A Flow Method for Determination of the Kinetic Parameters for Immobilized Enzymes T. T. NGO,P. S. BUNTING^, AND K. J . LAIDLER Chel~aistryDeportment, Unirtersity of Ottaw'a, Ottavc-a,Canada, K I N 6N5
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Received June 17, 1974 Ngo, T. T., Bunting, P. S. & Laidler, K. J. (1975) A Flow Method for Determination of the Kinetic Parameters for Immobilized Enzymes. Can. J. Bioclzern. 53, 11-14 A flow method is described for determination of the kinetic parameters (V, and K,,) for enzymes that are bound to particles, to membranes, and to the interior surfaces of tubes. Substrate solution is pumped through Tygon tubing to a microvolume flow cell and back into the reaction mixture, the flow rate being adjusted to be faster than the rate sf formation of product. To illustrate the technique, it is applied to the determination sf the parameters for electric-eel acetylcholinesterase attached to particles, to membranes, and to the inner surface of nylon tubing. Ngo, T. T., Bunting, P. S. & Laidler, K. J. (1975) A Flow Method for Determination of the Kinetic Parameters for Immobilized Enzymes. Can. J. Biochem. 53, 11-14 Une mCthode dlCcoulement est dCcrite pour la dttermination de paramkres cinCtiques (V, et K,) des enzymes likes aux particules, aux membranes et aux surfaces internes des tubes. La solution de substrat est pompke, B travers un tube de Tygon, vers une microcuve d'Ccoulement et ramenCe dans le milieu de rtaction, la vitesse d'kcoulement ktant ajustee B un niveau plus ClevC que celle de la formation du produit. Pour illustrer cette technique, nous l'appliquons a la dktermination des parametres de l'acktylcholinestkrase (anguille Clectrique) liCe aux particules, aux membranes et h la surface interne du tube de nylon. [Traduit par le journal]
Introduction Various theories of the kinetic behavior of immobilized enzymes have recently been formulated (1-4) and have been submitted to extensive experimental tests (5-9). There is need for an accurate and sensitive method of determining the kinetic parameters of immobilized enzyme systems, and two papers dealing with this problem have recently been published by Mort et al. (10) and Widmer et al. (1 1 ) . Mort et al. (18) have developed a continuous spectrophotometric assay for particle-bound enzyme using a spectrophotometer with a built-in magnetic stirrer. This method is convenient and sensitive but is not readily applicable to systems with enzyme immobilized on a sheet of membrane, such as papain immobilized on collodion membrane (9) and P-galactosidase trapped in polyacrylamide membrane ( 7 ) , because of the interference of these membranes with thc optical measurements. Also, it cannot be used to assay enzyme immobilized on the interior surfacer of tube. m e described by Widmer et a[. (11) requires the Illanufacture a apparatus and can-
'Present address: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A l .
not be used for enzyme attached to the interior surface of a tube. Recently, Bunting and Laidler (7) described a Row method to determine the kinetics sf Pgalactosidase immobilized in polyacrylamide. The present paper describes an extension of this method to the determination of the kinetic parameters of free soluble enzyme, particle-bound enzyme, membrane-bound enzyme, and enzyme immobilized on the interior surface of a tube. The technique involves pumping the substrate solution through Tygon tubing into a microvolume Aow cell and recirculating it into the original mixture in the case of particles and membranes; in the case of tubes, there is no recirculation. The enzyme used in the present studies to test the method is acetylcholinesterase from electric eel and the substrate is acetylthiocholine. Materials The enzyme, acetylcholinesterase from electric eel (type 111); was obtained from Sigma Chemical Co. The substrate acet~lthiocholine ( ASCh) and the chromogenic agent 5,S-d$hiobisnitrobenzoic acid (DTNB) were obtained from Sigma Chemical 450. N,N,N', N'-~etramethyleth~lenediahine (TEMED), N,N'-methylene bisacrylamide (BIS), and acrylamide were obtained from Eastman Organic Chemicals. The
82
CAN. J . BIOCHEM. VOL. 53, I975
Can. J. Biochem. Downloaded from www.nrcresearchpress.com by San Francisco (UCSF) on 01/04/15 For personal use only.
ammonium persulfate used in the polymerizations, and dipotassiunr hydrogen phosphate, were obtained from Fisher Scientific Co. Phthalic acid and boric acid were obtained from British Drug Houses, and sodium chloride from Canadian Laboratory Supplies Ltd.
