
LOi’C J. BLUM and PIERRE
R. COULET*
Laboratoire de Biologle et Technologie des Membmnes du CN R.S., UnwersitG Claude Bernard, Lyon 143, Boulevard du 11 Novembre 1918, 69622 Vdleurbanne Cedex (Fmnce) (Received 17th January 1984)
Summary. Bacterial luciferase and flavin mononucleotide oxidoreductase were co-immobilized on collagen strips. Reduced nicotinamide adenine dinucleotide was determined in the range 1 x lo*---2 x lo* M, with a precision of 5%. The immobilized system retained 70% of its initial activity after two weeks.
The determination of metabolites and measurements of enzymatic activities are usually achieved by using one or several auxiliary enzymes, and in a final step reduced nicotinamide adenine dinucleotide (NADH) is monitored spectrophotometricahy at 340 nm. The detection limit for the coenzyme is about 1 X lo6 M. Improvements have been obtained by using enzymatic cycling [l] but this requires the use of three auxiliary enzymes and an incubation time of at least 30 min. Luminescent bacteria contain enzymes which catalyze the following reactions [ 21 NAD(P)H + H’ + FMN + NAD(P)+ + FMNHz
(1)
FMNHz + O2 + RCHO + FMN + RCOOH + Hz0 + light
(2)
Reaction (1) is catalyzed by NAD(P)H: flavin mononucleotide (FMN) oxidoreductase and reaction (2), which produces light in the presence of oxygen and a long-chain aldehyde (R-C&IO), is catalyzed by bacterial luciferase. If NAD( P)H is the limiting substrate, the light intensity is proportional to the NAD(P)H concentration. With soluble enzymes, NADH concentrations as low as 1 X lo-” M can be measured [ 31. Considering the sensitivity of the bioluminescent bacterial system with respect to NADH, it appeared attractive to co-immobilize luciferase and the oxidoreductase on a suitable support. For this purpose, collagen membranes were chosen for their high mechanical strength and ease of use compared to the particulate and gel supports used by other groups [4-6]. The conditions 0003-2670/84/$03.00
0 1984 Elsevier Science Publishers B.V.
356
allowing sensitive measurements with such a reusable material are described in this communication. Experimental Reagents. The bacterial luciferase/oxidoreductase system from Photobacterium fischeri, lyophilized powder, and ZV-decylaldehyde (decanal) were from Sigma Chemical Co. Dithiothreitol (DTT) and NADH were obtained from Calbiochem and FMN from Boehringer Mannheim. All other reagents were of the highest grade commercially available. Immobilization procedure. Flat sheets (1.5 X 18 cm) of highly polymerized insoluble collagen were a gift of the Centre Technique du Cuir, Lyon, France. Strips of 1 X 4.5 cm cut from the collagen films were used for the immobilization of the enzymes. The collagen activation, which involves the conversion of surface-available carboxyl groups to acyl-azide groups, and the coupling of enzymes were done according to the general method described previously [ 71. The activated collagen strip was dipped into a standard cuvette (1 X 1 X 4.5 cm) containing 1.3 mg of lyophilized enzyme preparation dissolved in 1 ml of 0.1 M phosphate buffer, pH 7.8, containing 2 X 10m3 M DTT, to prevent luciferase inactivation. After coupling, the strips were washed in 0.1 M phosphate buffer, pH 6.4, containing 2 X 10m3M DTT and 1 M potassium chloride and stored at 4°C in the same solution but without potassium chloride. Appamtus. Light emission was measured with a Berthold Biolumat LB 9500 luminometer. There is no accepted light standard, thus all luminescence measurements are relative. The contents of the reaction vessel (a round flat-bottomed cuvette of 12 X 47 mm) were stirred with a small stirring bar (5 X 2 mm) by placing a magnetic stirrer close to the luminometer. The sample chamber was thermostated at 23°C. Bioluminescent assay procedure. The enzymatic strip was introduced into the reaction vessel and set close to the inner wall of the cuvette. Maximum light intensity was measured upon injection of 10 ~1 of aqueous NADH solution into 990 ~1 of 0.1 M phosphate buffer, pH 6.4, containing FMN and decanal at the appropriate concentrations and 2 X 10-j M DTT. Only part of the strip, which is 4.5 cm long, was immersed in the reaction mixture. The natural adherence of collagen onto smooth surfaces helped to maintain the strip motionless in the vessel. Under these conditions, only part of the inner wall was covered by the strip enabling the emitted light to reach the photomultiplier. An emulsion of decanal (0.2% v/v) in water and ethanol (0.8% v/v) was made fresh every 4 h and stored at 4°C. Aqueous FMN solutions were prepared each day and stored light-protected at 4°C. After each assay, the collagen strips were washed in 0.1 M phosphate buffer, pH 6.4, containing 2 X 10-j M DTT and 1 M potassium chloride.
