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A possible role for glass bead immobilized enzymes as therapeutic agents (immobilized uricase as enzyme therapy for hyperuricemia)
BIOCHEMICAL
MEDICINE
i2, 79-91 (1975)
A Possible Enzymes
Role
for Glass
as Therapeutic as Enzyme
J. CRAIG VENTER,
Agents
Therapy
for
of Medicine,
Immobilized
(Immobilized
Uricase
Hyperuricemia)
BARBARA R. VENTER, JACK E. DIXON,” ANDNATHAN
Departments
Bead
O.KAPLAN
Biology, and Chemistry, La Jolla, California
University 92037
oj. California,
San Diego.
Received September 25, I974
INTRODUCTION
Glass bead immobilized enzymes have been applied in this laboratory to study enzyme kinetics (1) and to study enzymatic hydrogen production (2). The technique of microencapsulation as developed by Chang and co-workers (3-6) has also been used with success with many enzymes and its potential for use in enzyme therapy established, although with this technique of enzyme insolubilization, enzyme stability is a significant problem (6). The stability of enzymes immobilized on glass beads, however, is tremendously enhanced to the effects of temperature, pH and antibody attack (1,7), and immobilized enzyme columns have been used for extended periods without significant loss of activity (8). We will discuss glass bead immobilized uricase as a possible therapeutic agent for chronic gout with renal insufficiency and immobilized glucose oxidase as a model enzyme system for the extension of studies to the development of semipermanent or permanent implantable shunt systems that will allow for continuous enzyme therapy. Renal failure is the eventual cause of death in 20-250/o of patients with gout. Recent evidence suggests that the incidence of gout is increasing. This is presumably due to the high purine content of modern diets. High urate levels are also produced, for example, as side effects of hemodialysis and of cortisone treatment for acute leukemia (9). The first reported use of uricase to decrease serum urate levels in animals was in 1949 ( 10). Uricase was first tested clinically in humans in 1957 (1 l), and again in 1968 (12) and as recently as 1972 ( 13); nevertheI This work was supported in part by grants from the National Institutes of Health (USPHS CA 1 I683-05), the American Cancer Society (BC-60-P). and Hoffman La Roche Inc. 2 Present Address: Department of Biochemistry, Purdue University, Lafayette, Indiana. 79 (-opyright All right,
Q 1975 by Academic Press, Inc. Printed ol reproduction in any form reserved.
in the United
States.
80
VENTER
ET
AL.
less, in all cases the treatment was eventually limited by antibody formation (1 O-l 3). However, even with this limitation, the effectiveness of uricase in lowering serum urate levels and the consequent reversal of tophi and other gout symptoms was of sufficient proportions for Brogard et al. (13) to recommend utilization of uricase as a possible treatment for acute hyperuricemia, hyperuricemia with uric lithiosis and hyperuricemia with renal insufficiency. Extracorporeal shunt systems are widely used and well characterized for hemodialysis and have been used with microencapsulated enzymes ( 14). Thus we propose such a system could be utilized in conjunction with glass bead immobilized enzymes for applying enzyme therapy to hyperuricemia and as a model system for further study in \vivo of immobilized enzyme systems. MATERIALS Enzyme
Immobilization
AND
and Activity
METHODS
Measurements