All solutitdns used to assay the enzyme and to prepare the gels were made up in Silman and Karlin buffer ( 12), which consists of 0.15 i W NaC1, 2 mA4 phthalate, 2 mM phosphate, and 2 rnM borate, pH 7.0. Enzymecontaining pslyacrylamide gels were made by polymerization sf acrylamide-monomer solution with BIS and TEAf ED 4 13, 14). The enzyme-polyacrylasni$e menlbranes were prepared by slicing a frozen gel with a microtome ('71, and the membrane thicknesses were measured with a Zeiss optical microscope with a calibrated 12.5X magnification eyepiece and a 18X magnification objective. Enzyme-polyacrylamide particles were prepared by homogenizing 1 ml of gel in a homogenizer consisting of a smooth-walled glass tube fitted with an electrically driven Teflon pestle, clearance 150-230 pm, capacity 50 ml. The gel was homogenized by 10 up-and-down strokes of the pestle and the homogenate was centrifuged in a clinical centrifuge for 1Bbm1in. One Rundrecl microliters of the packed homogenized gel was then diluted % O O X with bu8er solution, and 0.5 ml of the diluted homogenate was used in each assay. AcetyBcholinesterase was chemically attached to the interior surface sf a nylon tube (0.1 crn internal diameter, obtained from John Tullis. Tullibody, Alloa. Scotland) by a method similar to that of Sundarana and Hsrmby (151, of which details will be published elsewhere ( 16). Kinetic. Procedra~e Free and immobilized acetylcholinesterase were assayed calorimetrically with acetylthiocholine and DTNB, as described by Ellman et al. ( 1 7 ) . Aborbance at 412 nm was followed in a Unisarn SP 1800 recording spectrophotometer. Reaction rates were = 1 3 000 ,%fI. calsula ted from e I 2' Kinetic measurements on the enzyme-particles and enzyme-membranes were carried out by suspending the particles or membranes in 10 ml of bulFFered sukstrate solution. By means of a Watson-Marlow flow inducer (model MHWE 200), the solution was pumped through Tygon tubing to a microvolume flow cell (Hellma Co.) to determine the abssrbamce, and back into the reaction mixture. A nylon net, 40 ym mesh (Pharmacia Fine Chemicals Co.), was attached $0the inlet of the Tygon tubing to prevent the partides or membrane frown being pumped into the flow cell. The volume of the solution in the flow cell plus tubing was less than 10% "of the total volume of solution. The flow rate was adjusted so that the rate of enzyme reaction was independent of it. The sohation was stirred with a small magnetic stirrer, which kept the membranes or particles moving around in the vessel. The rates of reaction catalyzed by acetyHcholinesterase attached to nylon tube were measured by pumping thermostatically controlled substrate solution through one end of the enzyme tube, which was immersed in
TIME SCALE
F~;lci.1. Tracings of spectrophotsmetric progress curves for various systems (firm lines); the dashed lines are added to indicate the starting and stopping of the pumping. Arrows pointing down indicate commencement of pumping; arrows pointing up indicate cessation sf pumping. Flow rates. Vf, are in crnis. a temperature-controlled water bath, to the microvolume Wow cell; the absorbance was continuously recorded. Since there is continuous Row, at constant flow rate, with no mixing of substrate and product, the concentration of product is constant at a given flow rate.
Results and Discussion Traccs of the actual typical progress curves (change in absorbance V S . time) for soluble, particle-bound, membrane-bound, and tubebound enzyme systems are shown in Fig. 1. These recorder tracings show that the initial change in absorbance was directly proportional to the reaction time; the initial rates of the reaction can therefore be determined accurately from the initial slopes sf the recorder tracings. The tracings for enzyme-particles and enzymemembranes (Fig. Ib,c) show that when the pump was stopped, the absorbance remained unchanged. However, resumption of pumping Icd again to a linear increase in absorbance at the same rate as during the previous period. This is a convenient check for the presence of unbound enzyme in the reaction mixture; the presence of unbound enzyme would cause the absorbance to increase even when the pump is stopped. No such increase in absorbance was encountered in the
13
N 6 6 ET AL.: PARAMETERS FOR lMMOBILIZED ENZYMES
TABLE I . The Vn, and K , values for free and immobilized acetylcholinesterase State of enzyme
Assay method Static
Free enzyme
Flow
Enzyme trapped in polyacrylarnide particles
Flow
Enzyme trapped in polyacrylarnide membranes
Flow
Enzyme attached to inner surface of nylon tube
Flow
Can. J. Biochem. Downloaded from www.nrcresearchpress.com by San Francisco (UCSF) on 01/04/15 For personal use only.
