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Photochemical coupling of enzymes to mammalian cells
Pharmaco/ogica/ Research Communications, Vol. 9, No. 2, 1977
131
PHOTOCHEMICAL COUPLING OF ENZYMES TO MAM>~LIAN CELLS P. Guire, D. Fliger, and J. Hodgson Midwest Research Institute, 425 Volker Boulevard, Kansas City, Missouri 64110 9eceived 3 June 1976
Summary:
A thermochemical-photochemical bifunctional reagent has been prepared
and used for the stepwise coupling of catalase to Chinese hamster ovary cells, to polystyrene beads, and to glucose oxldase.
N-(4-Azido-2-nitrophenyl)-4-
aminobutryl N'-oxysuccinimide was reacted in the dark with catalase to prepare a catalytically active enzyme derivative containing da'rk-stable photoreactive side chains.
This enzyme derivative was then photocoupled with visible light
to tissue culture cells to yield predominantly intact, viable cells "armed" with an average activity equivalent of ~- 106 enzyme molecules per cell. The photoactivated enzyme derivative containing highly reactive aryl nltrene side chains was coupled under similar reaction conditions to uniform spherical polystyrene latex beads and, simultaneously, to native glucose oxldase.
The covalent stabilization (i.e., "fixing") for subsequent study and utilization, of the functional molecular interactions or spatial distributions critical to the functioning of biological systems, has met with only limited success through the use of thermochemical crosslinking reagents.
The design
and synthesis of crosslinking reagents containing two or more groups subject
Abbreviations:-
-
w
_ _
--
- _
•
ANP-GAB-NoS." N~(4-Azido-2-nitrophenyl)-4-amlnobutryl N'-oxysuccinimlde; ANP-GAB-Catalase, azldonltrophenyI gamma amlnobutryl-substltuted catalase.
Pharmacological Research Commun ations, VoL
132
No. 2, 1977
to independent activation, allows the controlled stepwise coupling of one molecular target to the other.
A combined thermochemical-photochemical
blfunctlonal reagent provides this independence of activation of the two reactive groups. A useful range of reactivity and target specificity is available in the choice of the thermochemically reactive group.
The photochemically activat-
able group may be selected to allow activation by the chosen wavelength of light, to produce under exceptionally mild reaction conditions, an intermediate of very high reactivity and very broad or low level of target specificity.
With
this type of stepwise crosslinking reagent, one may expect to prepare by dark reaction(s) photoreactive, biochemically functional derivatives of one member of the interacting system under study.
This functional photoreactlve derivative
may then be activated in the presence of the other component(s) of the system, using activation conditions which place negligible stress on the functional interactions, thereby completing the covalent crossllnking process with those targets within the space, time, and target specificity domain of the photoactivated group (s). The following is a report of the use of such a stepwlse crossllnking system for the covalent coupling or immobilization of enzymes for potential enzyme replacement therapy and enzyme chemotherapy applications, among others. A wide range of applications is expec=ed to be recognized and developed for this unique and improved crossllnking capability.
Ha terlals andMethods Fluoro-2-nltro-4-azldobenzene was prepared from the corresponding aniline (Aldrich Chem. Co.) by the dlazotization-azlde substitution methodology
Pharmacological Research Communications, Vol. 9, No. 2, 1977 described by Fleet, Knowles, and Porter (1972).
133
This compound was reacted with
4-aminobutyric acid to substitute the relatively poorly reactive fluoride with an aminoalkylcarboxy group.
This carboxy group was then esterlfled, by carbo-
diimide activation, with N-hydroxysuccinimide to yield the N-oxysuccinimide carboxylic ester (m.p. I14-I15°C, C14H14N606:Calc.-46.41°/~ C, 3.90% H, 23.20% N; found--46.21% C, 3.92% H, 22.86% _N) with thermochemical reactivity (Cuatrecasas and Parikh, 1972) satisfactory for this study.
A more detailed description of the
synthesis of this ANP-GAB-NOS and related photothermochemical stepwise crosslinking reagents will be reported elsewhere. The Chinese hamster ovary cell line CHO-KI (Kao and Puck, 1967) was used as the model cell target for the photocoupling of ANP-GAB-Catalase to mature8lian cells. The microbeads used as targets for the ANP-GAB-Catalase photocoupllng were suspensions obtained from the Bioproducts Department of DOW Chemical Co.: Polystyrene Latex, Run No. LS-464-E, 1.305 Dm particle diameter; and Polyvinyltoluene Latex, Run No. 642-6, 2.68 Dm particle diameter. The catalase used was a crystalline suspension from beef liver (Sigma, Lot 34C-8050).