357
Results and discussion Luminescent signal. Figure 1 shows the typical time-course of light production from the collagen-bound luciferase/oxidoreductase system on injection of NADH. After a transient phase, the light intensity reaches a steady state which signifies a constant reaction rate. The steady-state response is reached in 8 min when there is no DTT in the reaction medium, but in only 2 min in the presence of 2 X 10e3 M DTT. Optimum conditions for NADH determination. The concentrations of substrates (NADH, FMN, decanal) required to give maximum intensity were determined by measuring the intensity at different concentrations of one of the substrates at fixed concentrations of the other two. Maximum intensity was obtained with 1.3 X 1Oj M FMN, 0.004% decanal and 2 X lo4 M NADH. Figure 2 shows the pH/activity profile of the immobilized and soluble bacterial systems. For the soluble bienzymatic system the optimum pH value of 6.8 is a compromise between the optimum pH values for the oxidoreductase and luciferase which are respectively 6.5 and 7.0 [8]. After immobilization, the pH activity profile is modified. The optimum value becomes 6.4 (close to the value for the oxidoreductase) and the shoulder at pH 7.0 corresponds to the optimum pH for the luciferase. Thus after immobilization the activity of the luciferase/oxidoreductase system seems to be limited by the oxidoreductase. Calibration graph and precision. The calibration graph (log-log plot) for NADH obtained by measurements of the maximum light intensity is linear from 1 X lO+ M to 2 X lO* M NADH. At higher concentrations, the response levels off. The relative standard deviation for 10 replicate assays of 1 X lO* M NADH was 5.6%. Stability of the immobilized system. The stability of the system was
0
2
4 TIME
6
8
10
6
(mm)
1
0
PH
Fig. 1. Time-course of light emission from the immobilized luciferaseloxidoreductase system. The final NADH concentration was 1 X 10 q M; reaction conditions as in the experimental section. Intensity in arbitrary units. Fig. 2. pH/activity profiles for: (0) immobilized; (Phosphate buffer (0.1 M) was used throughout.)
(0) soluble luciferase/oxidoreductase.
358
greatly improved by immobilization. After two weeks of storage under the conditions described, 70% of the initial activity remained whereas the soluble preparation was not usable after this period. In conclusion, the collagen-bound two-enzyme system appears to be superior to the soluble system by being more stable and reusable. Moreover, the film form of the collagen support is very easy to handle, particularly for repeated use. REFERENCES 1 J. V. Passonneau and 0. H. Lowry, in H. U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, Vol. 4, Academic Press, 1974, p. 2059. 2 J. W. Hastings, in M. A. Deluca (Ed.), Methods in Enzymology, Bioluminescence and Chemiluminescence, Vol. 57, Academic Press, New York, 1978, p. 125. 3 P. E. Stanley, Anal. Biochem., 39 (1971) 441. 4 E. Jablonski and M. Deluca, Proc. Natl. Acad. Sci. USA, 73 (1976) 3848. 5 0. Rodriguez and G. G. Guilbault, Enzyme Microb. Technol., 3 (1981) 69. 6 G. K. Wienhausen, L. J. Kricka, J. E. Hinkley and M. Deluca, Appl. Biochem. Biotechnol., 7 (1982) 463. 7 P. R. Coulet, J. H. Julliard and D. C. Gautheron, Biotechnol. Bioeng., 16 (1974) 1055. 8 K. Puget and A. M. Michelson, Biochimie, 54 (1972) 1197.