Free enzyme
2.86 (mmolimin per 2.13 (mmol/min per 0.27 (mrnol,/min per 0.057 (mmol/min per 13.2 X 18-9 (mol/s)*
nag enzyme)
1.2
mg enzyme)
1 .I
mg enzyme)
2 "4
mg enzyme)
10.8 23.4*
-
*Data obtaincd with a 50-crn tube at a flow rate (Vf]of 8 cm/s.
present studies, indicating that the reactions are soleIy catalyzed by the immobilized enzyme. Since this method can distinguish between soluble and immobilized enzyme in a single sample, it can be used to follow the kinetics sf coupling sf enzyme to a support, as in the study of the kinetics of coupling of aldolase to CNBr-activated scpkarose, investigated by Mort et a&.(10). Figure Id shows the change in absorbance at 412 nrn plotted against the time that the reaction mixture flows through a nylon tube having enzyme attached to its inner surf ace; three different flow rates (V,) are used. AS expected, the absorbance increases with decreasing Wow rate, the substrate resicience time in the tube increasing as the Wow rate decreases. The maximal change in absorbance (OD,,,,) can be obtained by ( a ) recording the progress curve until no more change in absorbance is observed, or ( b ) plotting the reciprocal sf absorbance ( 1/ACID4 2 ) against the reciprocal of the reaction time, as shown in Fig. 2. The intercept on the 1/AOD412 axis gives f /Ok,,. The maximal change in absorbance can therefore be obtained consistently. The Lineweaver-Burk plots of free enzyme assayed in an ordinary static system and of free enzyme in a Wow system (Fig. 3a,b) give similar values of V , , and K,, thus verifying the validity of the flow technique. The kineweaver-Burk plots of particle-bound, membrane-bound, and tube-bound enzyme are shown in Fig. 3e,d,e. The various values of V , and K,,are listed in Table I. The results show that some diffusional effects are observed with the immobilized enzymes, the particle-bound enzyme being the least diffusion-controlled, as expected, with a K,,
30
20
L noD412
to
0 02
iI 0 4
i / a (s)
o 06
0 08
0.1
Frc. Z* Double reciprocal ,$lot of absorbance against time.
sf 2.4 X 1 8-4 M and a linear Lineweaver-Burk plot. More diffusional effects are observed with the membrane and tube systems, the K,'s being 10.8 X 1 0 - W and 23.4 X 10-%, respectively; such enhanced values are typical of processes involving substantial diffusion control ( 1, 7, 8). Furthermore, curvatures are seen in the Lineweaver-Burk plots for the membrane and tube systems; such curvatures are predicted by the theoretical treatment of Kobayashi and kaidler ( 4 ) when reactions are diffusion-controlled. A detailed study of the diffusional effects on the immobilized acetylcholinesterase system will be published elsewhere. It should be noted that when this technique is employed, care should be taken to ensure that product is not adsorbed on the surface of the Tygon tubing. The somewhat smaller V , for the flow system, as compared with the static
Can. J. Biochem. Downloaded from www.nrcresearchpress.com by San Francisco (UCSF) on 01/04/15 For personal use only.
CAN. J. BIOCHEM. VOL. 53. 8975
FREE ENZYME
G CP. b
6.0 4.0
RTICLE-BOUND 2.o
FIG. 3. Lineweaver-Burk plots for eke five systems. Units of l,'rate and l:'[S] are the same in all cases except for lj'rate in the case of enzyme attached to tubing. For all systems, [El, = 91 ,ug enzyme per mijliiiter of gel. For the membrane studies, the thickness was 430 pm and the diameter 1 cm.