The glucose oxidase was provided in solution from
AsPergillu s niger by Niles Laboratories (Lot 37, Code 31-617). The thermochemical step or "dark" reaction for the preparation of photoreactive enzyme derivatives was carried out in dim daylight at 10°C for 60-90 minutes.
To approximately I00 mg catalase in 5.5 ml 0 . 0 1 M sodium
borate buffer, pH 9.0, was added with continuous stirring a 0.1 M solution of ANP-GAB-NOS in dimethyl formamide by continuous delivery from a syringe driven by an infusion pump.
The final organic solvent concentration in the reaction
Pharmacolog~al Research Cornrnun~ations, VoL 9, No. 2, 1977
134
mixture did not exceed I0%. The reaction mixture was dialyzed overnight against a flowing stream of sodium phosphate buffer (0.05 M, pH 7.0).
Portions of this
enzyme derivative were sterilized by passage through 0.45 Dm filters. The dialyzed photoreactive catalase preparation was photocoupled to cells, beads, and glucose oxidase by mixing with the target suspension in a Warburg apparatus and illuminating with a parallel beam tungsten halide projector lamp (i00 watt, 12 volt) operated at i0 volts.
The photoreaction was maintained
under nitrogen at 0°C with continuous reciprocal shaking for 90 minutes.
The
photocoupling to cells was done in Saline F (Puck, Cieciura, and Robinson, 1958) with sterilized container and solutions.
The beads and glucose oxldase were
placed in the sodium phosphate buffer containing the catalase. The photocoupled cells and beads were washed 6 times with the type solvent used in the photoreaction and assayed for enzyme activity.
Catalase
activity was measured by the rate of decrease in A240, after the method of Beers and Sizer (1952).
The glucose oxidase activity was determined manometrically
(Swoboda and Massey, 1965).
The plating efficiency of the washed cells and the
stability of the enzyme-cell couple were measured by incubating the cells under growth conditions (Ham's Nutrient Mixture FI2 supplemented with 2% dialyzed fetal calf serum - FI2FCD2, 37°C). methods of Ham and Puck (1962).
Plating effi¢iencies were measured by the
Coupling stability was measured by determining
the enzyme activity in the soluble and the cell-bound phases after various times of incubation under growth conditions. Results and Discussion The acylatlon of enzymes by dark reaction with N-oxysuccinlmlde esters at slightly alkaline pH is a relatively gentle chemical modification
Pharmacological Research Communications, Vol. 9, No. 2, 1977
135
process expected to yield good catalytic activity recovery for those enzymes which do not contain a reactive free amino group critical to the catalytic function (Cuatrecasas and Parikh, 1972).
Beef liver catalase substituted in
this manner with ANP- AB groups to the extent of 8 groups per enzyme subunit, retained a total catalytic activity in the dialyzed reaction mixture of 70-80% of the activity present at the initiation of the dark reaction (Table I).
The
enzyme subjected to the reaction and dialysis conditions without exposure to the reagent (ANP-GAB-NOS) yielded a slightly lower activity recovery.
Appreciable
variation was seen in the initial activities of the "native" enzyme dilutions prepared at different times, but the activity recovery from the dark reaction was consistently good.
Table I .
Rxn°
Dark Reaction: Photoreactive Catalase PreParation~
ANP- GAB-NOS Enzyme Subunit _(mole/mole)
1 2 3 4 5 6 Control
6 i0 15 20 20 20 0
_ ANP-Groups Enzyme Subunit _(mole/mole) 3.0 3.9 4.2 5. i 7.5 8.3 0.0
Enzyme Activity (l.U./mg) 23,700 26,750 21,200 19,700 27,250 36,300 33,000
Activity Recovery ~ (%! 92 104 82 77 70 79 61
The substitution of AN-P-groups onto the enzyme molecule is readily measured by the change in the enzyme's absorbance spectrum (Figure i).
The
values given in the 3rd column of Table I were calculated from the measured A480 values, using a molar absorptivity value of 4800 M-Icm -I (Fleet, Knowles, and Porter, 1972). The dialyzed AN'P-GAB-Catalase solution was stored in the dark at about
136
Pharmacological Research Communications, VoL 9, No. 2, 1977
SCALE0- 2.0 OFFSETI .0 o!
i. :~
ol
SCALE0-1.0 ABSORBANCE
8-
Figure I.