Short Communication
BIOLUMINESCENT DETERMINATION OF REDUCED NICOTINAMIDE ADENINE DINUCLEOTIDE WITH IMMOBILIZED BACTERIAL LUCIFERASE AND FLAVIN MONONUCLEOTIDE OXIDOREDUCTASE ON COLLAGEN FILM
LOi’C J. BLUM and PIERRE
R. COULET*
Laboratoire de Biologle et Technologie des Membmnes du CN R.S., UnwersitG Claude Bernard, Lyon 143, Boulevard du 11 Novembre 1918, 69622 Vdleurbanne Cedex (Fmnce) (Received 17th January 1984)
Summary. Bacterial luciferase and flavin mononucleotide oxidoreductase were co-immobilized on collagen strips. Reduced nicotinamide adenine dinucleotide was determined in the range 1 x lo*---2 x lo* M, with a precision of 5%. The immobilized system retained 70% of its initial activity after two weeks.
The determination of metabolites and measurements of enzymatic activities are usually achieved by using one or several auxiliary enzymes, and in a final step reduced nicotinamide adenine dinucleotide (NADH) is monitored spectrophotometricahy at 340 nm. The detection limit for the coenzyme is about 1 X lo6 M. Improvements have been obtained by using enzymatic cycling [l] but this requires the use of three auxiliary enzymes and an incubation time of at least 30 min. Luminescent bacteria contain enzymes which catalyze the following reactions [ 21 NAD(P)H + H’ + FMN + NAD(P)+ + FMNHz
(1)
FMNHz + O2 + RCHO + FMN + RCOOH + Hz0 + light
(2)
Reaction (1) is catalyzed by NAD(P)H: flavin mononucleotide (FMN) oxidoreductase and reaction (2), which produces light in the presence of oxygen and a long-chain aldehyde (R-C&IO), is catalyzed by bacterial luciferase. If NAD( P)H is the limiting substrate, the light intensity is proportional to the NAD(P)H concentration. With soluble enzymes, NADH concentrations as low as 1 X lo-” M can be measured [ 31. Considering the sensitivity of the bioluminescent bacterial system with respect to NADH, it appeared attractive to co-immobilize luciferase and the oxidoreductase on a suitable support. For this purpose, collagen membranes were chosen for their high mechanical strength and ease of use compared to the particulate and gel supports used by other groups [4-6]. The conditions 0003-2670/84/$03.00
0 1984 Elsevier Science Publishers B.V.
356
allowing sensitive measurements with such a reusable material are described in this communication. Experimental Reagents. The bacterial luciferase/oxidoreductase system from Photobacterium fischeri, lyophilized powder, and ZV-decylaldehyde (decanal) were from Sigma Chemical Co. Dithiothreitol (DTT) and NADH were obtained from Calbiochem and FMN from Boehringer Mannheim. All other reagents were of the highest grade commercially available. Immobilization procedure. Flat sheets (1.5 X 18 cm) of highly polymerized insoluble collagen were a gift of the Centre Technique du Cuir, Lyon, France. Strips of 1 X 4.5 cm cut from the collagen films were used for the immobilization of the enzymes. The collagen activation, which involves the conversion of surface-available carboxyl groups to acyl-azide groups, and the coupling of enzymes were done according to the general method described previously [ 71. The activated collagen strip was dipped into a standard cuvette (1 X 1 X 4.5 cm) containing 1.3 mg of lyophilized enzyme preparation dissolved in 1 ml of 0.1 M phosphate buffer, pH 7.8, containing 2 X 10m3 M DTT, to prevent luciferase inactivation. After coupling, the strips were washed in 0.1 M phosphate buffer, pH 6.4, containing 2 X 10m3M DTT and 1 M potassium chloride and stored at 4°C in the same solution but without potassium chloride. Appamtus. Light emission was measured with a Berthold Biolumat LB 9500 luminometer. There is no accepted light standard, thus all luminescence measurements are relative. The contents of the reaction vessel (a round flat-bottomed cuvette of 12 X 47 mm) were stirred with a small stirring bar (5 X 2 mm) by placing a magnetic stirrer close to the luminometer. The sample chamber was thermostated at 23°C. Bioluminescent assay procedure. The enzymatic strip was introduced into the reaction vessel and set close to the inner wall of the cuvette. Maximum light intensity was measured upon injection of 10 ~1 of aqueous NADH solution into 990 ~1 of 0.1 M phosphate buffer, pH 6.4, containing FMN and decanal at the appropriate concentrations and 2 X 10-j M DTT. Only part of the strip, which is 4.5 cm long, was immersed in the reaction mixture. The natural adherence of collagen onto smooth surfaces helped to maintain the strip motionless in the vessel. Under these conditions, only part of the inner wall was covered by the strip enabling the emitted light to reach the photomultiplier. An emulsion of decanal (0.2% v/v) in water and ethanol (0.8% v/v) was made fresh every 4 h and stored at 4°C. Aqueous FMN solutions were prepared each day and stored light-protected at 4°C. After each assay, the collagen strips were washed in 0.1 M phosphate buffer, pH 6.4, containing 2 X 10-j M DTT and 1 M potassium chloride.