7. Bunting, P. S. & Laidler, K. J. (1972) Biochemistry I 1, 4477-448 3 8. Bunting, P. S. & Laidler. K. J . (8974) Blorschnol. Bioeng. 16, 119-134 This work was supported by a grant from the 9. Goldman, W., Kedem, O . , Silman, I., Caplan, S. R.& National Research Council of Canada. The authors Katchalski. E. (1968) Biockernistry 7, 486-500 thank Dr. R. Kretz, Mr. M. Jackson, and Mr. T. P. Eim 10. Mort, 9. S., Chong, D. K. K. & Chan, W. C.-C. for assistance. (1973) Anal. Biockem. 52, 162-168 1. Laidler, K. J. & Sundaram, 1.".V. (1978) in Clzemhstry 11. Widmer, F., Dixon, 9. E. & Kaplan, N. 0. (1973) Anal. Biochem. 55, 282-287 of the Cell Ptlterface, part A, chapt. V. Academic 12. Silman, PI. 8 . & Karlin, '4. (1965) Prsc. Natl. Acad. Press Inc.. New York, N.Y. Sci. U.S. 58, 8664-1668 2. Goldman, R., Goldstein, L. & Katchalski. E. (1971) in Biochemical Aspects of Reactions on Solid Supports, 13. Davis, B. J . (1964) Atrrt. N.Y. Acud. Sci. 121, 404-427 14. Reisfeld, R. A., Lewis, U. J. 86 Williams, D. E. (1962) chapt. I , Academic Press Inc., New York. N.Y. Nature 193, 281-283 3. Katchalski, E., Sifiman, 1. 86 Goldman, W. (1971) 15. Sundaram, P. V. & Hornby, W. E. (1970) FEBS (Fed. Ah?. Enayrnob. 34, 445-536 Enr. Biochem. Soc,) Left. 10, 325-327 4. Kobayashi, T. & Laidler, #. J. (1974) Biotecht~ol. 16. Ngo, T. T. & Laidler, K. J. (1975) Biochim. Biophys. Bioeng. Id, 77-97 Acfa in press 5 . Gsldtnan, R., Kedem, 8.& KatchaIski, E. (1968) 17. Ellman, G. k., Courtnay, M. D., Andres, V., Jr. & Bioc/~pmistry7, 45 18-4532 Featherstone, R. M. (1961) Biochem. Pharmacol. 7, 6. ~ d h a n R., , Kedem. 8.& Katchalski, E. (1971) 88-95 BimkePnistrv 10, 165-8 72
system (Table I ) , may be due to some adsorption of the product.
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Received June 17, 1974 Ngo, T. T., Bunting, P. S. & Laidler, K. J. (1975) A Flow Method for Determination of the Kinetic Parameters for Immobilized Enzymes. Can. J. Bioclzern. 53, 11-14 A flow method is described for determination of the kinetic parameters (V, and K,,) for enzymes that are bound to particles, to membranes, and to the interior surfaces of tubes. Substrate solution is pumped through Tygon tubing to a microvolume flow cell and back into the reaction mixture, the flow rate being adjusted to be faster than the rate sf formation of product. To illustrate the technique, it is applied to the determination sf the parameters for electric-eel acetylcholinesterase attached to particles, to membranes, and to the inner surface of nylon tubing. Ngo, T. T., Bunting, P. S. & Laidler, K. J. (1975) A Flow Method for Determination of the Kinetic Parameters for Immobilized Enzymes. Can. J. Biochem. 53, 11-14 Une mCthode dlCcoulement est dCcrite pour la dttermination de paramkres cinCtiques (V, et K,) des enzymes likes aux particules, aux membranes et aux surfaces internes des tubes. La solution de substrat est pompke, B travers un tube de Tygon, vers une microcuve d'Ccoulement et ramenCe dans le milieu de rtaction, la vitesse d'kcoulement ktant ajustee B un niveau plus ClevC que celle de la formation du produit. Pour illustrer cette technique, nous l'appliquons a la dktermination des parametres de l'acktylcholinestkrase (anguille Clectrique) liCe aux particules, aux membranes et h la surface interne du tube de nylon. [Traduit par le journal]
Introduction Various theories of the kinetic behavior of immobilized enzymes have recently been formulated (1-4) and have been submitted to extensive experimental tests (5-9). There is need for an accurate and sensitive method of determining the kinetic parameters of immobilized enzyme systems, and two papers dealing with this problem have recently been published by Mort et al. (10) and Widmer et al. (1 1 ) . Mort et al. (18) have developed a continuous spectrophotometric assay for particle-bound enzyme using a spectrophotometer with a built-in magnetic stirrer. This method is convenient and sensitive but is not readily applicable to systems with enzyme immobilized on a sheet of membrane, such as papain immobilized on collodion membrane (9) and P-galactosidase trapped in polyacrylamide membrane ( 7 ) , because of the interference of these membranes with thc optical measurements. Also, it cannot be used to assay enzyme immobilized on the interior surfacer of tube. m e described by Widmer et a[. (11) requires the Illanufacture a apparatus and can-
'Present address: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A l .