Absorbance spectrum of ANP-GAB-Catalase in 0.05 M phosphate, pff 7.0. The dialyzed reaction mixture of Rxn. I, Table I, was measured against the dialysis buffer.
Pharmacological Research Communications, Vol. 9, No. 2, 1977
137
4°C until used for the photocoupling experiments to cells, polystyrene beads, and glucose oxidase. The use of enzymes for the alleviation of inherited enzyme defect diseases (e.g., ceramide trihexosidases for Fabry's and related diseases) or for enzyme chemotherapy (e.g., asparaginase for some forms of leukemia) has been hindered by the propensity of the applied enzyme to be recognized as a foreign protein.
This results in its rapid clearance from the circulating
fluids and the production of antibodies against the applied enzyme.
The stable
attachment of the enzyme to the patient's own cells may prove to be a process useful for increasing significantly the functional lifetime of the applied enzyme in vivo, in controlling the location of the enzyme in vlvo, and in diminishing, delaying, or otherwise altering the antigenic response.
Reports
of the use of a class of thermochemical bifunctional reagents for this goal were published (then and Richardson, 1974) while this work was underway.
The step-
wise coupling of alpha amylase to human erythrocytes by means of the surface sulfhydryl groups leaves one with problems of the instability of the cell linkage and hindered applicability to sulfhydryl-contalning enzymes.
The
relative thermochemical and metabolic stability of the nitrophenylazide group (Knowles, 1972 and Fleet, Knowles, and Porter, 1972) and the propensity of the aryl nitrene generated from it by visible light, to form stable addition linkages to a wide variety of chemical target groups under very mild reaction conditions, recommends this photochemical reaction as the terminal step in the desired stepwise crossllnking of enzymes to cells and other materials. Table II contains data from four attempts to photocouple catalase derivatives to Chinese hamster ovary cells under conditions compatible with cell
Pharmacological Research Communications, VoL 9, No. 2, 1977
138 viability.
Each of the four reactions included a photoreactive enzyme deriva-
rive (Rxns. 5 and 6 of Table I) and a native enzyme control for the enzyme activity and cell viability measurements.
While these data do not provide a
high level of quantitative precision, the photoreactive catalase activity still sssociated with the cells after extensive washing is equivalent to approximately 1.5-4 x 106 soluble catalase subunits per cell.
The control or native catalase
activity remaining with the cells after similar illumination and washing conditions was approximately 20 to 25% the activity obtained with the similar but somewhat lower photoreactive enzyme concentrations in the illuminated enzyme-cell suspension.
_.~__~. I Control 2 Control 3 Control 4a 4b Control
Table II.
Photochemical Couplin~ of Catalase to Cells
[Enzyme] (m~/ml)
[Cell] (#/ml) (xl06)
Activity Recovery Washed Cells Rxn. Mix. (subunits/cell) ~7o) (xl06)
~5.5 "~3.3 5.9 8.2 7.2 i0 9.6 4.8 I0.0
~3.0 -~7.8 "~300•0 ~-I00.0 6.5 6.5 50.0 I00.0 80.0
86 79 -- i. 54 -~ O. 27 I. 59 . . 3.8 2.3 I. 0
.
Cell Viability Viable Stain > 70% Viable Stain > 70% --Plating -~90% . Plating --30% Plating --9% Plating --6%
The overall catalase activity recovery which can be expected from the two steps of the coupling process is indicated to be greater than 50%, since 70 to 80% activity recovery is obtained from the thermochemical reaction step (Table I) and at least 80% from the photochemical step (Reaction i of Table II). The fraction of the enzyme activity present before illumination which was
Pharmacological Research Cornmunications~ Vol. 9, No. 2, 1977 coupled to the washed cells was at most 0.18%.
139
Since Reaction 4b produced an
approximately 2.5-fold greater fraction of enzyme activity bound than did 4a, one can expect to improve the enzyme coupling activity yield by optimizing the enzyme and cell concentrations in the illumination suspension.
Other options
which may reasonably be expected to improve the enzyme activity coupling yield are also feasible with this coupling procedure and are under development in this laboratory. While the chemical nature of the bonds formed photochemically with the cells has not yet been investigated by us, quite satisfactory stability of these bonds under cell growth conditions was indicated by measurements of the time-dependence of the cell-bound enzyme activity during subsequent incubation of the washed cells.