Glucose oxidase (EC 1.1.3.4 @D-glucose oxygen oxidoreductase) was coupled to activated aryl amine glass as described in (15) and as follows: One gram of aryl amine glass (16) was added to 10 ml of 2 N HCl and cooled in an ice salt bath to 0°C; 250 mg sodium nitrite were added and the reaction placed under vacuum for 20 min at 0°C. The activated diazonium glass beads were then washed with 80 ml ice cold 1% sulfamic acid followed immediately with 200 ml ice cold distilled water utilizing a sintered glass funnel. The glass beads were then rapidly added to 50 mg of the enzyme in 10 ml 0.1 M phosphate buffer pH 7.4 at 4°C. The reaction was continued for 1 hr. The orange-red glass beads were then filtered and washed slowly with 1 liter of phosphate buffer. The glucose oxidase beads were then allowed to stand overnight in either 1% albumin or 0.1 M pyrophosphate solution to remove noncovalently attached enzyme. Bacterial uricase (urate : oxygen oxidoreductase, EC I .7.3.3) (Novo Enzymes) was used without further purification for direct coupling to alkyl and aryl amine glass beads (16) as follows. Gluteraldehyde coupling. To each gram of alkyl amine glass were added 5 ml of a 5% solution of gluteraldehyde in 0.1 M phosphate buffer, pH 7.5. The mixture was placed under vacuum in a dessicator for 60 min at room temperature. The orange gluteraldehyde glass was then washed twice with 100 ml of ice cold phosphate buffer, 0.1 M, pH 7.5 and immediately added to 20 IU of uricase dissolved in 10 ml of a saturated solution of uric acid in ice cold 0.1 M phosphate buffer, pH 7.5. The reaction was continued overnight at 4°C. Uricase and catalase (EC
IMMOBILIZED
ENZYME
THERAPY
81
1.11.1.6 hydrogen peroxide : hydrogen peroxide oxidoreductase) were coupled together to the aryl amine glass by the gluteraldehyde procedure as above, catalase (20 IU/ml) being added to the uricase/uric acid solution prior to the gluteraldehyde glass addition. Diazo coupling. One gram of aryl amine glass was added to IO ml of 2 N HCl and cooled to 0°C: 250 mg of sodium nitrite were added and the reaction placed under vacuum for 20 min at 0°C. The activated diazonium glass beads were then washed with 80 ml ice cold 1% sulfamic acid followed immediately with 200 ml ice cold distilled water, then rapidly added to 20 IU uricase in 10 ml phosphate buffer, pH 7.5, saturated with uric acid and the reaction continued overnight at 4°C. The uricase-glass and the uricase-catalase-glass beads were washed slowly with 2 liters of ice cold phosphate buffer and placed in a 1% albumin solution overnight at 4°C. They were then washed slowly with 2 liters of ice cold phosphate buffer. Enzyme activities were measured on a Perkin-Elmer Model 46 spectrophotometer equipped with an immobilized enzyme reactor system (17). Uricase reactions were run in glycine buffer, 0.1 M, pH 9.4, and the oxidation of uric acid to allantoin followed as a decrease in OD at 292 nm. For storage, uricase-glass beads and glucose oxidase beads were kept at 4°C in 0.1 M glycine buffer, pH 9.4, and phosphate buffer, 0.1 M, pH 7.4, respectively. In vitro blood assays. Canine blood (4 liters) was obtained fresh from dogs anesthetized with 50 mg/kg pentobarbitol and heparinized with sodium heparin. The blood was added to a glass reservoir in a 37°C water bath. The blood was circulated through Tygon tubing into the immobilized enzyme reactor (a cardiotomy reservoir (Model Ql20, Bentley Laboratories, Inc.)) then back to the starting pool with the aid of a peristaltic pump. Ninety-five percent 02, 5% CO, was continuously bubbled into the inner chamber of the cardiotomy reservoir. Samples for substrate analysis were taken from the blood pool. A typical experiment to study the effectiveness of immobilized glucose oxidase or uricase involved circulating the blood for at least 2 hr through the entire system prior to addition of the enzyme-glass beads to the cardiotomy reservoir. An accurate control level of glucose, uric acid or allantoin was thereby established. One to 5 g of enzyme-glass beads were added to the inner chamber of the cardiotomy reservoir and the concentration of the appropriate substrate followed with time. Experiments on human blood were modified from those described for the canine blood because a smaller starting volume was utilized. Two pints of fresh blood (obtained from the San Diego Blood Bank) were warmed to 37°C and pumped over a small column of uricase-glass beads. For
82
VENTER
ET
AL.