not be used for enzyme attached to the interior surface of a tube. Recently, Bunting and Laidler (7) described a Row method to determine the kinetics sf Pgalactosidase immobilized in polyacrylamide. The present paper describes an extension of this method to the determination of the kinetic parameters of free soluble enzyme, particle-bound enzyme, membrane-bound enzyme, and enzyme immobilized on the interior surface of a tube. The technique involves pumping the substrate solution through Tygon tubing into a microvolume Aow cell and recirculating it into the original mixture in the case of particles and membranes; in the case of tubes, there is no recirculation. The enzyme used in the present studies to test the method is acetylcholinesterase from electric eel and the substrate is acetylthiocholine. Materials The enzyme, acetylcholinesterase from electric eel (type 111); was obtained from Sigma Chemical Co. The substrate acet~lthiocholine ( ASCh) and the chromogenic agent 5,S-d$hiobisnitrobenzoic acid (DTNB) were obtained from Sigma Chemical 450. N,N,N', N'-~etramethyleth~lenediahine (TEMED), N,N'-methylene bisacrylamide (BIS), and acrylamide were obtained from Eastman Organic Chemicals. The
82
CAN. J . BIOCHEM. VOL. 53, I975
Can. J. Biochem. Downloaded from www.nrcresearchpress.com by San Francisco (UCSF) on 01/04/15 For personal use only.
ammonium persulfate used in the polymerizations, and dipotassiunr hydrogen phosphate, were obtained from Fisher Scientific Co. Phthalic acid and boric acid were obtained from British Drug Houses, and sodium chloride from Canadian Laboratory Supplies Ltd.
All solutitdns used to assay the enzyme and to prepare the gels were made up in Silman and Karlin buffer ( 12), which consists of 0.15 i W NaC1, 2 mA4 phthalate, 2 mM phosphate, and 2 rnM borate, pH 7.0. Enzymecontaining pslyacrylamide gels were made by polymerization sf acrylamide-monomer solution with BIS and TEAf ED 4 13, 14). The enzyme-polyacrylasni$e menlbranes were prepared by slicing a frozen gel with a microtome ('71, and the membrane thicknesses were measured with a Zeiss optical microscope with a calibrated 12.5X magnification eyepiece and a 18X magnification objective. Enzyme-polyacrylamide particles were prepared by homogenizing 1 ml of gel in a homogenizer consisting of a smooth-walled glass tube fitted with an electrically driven Teflon pestle, clearance 150-230 pm, capacity 50 ml. The gel was homogenized by 10 up-and-down strokes of the pestle and the homogenate was centrifuged in a clinical centrifuge for 1Bbm1in. One Rundrecl microliters of the packed homogenized gel was then diluted % O O X with bu8er solution, and 0.5 ml of the diluted homogenate was used in each assay. AcetyBcholinesterase was chemically attached to the interior surface sf a nylon tube (0.1 crn internal diameter, obtained from John Tullis. Tullibody, Alloa. Scotland) by a method similar to that of Sundarana and Hsrmby (151, of which details will be published elsewhere ( 16). Kinetic. Procedra~e Free and immobilized acetylcholinesterase were assayed calorimetrically with acetylthiocholine and DTNB, as described by Ellman et al. ( 1 7 ) . Aborbance at 412 nm was followed in a Unisarn SP 1800 recording spectrophotometer. Reaction rates were = 1 3 000 ,%fI. calsula ted from e I 2' Kinetic measurements on the enzyme-particles and enzyme-membranes were carried out by suspending the particles or membranes in 10 ml of bulFFered sukstrate solution. By means of a Watson-Marlow flow inducer (model MHWE 200), the solution was pumped through Tygon tubing to a microvolume flow cell (Hellma Co.) to determine the abssrbamce, and back into the reaction mixture. A nylon net, 40 ym mesh (Pharmacia Fine Chemicals Co.), was attached $0the inlet of the Tygon tubing to prevent the partides or membrane frown being pumped into the flow cell. The volume of the solution in the flow cell plus tubing was less than 10% "of the total volume of solution. The flow rate was adjusted so that the rate of enzyme reaction was independent of it. The sohation was stirred with a small magnetic stirrer, which kept the membranes or particles moving around in the vessel. The rates of reaction catalyzed by acetyHcholinesterase attached to nylon tube were measured by pumping thermostatically controlled substrate solution through one end of the enzyme tube, which was immersed in
TIME SCALE
F~;lci.1. Tracings of spectrophotsmetric progress curves for various systems (firm lines); the dashed lines are added to indicate the starting and stopping of the pumping. Arrows pointing down indicate commencement of pumping; arrows pointing up indicate cessation sf pumping. Flow rates. Vf, are in crnis. a temperature-controlled water bath, to the microvolume Wow cell; the absorbance was continuously recorded. Since there is continuous Row, at constant flow rate, with no mixing of substrate and product, the concentration of product is constant at a given flow rate.