While the growth medium used in Experiment 4 of Table II
and the subsequent stability testing of the enzyme-loaded washed cells, supported suboptimal growth rate and plating efficiency, the illuminated, washed cells from this experiment released no measurable catalase activity into solution during a 51-hour period of growth.
Approximately 50% of the original cell-
bound activity remained on the cells at the end of this growth period. Because of the high reactivity of the aryl nltrene photogenerated from the azidonitrophenyl group and the usefulness for phagocytosis studies of polystyrene microbeads, the coupling efficiency of the photoreactive catalase to these spheres was examined.
In addition, since catalase functions as a
stabilizer or "protector" for glucose oxidase activity, this latter unmodified enzyme was included in approximately 50Z weight concentration to the photoreactive catalase in some of the photocoupling mixtures with polystyrene beads. The data in Table III indicate a relatively high level of efficiency in the
III_.
CouplinR
43.0
28.6 28.6 28.6 74.0
9.7 70.0 40.0
-
-
-
11,400
865 8,430 28,500
6,200
40,800
308,000
2.5
0.15 1.8 6.3
0.14
1.3
9.7
590 < 300 1,950
< 300
< 300
57,300
Enzyme Activity.on Beads ,, Catalase . . . . . Glucose Oxldase (tetramer/bead) .(% yield) (.molecules/bead)
....
The beads used in Exp. 1 were 2.68 Pm diameter polyvlnyltoluene; those of Exp. 2 were 1.3 pm diameter polystyrene latex. Native glucose oxidase was present at weight concentrations approximately 50% the catalase in all the r e a c t i o n mixtures wlth I designated exception.
8.1
6.6 5.4 5.4
3.7
3.7
4.8
6,6
3.7
4.8
i
lo9>
Catalase Activity [Catalase] (per ml) Exn mixt (x = (% r e c o v e r y )
[Bead]
of PhotoKeactlve ' Ca talase to Polyaromatic Microbeads and to Glucose Oxidase
Catalase. without
Glucose Oxidase, Photolyzed
Photoreactive
Photolyzed
PhotoreacCive Catalase,
EXperiment 2* Native Catalase, Photolyzed Photoreactlve Catalase, Dark
Native Catalase, Photolyzed
Dark
EXperiment 1 Photoreactlve Catalase, Photolyzed Photoreactive Catalase,
Table
to ~4
0
Pharmacolo#ical Research Communications, Vol. 9, No. 2, 1977
141
photocoupling of catalase to aromatic beads and simultaneously, to glucose oxidase.
The presence of the glucose oxidase in the coupling reaction seems
to enhance the catalase activity yield on the polystyrene beads.
Further
measurements including protein analyses for the immobilized enzyme(s) and the effects of other proteins on the photoreactive catalase coupling yields, are expected to distinguish between alternative explanations for this observation. Research plans and efforts are underway for the demonstration of usefulness of this class of stepwise thermophotochemical crosslinking reagents and reaction processes in basic research, medical, and commercial applications.
Acknowled,~ement This work was supported in part by a grant from the National Science Foundation - RANN.
The authors acknowledge with appreciation the very valuable
contributions of Ray Blake and Dr. Eugene Podrebarac in the preparation of the bifunctional reagent used and of blary Breen in the cell culture aspects of this work.
Referenc.es Beers, R. F., Jr., and Sizer, I. W. (1952) J. Biol. Chem., 195, 133-140. Chen, Li-Fu and Richardson, T. (1974) Pharmacological Research Communications, 6, 273-279, 581-593. Cuatrecasas, P. and Parlkh, I. (1972) Biochemlstry, l!, 2291-2299. Fleet, G. W. J., Knowles, J. R., and Porter, R. R. (1972) Biochem. J.,
128, 499-508. Ham, R. G° and Puck, T. T. (1962) He,hods in Enzymology, 5, 90-119. Colowick and Kaplan, editors, Academic Press, New York. Kao, F. T. and Puck, T. T. (1967) Genetics, 55, 513-524. Knowles, J. R. (1972) Accounts of Chemical Research, 5, 155-160. Puck, T. T., Cleciura, J., and Robinson, A. (1958) J. Exp. Med., I08, 945-956. Svoboda, Bo E. P. and b~ssey, V. (1965) J. B£ol° Chem., 240, 2209-2215o
131
PHOTOCHEMICAL COUPLING OF ENZYMES TO MAM>~LIAN CELLS P. Guire, D. Fliger, and J. Hodgson Midwest Research Institute, 425 Volker Boulevard, Kansas City, Missouri 64110 9eceived 3 June 1976
Summary:
A thermochemical-photochemical bifunctional reagent has been prepared
and used for the stepwise coupling of catalase to Chinese hamster ovary cells, to polystyrene beads, and to glucose oxldase.