glucose oxidase experiments, glucose was added to the blood to bring the initial concentration to 400 mg%. For uricase experiments uric acid was added when necessary to bring initial blood levels to 5 mg%. In vivo blood assays. Conditioned dogs were anesthetized with 30 mg/kg pentobarbitol and subjected to artificial respiration. The dogs were heparinized with 10,000 units heparin. The right carotid artery was canulated and connected to a transducer for the continual monitoring of blood pressure. ECG was also continuously monitored. The right femoral vein and artery were canulated to establish an extracorporeal shunt. The femoral artery catheter was connected directly to the cardiotomy reservoir which had been primed with 1 liter of Ringers lactate with 1% albumin. A peristaltic pump was used between the reservoir and the femoral vein. When the lines were opened to blood flow, the pump was set at a speed to match arterial blood flow in order to maintain the arterial pressure and the blood level in the reservoir constant. Volume in the cardiotomy reservoir was 900 ml. Flow rates of 500 to 1,000 ml per minute were used. Blood samples for substrate analysis were taken from the femoral artery proximal to the cardiotomy reservoir. In experiments where urine samples were desired, a Folly catheter was inserted and urine flow rates monitored for each sample. As in the in vitro systems, the extracorporeal shunts were run for l-2 hr prior to the addition of the enzyme glass beads to the system in order to obtain adequate base lines for the substrate to be monitored. Blood glucose levels were measured using glucose Stat Packs (Calbiochem). Blood and urine uric acid were measured by the method of Liddle et al. (18) and blood and urine allantoin by the method of (19). Antibody preparation. A 2 kg New Zealand white rabbit was immunized with intradermal uricase (20 IU) in Freunds complete adjuvant (Dilco). Secondary immunization was carried out after 6 wk with 20 IU uricase in incomplete adjuvant (Dilco). Ten days prior to serum collection, 7.5 IU uricase were given intravenously. The collected rabbit serum was treated with 50% saturated ammonium sulfate overnight at 4”C, centrifuged at 5000 g for 15 min and the supernatant discarded. The pellet was resuspended and then dialyzed against 0.8% sodium chloride for 48 hr with two volume changes. Control serum from an unimmunized rabbit was prepared in an identical manner. RESULTS
Yields and Stability of the Immobilized Uricase When uricase is covalently coupled to porous glass beads by the diazo and gluteraldehyde linkages described (Fig. I) and stored at 4°C in glycine buffer, 0.1 M, pH 9.4, the enzyme is stable for several months.
IMMOBILIZED
ENZYME DIAZO
83
THERAPY
COUPLING
Si- CHz-CH2-CH2-Ntl-EeNH2
-
(~0~0~ + HCI) + Enzyme
glass
Si-CH,-Cn,-,‘,,-N~-e~N=~
-Enzyme
GLUTERALDEHYDE
glass
COUPLING
Si-CH,-CH,-CH,-NH, + CHO WI,), + Enzyme
3
CHO
I I-CH,-CH,-CH,-
ms
N = CH - (CH,),-
CH=N - Enzyme
J I. Diazo and gluteraldehyde enzyme coupling to aryl amine and alkyl amine glass beads. Diazo coupling is presumably to aromatic residues such as tyrosine: gluteraldehyde coupling probably involves lysine residues (I). FIG.
The differences in yields and activities of the immobilized enzymes prepared by the various methods outlined are summarized in Table 1. Due to enzyme inactivation during the coupling process, only IO- 12% of the initial uricase activity in solution was recovered on the glass beads. The uricase coupled to the glass via an azo linkage appears to be the most stable preparation, maintaining 90% of the initial activity over a 50 day period (Fig. 2). The gluteraldehyde coupling yielded a higher TABLE STABILITY
Uricase immobilization procedure Diazo linkage Gluteraldehyde Combined uricase + catalase -both linked through gluteraldehyde
1
OF URICASE-GLASS
BEADS”
Initial activity Activityafter5Odays (I units/g glass) (I units/g glass)
% Activity after 4 hr in blood EL 37°C
2.0 2.1
1.8 1.7
>95%
2.4
1.5
<75%
82%
n The activity and stability of uricase immobilized on glass beads via the indicated linkages: “Initial activity” was assessed immediately following coupling and washing procedures listed in Methods: “Activity after 50 days” was measured following 50 days storage at 4°C in 0.1 M glycine buffer, pH 9.4; “Percent activity in blood” was measured following four hours of incubation in canine blood at 37°C.
84
VENTER
ET
AL.