357
Results and discussion Luminescent signal. Figure 1 shows the typical time-course of light production from the collagen-bound luciferase/oxidoreductase system on injection of NADH. After a transient phase, the light intensity reaches a steady state which signifies a constant reaction rate. The steady-state response is reached in 8 min when there is no DTT in the reaction medium, but in only 2 min in the presence of 2 X 10e3 M DTT. Optimum conditions for NADH determination. The concentrations of substrates (NADH, FMN, decanal) required to give maximum intensity were determined by measuring the intensity at different concentrations of one of the substrates at fixed concentrations of the other two. Maximum intensity was obtained with 1.3 X 1Oj M FMN, 0.004% decanal and 2 X lo4 M NADH. Figure 2 shows the pH/activity profile of the immobilized and soluble bacterial systems. For the soluble bienzymatic system the optimum pH value of 6.8 is a compromise between the optimum pH values for the oxidoreductase and luciferase which are respectively 6.5 and 7.0 [8]. After immobilization, the pH activity profile is modified. The optimum value becomes 6.4 (close to the value for the oxidoreductase) and the shoulder at pH 7.0 corresponds to the optimum pH for the luciferase. Thus after immobilization the activity of the luciferase/oxidoreductase system seems to be limited by the oxidoreductase. Calibration graph and precision. The calibration graph (log-log plot) for NADH obtained by measurements of the maximum light intensity is linear from 1 X lO+ M to 2 X lO* M NADH. At higher concentrations, the response levels off. The relative standard deviation for 10 replicate assays of 1 X lO* M NADH was 5.6%. Stability of the immobilized system. The stability of the system was
0
2
4 TIME
6
8
10
6
(mm)
1
0
PH
Fig. 1. Time-course of light emission from the immobilized luciferaseloxidoreductase system. The final NADH concentration was 1 X 10 q M; reaction conditions as in the experimental section. Intensity in arbitrary units. Fig. 2. pH/activity profiles for: (0) immobilized; (Phosphate buffer (0.1 M) was used throughout.)
(0) soluble luciferase/oxidoreductase.
358
greatly improved by immobilization. After two weeks of storage under the conditions described, 70% of the initial activity remained whereas the soluble preparation was not usable after this period. In conclusion, the collagen-bound two-enzyme system appears to be superior to the soluble system by being more stable and reusable. Moreover, the film form of the collagen support is very easy to handle, particularly for repeated use. REFERENCES 1 J. V. Passonneau and 0. H. Lowry, in H. U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, Vol. 4, Academic Press, 1974, p. 2059. 2 J. W. Hastings, in M. A. Deluca (Ed.), Methods in Enzymology, Bioluminescence and Chemiluminescence, Vol. 57, Academic Press, New York, 1978, p. 125. 3 P. E. Stanley, Anal. Biochem., 39 (1971) 441. 4 E. Jablonski and M. Deluca, Proc. Natl. Acad. Sci. USA, 73 (1976) 3848. 5 0. Rodriguez and G. G. Guilbault, Enzyme Microb. Technol., 3 (1981) 69. 6 G. K. Wienhausen, L. J. Kricka, J. E. Hinkley and M. Deluca, Appl. Biochem. Biotechnol., 7 (1982) 463. 7 P. R. Coulet, J. H. Julliard and D. C. Gautheron, Biotechnol. Bioeng., 16 (1974) 1055. 8 K. Puget and A. M. Michelson, Biochimie, 54 (1972) 1197.