N-(4-Azido-2-nitrophenyl)-4-
aminobutryl N'-oxysuccinimide was reacted in the dark with catalase to prepare a catalytically active enzyme derivative containing da'rk-stable photoreactive side chains.
This enzyme derivative was then photocoupled with visible light
to tissue culture cells to yield predominantly intact, viable cells "armed" with an average activity equivalent of ~- 106 enzyme molecules per cell. The photoactivated enzyme derivative containing highly reactive aryl nltrene side chains was coupled under similar reaction conditions to uniform spherical polystyrene latex beads and, simultaneously, to native glucose oxldase.
The covalent stabilization (i.e., "fixing") for subsequent study and utilization, of the functional molecular interactions or spatial distributions critical to the functioning of biological systems, has met with only limited success through the use of thermochemical crosslinking reagents.
The design
and synthesis of crosslinking reagents containing two or more groups subject
Abbreviations:-
-
w
_ _
--
- _
•
ANP-GAB-NoS." N~(4-Azido-2-nitrophenyl)-4-amlnobutryl N'-oxysuccinimlde; ANP-GAB-Catalase, azldonltrophenyI gamma amlnobutryl-substltuted catalase.
Pharmacological Research Commun ations, VoL
132
No. 2, 1977
to independent activation, allows the controlled stepwise coupling of one molecular target to the other.
A combined thermochemical-photochemical
blfunctlonal reagent provides this independence of activation of the two reactive groups. A useful range of reactivity and target specificity is available in the choice of the thermochemically reactive group.
The photochemically activat-
able group may be selected to allow activation by the chosen wavelength of light, to produce under exceptionally mild reaction conditions, an intermediate of very high reactivity and very broad or low level of target specificity.
With
this type of stepwise crosslinking reagent, one may expect to prepare by dark reaction(s) photoreactive, biochemically functional derivatives of one member of the interacting system under study.
This functional photoreactlve derivative
may then be activated in the presence of the other component(s) of the system, using activation conditions which place negligible stress on the functional interactions, thereby completing the covalent crossllnking process with those targets within the space, time, and target specificity domain of the photoactivated group (s). The following is a report of the use of such a stepwlse crossllnking system for the covalent coupling or immobilization of enzymes for potential enzyme replacement therapy and enzyme chemotherapy applications, among others. A wide range of applications is expec=ed to be recognized and developed for this unique and improved crossllnking capability.
Ha terlals andMethods Fluoro-2-nltro-4-azldobenzene was prepared from the corresponding aniline (Aldrich Chem. Co.) by the dlazotization-azlde substitution methodology
Pharmacological Research Communications, Vol. 9, No. 2, 1977 described by Fleet, Knowles, and Porter (1972).
133
This compound was reacted with
4-aminobutyric acid to substitute the relatively poorly reactive fluoride with an aminoalkylcarboxy group.
This carboxy group was then esterlfled, by carbo-
diimide activation, with N-hydroxysuccinimide to yield the N-oxysuccinimide carboxylic ester (m.p. I14-I15°C, C14H14N606:Calc.-46.41°/~ C, 3.90% H, 23.20% N; found--46.21% C, 3.92% H, 22.86% _N) with thermochemical reactivity (Cuatrecasas and Parikh, 1972) satisfactory for this study.
A more detailed description of the
synthesis of this ANP-GAB-NOS and related photothermochemical stepwise crosslinking reagents will be reported elsewhere. The Chinese hamster ovary cell line CHO-KI (Kao and Puck, 1967) was used as the model cell target for the photocoupling of ANP-GAB-Catalase to mature8lian cells. The microbeads used as targets for the ANP-GAB-Catalase photocoupllng were suspensions obtained from the Bioproducts Department of DOW Chemical Co.: Polystyrene Latex, Run No. LS-464-E, 1.305 Dm particle diameter; and Polyvinyltoluene Latex, Run No. 642-6, 2.68 Dm particle diameter. The catalase used was a crystalline suspension from beef liver (Sigma, Lot 34C-8050).