Results and Discussion Traccs of the actual typical progress curves (change in absorbance V S . time) for soluble, particle-bound, membrane-bound, and tubebound enzyme systems are shown in Fig. 1. These recorder tracings show that the initial change in absorbance was directly proportional to the reaction time; the initial rates of the reaction can therefore be determined accurately from the initial slopes sf the recorder tracings. The tracings for enzyme-particles and enzymemembranes (Fig. Ib,c) show that when the pump was stopped, the absorbance remained unchanged. However, resumption of pumping Icd again to a linear increase in absorbance at the same rate as during the previous period. This is a convenient check for the presence of unbound enzyme in the reaction mixture; the presence of unbound enzyme would cause the absorbance to increase even when the pump is stopped. No such increase in absorbance was encountered in the
13
N 6 6 ET AL.: PARAMETERS FOR lMMOBILIZED ENZYMES
TABLE I . The Vn, and K , values for free and immobilized acetylcholinesterase State of enzyme
Assay method Static
Free enzyme
Flow
Enzyme trapped in polyacrylarnide particles
Flow
Enzyme trapped in polyacrylarnide membranes
Flow
Enzyme attached to inner surface of nylon tube
Flow
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Free enzyme
2.86 (mmolimin per 2.13 (mmol/min per 0.27 (mrnol,/min per 0.057 (mmol/min per 13.2 X 18-9 (mol/s)*
nag enzyme)
1.2
mg enzyme)
1 .I
mg enzyme)
2 "4
mg enzyme)
10.8 23.4*
-
*Data obtaincd with a 50-crn tube at a flow rate (Vf]of 8 cm/s.
present studies, indicating that the reactions are soleIy catalyzed by the immobilized enzyme. Since this method can distinguish between soluble and immobilized enzyme in a single sample, it can be used to follow the kinetics sf coupling sf enzyme to a support, as in the study of the kinetics of coupling of aldolase to CNBr-activated scpkarose, investigated by Mort et a&.(10). Figure Id shows the change in absorbance at 412 nrn plotted against the time that the reaction mixture flows through a nylon tube having enzyme attached to its inner surf ace; three different flow rates (V,) are used. AS expected, the absorbance increases with decreasing Wow rate, the substrate resicience time in the tube increasing as the Wow rate decreases. The maximal change in absorbance (OD,,,,) can be obtained by ( a ) recording the progress curve until no more change in absorbance is observed, or ( b ) plotting the reciprocal sf absorbance ( 1/ACID4 2 ) against the reciprocal of the reaction time, as shown in Fig. 2. The intercept on the 1/AOD412 axis gives f /Ok,,. The maximal change in absorbance can therefore be obtained consistently. The Lineweaver-Burk plots of free enzyme assayed in an ordinary static system and of free enzyme in a Wow system (Fig. 3a,b) give similar values of V , , and K,, thus verifying the validity of the flow technique. The kineweaver-Burk plots of particle-bound, membrane-bound, and tube-bound enzyme are shown in Fig. 3e,d,e. The various values of V , and K,,are listed in Table I. The results show that some diffusional effects are observed with the immobilized enzymes, the particle-bound enzyme being the least diffusion-controlled, as expected, with a K,,
30
20
L noD412
to
0 02
iI 0 4
i / a (s)
o 06
0 08
0.1
Frc. Z* Double reciprocal ,$lot of absorbance against time.