The glucose oxidase was provided in solution from
AsPergillu s niger by Niles Laboratories (Lot 37, Code 31-617). The thermochemical step or "dark" reaction for the preparation of photoreactive enzyme derivatives was carried out in dim daylight at 10°C for 60-90 minutes.
To approximately I00 mg catalase in 5.5 ml 0 . 0 1 M sodium
borate buffer, pH 9.0, was added with continuous stirring a 0.1 M solution of ANP-GAB-NOS in dimethyl formamide by continuous delivery from a syringe driven by an infusion pump.
The final organic solvent concentration in the reaction
Pharmacolog~al Research Cornrnun~ations, VoL 9, No. 2, 1977
134
mixture did not exceed I0%. The reaction mixture was dialyzed overnight against a flowing stream of sodium phosphate buffer (0.05 M, pH 7.0).
Portions of this
enzyme derivative were sterilized by passage through 0.45 Dm filters. The dialyzed photoreactive catalase preparation was photocoupled to cells, beads, and glucose oxidase by mixing with the target suspension in a Warburg apparatus and illuminating with a parallel beam tungsten halide projector lamp (i00 watt, 12 volt) operated at i0 volts.
The photoreaction was maintained
under nitrogen at 0°C with continuous reciprocal shaking for 90 minutes.
The
photocoupling to cells was done in Saline F (Puck, Cieciura, and Robinson, 1958) with sterilized container and solutions.
The beads and glucose oxldase were
placed in the sodium phosphate buffer containing the catalase. The photocoupled cells and beads were washed 6 times with the type solvent used in the photoreaction and assayed for enzyme activity.
Catalase
activity was measured by the rate of decrease in A240, after the method of Beers and Sizer (1952).
The glucose oxidase activity was determined manometrically
(Swoboda and Massey, 1965).
The plating efficiency of the washed cells and the
stability of the enzyme-cell couple were measured by incubating the cells under growth conditions (Ham's Nutrient Mixture FI2 supplemented with 2% dialyzed fetal calf serum - FI2FCD2, 37°C). methods of Ham and Puck (1962).
Plating effi¢iencies were measured by the
Coupling stability was measured by determining
the enzyme activity in the soluble and the cell-bound phases after various times of incubation under growth conditions. Results and Discussion The acylatlon of enzymes by dark reaction with N-oxysuccinlmlde esters at slightly alkaline pH is a relatively gentle chemical modification
Pharmacological Research Communications, Vol. 9, No. 2, 1977
135
process expected to yield good catalytic activity recovery for those enzymes which do not contain a reactive free amino group critical to the catalytic function (Cuatrecasas and Parikh, 1972).
Beef liver catalase substituted in
this manner with ANP- AB groups to the extent of 8 groups per enzyme subunit, retained a total catalytic activity in the dialyzed reaction mixture of 70-80% of the activity present at the initiation of the dark reaction (Table I).
The
enzyme subjected to the reaction and dialysis conditions without exposure to the reagent (ANP-GAB-NOS) yielded a slightly lower activity recovery.
Appreciable
variation was seen in the initial activities of the "native" enzyme dilutions prepared at different times, but the activity recovery from the dark reaction was consistently good.
Table I .
Rxn°
Dark Reaction: Photoreactive Catalase PreParation~
ANP- GAB-NOS Enzyme Subunit _(mole/mole)
1 2 3 4 5 6 Control
6 i0 15 20 20 20 0
_ ANP-Groups Enzyme Subunit _(mole/mole) 3.0 3.9 4.2 5. i 7.5 8.3 0.0
Enzyme Activity (l.U./mg) 23,700 26,750 21,200 19,700 27,250 36,300 33,000
Activity Recovery ~ (%! 92 104 82 77 70 79 61
The substitution of AN-P-groups onto the enzyme molecule is readily measured by the change in the enzyme's absorbance spectrum (Figure i).
The
values given in the 3rd column of Table I were calculated from the measured A480 values, using a molar absorptivity value of 4800 M-Icm -I (Fleet, Knowles, and Porter, 1972). The dialyzed AN'P-GAB-Catalase solution was stored in the dark at about
136
Pharmacological Research Communications, VoL 9, No. 2, 1977
SCALE0- 2.0 OFFSETI .0 o!
i. :~
ol
SCALE0-1.0 ABSORBANCE
8-
Figure I.