sf 2.4 X 1 8-4 M and a linear Lineweaver-Burk plot. More diffusional effects are observed with the membrane and tube systems, the K,'s being 10.8 X 1 0 - W and 23.4 X 10-%, respectively; such enhanced values are typical of processes involving substantial diffusion control ( 1, 7, 8). Furthermore, curvatures are seen in the Lineweaver-Burk plots for the membrane and tube systems; such curvatures are predicted by the theoretical treatment of Kobayashi and kaidler ( 4 ) when reactions are diffusion-controlled. A detailed study of the diffusional effects on the immobilized acetylcholinesterase system will be published elsewhere. It should be noted that when this technique is employed, care should be taken to ensure that product is not adsorbed on the surface of the Tygon tubing. The somewhat smaller V , for the flow system, as compared with the static
Can. J. Biochem. Downloaded from www.nrcresearchpress.com by San Francisco (UCSF) on 01/04/15 For personal use only.
CAN. J. BIOCHEM. VOL. 53. 8975
FREE ENZYME
G CP. b
6.0 4.0
RTICLE-BOUND 2.o
FIG. 3. Lineweaver-Burk plots for eke five systems. Units of l,'rate and l:'[S] are the same in all cases except for lj'rate in the case of enzyme attached to tubing. For all systems, [El, = 91 ,ug enzyme per mijliiiter of gel. For the membrane studies, the thickness was 430 pm and the diameter 1 cm.
7. Bunting, P. S. & Laidler, K. J. (1972) Biochemistry I 1, 4477-448 3 8. Bunting, P. S. & Laidler. K. J . (8974) Blorschnol. Bioeng. 16, 119-134 This work was supported by a grant from the 9. Goldman, W., Kedem, O . , Silman, I., Caplan, S. R.& National Research Council of Canada. The authors Katchalski. E. (1968) Biockernistry 7, 486-500 thank Dr. R. Kretz, Mr. M. Jackson, and Mr. T. P. Eim 10. Mort, 9. S., Chong, D. K. K. & Chan, W. C.-C. for assistance. (1973) Anal. Biockem. 52, 162-168 1. Laidler, K. J. & Sundaram, 1.".V. (1978) in Clzemhstry 11. Widmer, F., Dixon, 9. E. & Kaplan, N. 0. (1973) Anal. Biochem. 55, 282-287 of the Cell Ptlterface, part A, chapt. V. Academic 12. Silman, PI. 8 . & Karlin, '4. (1965) Prsc. Natl. Acad. Press Inc.. New York, N.Y. Sci. U.S. 58, 8664-1668 2. Goldman, R., Goldstein, L. & Katchalski. E. (1971) in Biochemical Aspects of Reactions on Solid Supports, 13. Davis, B. J . (1964) Atrrt. N.Y. Acud. Sci. 121, 404-427 14. Reisfeld, R. A., Lewis, U. J. 86 Williams, D. E. (1962) chapt. I , Academic Press Inc., New York. N.Y. Nature 193, 281-283 3. Katchalski, E., Sifiman, 1. 86 Goldman, W. (1971) 15. Sundaram, P. V. & Hornby, W. E. (1970) FEBS (Fed. Ah?. Enayrnob. 34, 445-536 Enr. Biochem. Soc,) Left. 10, 325-327 4. Kobayashi, T. & Laidler, #. J. (1974) Biotecht~ol. 16. Ngo, T. T. & Laidler, K. J. (1975) Biochim. Biophys. Bioeng. Id, 77-97 Acfa in press 5 . Gsldtnan, R., Kedem, 8.& KatchaIski, E. (1968) 17. Ellman, G. k., Courtnay, M. D., Andres, V., Jr. & Bioc/~pmistry7, 45 18-4532 Featherstone, R. M. (1961) Biochem. Pharmacol. 7, 6. ~ d h a n R., , Kedem. 8.& Katchalski, E. (1971) 88-95 BimkePnistrv 10, 165-8 72
system (Table I ) , may be due to some adsorption of the product.