Absorbance spectrum of ANP-GAB-Catalase in 0.05 M phosphate, pff 7.0. The dialyzed reaction mixture of Rxn. I, Table I, was measured against the dialysis buffer.
Pharmacological Research Communications, Vol. 9, No. 2, 1977
137
4°C until used for the photocoupling experiments to cells, polystyrene beads, and glucose oxidase. The use of enzymes for the alleviation of inherited enzyme defect diseases (e.g., ceramide trihexosidases for Fabry's and related diseases) or for enzyme chemotherapy (e.g., asparaginase for some forms of leukemia) has been hindered by the propensity of the applied enzyme to be recognized as a foreign protein.
This results in its rapid clearance from the circulating
fluids and the production of antibodies against the applied enzyme.
The stable
attachment of the enzyme to the patient's own cells may prove to be a process useful for increasing significantly the functional lifetime of the applied enzyme in vivo, in controlling the location of the enzyme in vlvo, and in diminishing, delaying, or otherwise altering the antigenic response.
Reports
of the use of a class of thermochemical bifunctional reagents for this goal were published (then and Richardson, 1974) while this work was underway.
The step-
wise coupling of alpha amylase to human erythrocytes by means of the surface sulfhydryl groups leaves one with problems of the instability of the cell linkage and hindered applicability to sulfhydryl-contalning enzymes.
The
relative thermochemical and metabolic stability of the nitrophenylazide group (Knowles, 1972 and Fleet, Knowles, and Porter, 1972) and the propensity of the aryl nitrene generated from it by visible light, to form stable addition linkages to a wide variety of chemical target groups under very mild reaction conditions, recommends this photochemical reaction as the terminal step in the desired stepwise crossllnking of enzymes to cells and other materials. Table II contains data from four attempts to photocouple catalase derivatives to Chinese hamster ovary cells under conditions compatible with cell
Pharmacological Research Communications, VoL 9, No. 2, 1977
138 viability.
Each of the four reactions included a photoreactive enzyme deriva-
rive (Rxns. 5 and 6 of Table I) and a native enzyme control for the enzyme activity and cell viability measurements.
While these data do not provide a
high level of quantitative precision, the photoreactive catalase activity still sssociated with the cells after extensive washing is equivalent to approximately 1.5-4 x 106 soluble catalase subunits per cell.
The control or native catalase
activity remaining with the cells after similar illumination and washing conditions was approximately 20 to 25% the activity obtained with the similar but somewhat lower photoreactive enzyme concentrations in the illuminated enzyme-cell suspension.
_.~__~. I Control 2 Control 3 Control 4a 4b Control
Table II.
Photochemical Couplin~ of Catalase to Cells
[Enzyme] (m~/ml)
[Cell] (#/ml) (xl06)
Activity Recovery Washed Cells Rxn. Mix. (subunits/cell) ~7o) (xl06)
~5.5 "~3.3 5.9 8.2 7.2 i0 9.6 4.8 I0.0
~3.0 -~7.8 "~300•0 ~-I00.0 6.5 6.5 50.0 I00.0 80.0
86 79 -- i. 54 -~ O. 27 I. 59 . . 3.8 2.3 I. 0
.
Cell Viability Viable Stain > 70% Viable Stain > 70% --Plating -~90% . Plating --30% Plating --9% Plating --6%
The overall catalase activity recovery which can be expected from the two steps of the coupling process is indicated to be greater than 50%, since 70 to 80% activity recovery is obtained from the thermochemical reaction step (Table I) and at least 80% from the photochemical step (Reaction i of Table II). The fraction of the enzyme activity present before illumination which was
Pharmacological Research Cornmunications~ Vol. 9, No. 2, 1977 coupled to the washed cells was at most 0.18%.
139
Since Reaction 4b produced an
approximately 2.5-fold greater fraction of enzyme activity bound than did 4a, one can expect to improve the enzyme coupling activity yield by optimizing the enzyme and cell concentrations in the illumination suspension.
Other options
which may reasonably be expected to improve the enzyme activity coupling yield are also feasible with this coupling procedure and are under development in this laboratory. While the chemical nature of the bonds formed photochemically with the cells has not yet been investigated by us, quite satisfactory stability of these bonds under cell growth conditions was indicated by measurements of the time-dependence of the cell-bound enzyme activity during subsequent incubation of the washed cells.
While the growth medium used in Experiment 4 of Table II
and the subsequent stability testing of the enzyme-loaded washed cells, supported suboptimal growth rate and plating efficiency, the illuminated, washed cells from this experiment released no measurable catalase activity into solution during a 51-hour period of growth.
Approximately 50% of the original cell-
bound activity remained on the cells at the end of this growth period. Because of the high reactivity of the aryl nltrene photogenerated from the azidonitrophenyl group and the usefulness for phagocytosis studies of polystyrene microbeads, the coupling efficiency of the photoreactive catalase to these spheres was examined.
In addition, since catalase functions as a
stabilizer or "protector" for glucose oxidase activity, this latter unmodified enzyme was included in approximately 50Z weight concentration to the photoreactive catalase in some of the photocoupling mixtures with polystyrene beads. The data in Table III indicate a relatively high level of efficiency in the
III_.
CouplinR
43.0
28.6 28.6 28.6 74.0
9.7 70.0 40.0
-
-
-
11,400
865 8,430 28,500
6,200
40,800
308,000
2.5
0.15 1.8 6.3
0.14
1.3
9.7
590 < 300 1,950
< 300
< 300
57,300
Enzyme Activity.on Beads ,, Catalase . . . . . Glucose Oxldase (tetramer/bead) .(% yield) (.molecules/bead)
....
The beads used in Exp. 1 were 2.68 Pm diameter polyvlnyltoluene; those of Exp. 2 were 1.3 pm diameter polystyrene latex. Native glucose oxidase was present at weight concentrations approximately 50% the catalase in all the r e a c t i o n mixtures wlth I designated exception.
8.1
6.6 5.4 5.4
3.7
3.7
4.8
6,6
3.7
4.8
i
lo9>
Catalase Activity [Catalase] (per ml) Exn mixt (x = (% r e c o v e r y )
[Bead]
of PhotoKeactlve ' Ca talase to Polyaromatic Microbeads and to Glucose Oxidase
Catalase. without
Glucose Oxidase, Photolyzed
Photoreactive
Photolyzed
PhotoreacCive Catalase,
EXperiment 2* Native Catalase, Photolyzed Photoreactlve Catalase, Dark
Native Catalase, Photolyzed
Dark
EXperiment 1 Photoreactlve Catalase, Photolyzed Photoreactive Catalase,
Table
to ~4
0
Pharmacolo#ical Research Communications, Vol. 9, No. 2, 1977
141
photocoupling of catalase to aromatic beads and simultaneously, to glucose oxidase.
The presence of the glucose oxidase in the coupling reaction seems
to enhance the catalase activity yield on the polystyrene beads.
Further
measurements including protein analyses for the immobilized enzyme(s) and the effects of other proteins on the photoreactive catalase coupling yields, are expected to distinguish between alternative explanations for this observation. Research plans and efforts are underway for the demonstration of usefulness of this class of stepwise thermophotochemical crosslinking reagents and reaction processes in basic research, medical, and commercial applications.
Acknowled,~ement This work was supported in part by a grant from the National Science Foundation - RANN.
The authors acknowledge with appreciation the very valuable
contributions of Ray Blake and Dr. Eugene Podrebarac in the preparation of the bifunctional reagent used and of blary Breen in the cell culture aspects of this work.
Referenc.es Beers, R. F., Jr., and Sizer, I. W. (1952) J. Biol. Chem., 195, 133-140. Chen, Li-Fu and Richardson, T. (1974) Pharmacological Research Communications, 6, 273-279, 581-593. Cuatrecasas, P. and Parlkh, I. (1972) Biochemlstry, l!, 2291-2299. Fleet, G. W. J., Knowles, J. R., and Porter, R. R. (1972) Biochem. J.,
128, 499-508. Ham, R. G° and Puck, T. T. (1962) He,hods in Enzymology, 5, 90-119. Colowick and Kaplan, editors, Academic Press, New York. Kao, F. T. and Puck, T. T. (1967) Genetics, 55, 513-524. Knowles, J. R. (1972) Accounts of Chemical Research, 5, 155-160. Puck, T. T., Cleciura, J., and Robinson, A. (1958) J. Exp. Med., I08, 945-956. Svoboda, Bo E. P. and b~ssey, V. (1965) J. B£ol° Chem., 240, 2209-2215o