Time (days) FIG. 2. Glass bead immobilized uricase stability at 4°C in glycine buffer, 0.1 M, pH 9.4: Diazo coupled uricase (closed circles); gluteraldehyde coupled uricase (open triangles); gluteraldehyde coupled uricase + catalase (open circles). One hundred milligrams (dry weight) of the appropriate immobilized uricase-glass beads were assayed at the indicated times utilizing an immobilized enzyme reactor (17). Enzyme activities are given as the percentage of initial uricase activity obtained following albumin stripping and washing procedures as described in Methods.
initial specific activity but was less stable than the diazotized uricase preparation. When uricase and catalase were coupled to the same glass beads, the initial uricase activity was 12.5% greater than the gluteraldehyde uricase alone, however, this preparation was found to be the least stable with only 68% of the starting activity remaining after 50 days. To assess the immobilized enzymes’ stability under in vivo conditions. each type of uricase-glass complex was incubated in oxygenated human or canine blood at 37°C in a continuously flowing system. It was found that greater than 95% of the diazo-uricase-glass enzymatic activity was recoverable, whereas for the uricase-gluteraldehyde-glass, only 82% of the initial activity was recoverable following four hours of incubation. The glucose oxidase-gluteraldehyde-glass was stable under these conditions with more than 95% of the starting enzymatic activity recoverable after four hours of incubation. Immobilized-Glucose
Oxidase (In Vitro and In Vivo Assays)
To assess the ability of the glass bead immobilized enzymes to remove substrates from blood, 1 g of the glucose oxidase-glass beads, representing 50 IU of glucose oxidase activity, was added to the inner chamber of the cardiotomy reservoir and 4 liters of canine blood initially containing 400 mg% glucose were pumped continuously through the chamber for three hours. This resulted in the removal of 100% of the glucose, as shown in Fig. 3. This experiment was repeated three times with the same gram of glucose oxidase-glass beads. Each experiment
IMMOBILIZED
ENZYME
8.5
THERAPY
FIG. 3. Four liters of canine blood with 400 mg% blood glucose were continuously circulated through a cardiotomy reservoir at 37°C for three hours. The inner chamber of the cardiotomy reservoir contained 1 g of glucose oxidase glass beads, representing 50 IU of glucose oxidase. Blood samples were removed at the indicated times and assayed for glucose levels (Methods). These data represent the oxidation of 16 g of glucose in the three hour period.
represents the oxidation of 16 g of glucose in a three hour period, When the glucose oxidase glass beads were recovered after the three experiments, more than 95% of the activity on the glass remained. The glucose oxidase-glass beads were then tested in vivo for their ability to oxidize blood glucose. One gram of glucose oxidase glass beads was added to a cardiotomy reservoir in an extracorporeal shunt system established on an anesthetized dog. As Fig. 4 shows, there was a significant and rapid drop in the blood glucose level, resulting in the death of
I
IO
4
20 40 50 Tirne3?min~ FIG. 4. Glass bead immobilized glucose oxidase lowering of blood glucose levels in an anesthetized dog. Glucose-oxidase-glass beads were added to the inner chamber of a cardiotomy reservoir on line in an extracorporeal shunt at time 0. Blood glucose samples were withdrawn from the femoral artery proximal to the cardiotomy reservoir.
0
86
VENTER
the dog within 40 minutes. almost full activity. Immobilized
Uricase-
ET AL.
Again the recovered glass bead enzyme had
In Vitro and In Vivo Assays
Due to the low uric acid levels and the presence of allantoin in canine blood, human blood was chosen to test the effectiveness of the uricaseglass beads in lowering serum urate levels. One liter of fresh whole human blood was continuously circulated over a column of 2 g of uricase-diazo-glass beads at 37°C. Samples were removed throughout the four hours of incubation and assayed for uric acid and allantoin levels. As Fig. 5 shows, the serum urate was stoichiometrically converted to allantoin, the conversion rate being 5 mg urate/hr/liter of blood/g of uricase glass beads. This represents the oxidation of 40 mg urate in the four hours of incubation. Full uricase activity was recovered on the glass beads from these experiments. The glass bead immobilized uricase was tested on a Dalmatian dog for its ability to lower serum urate levels under in vivo conditions. The Dalmatian was placed on a high meat diet and probenecid (500 mg three times a day) for one week prior to the experiment. This combination resulted in an increase in the urate level from l/2 mg% to 2.4 mg% and it was maintained at this level. An extracorporeal shunt was established as previously described. Following the addition of 6 g of uricase-glass beads to the extracorporeal shunt reaction chamber, there was a substantial decrease in both serum and urinary uric acid levels and a corresponding increase in the urinary allantoin levels. These results are summarized in Fig. 6. The postoperative period was unremarkable with no
FIG. 5. Stoichiometric conversion of blood uric acid (open circles) to blood allantoin (closed circles) catalyzed by uricase-glass beads. One liter of fresh whole human blood at 37°C was continuously pumped over a small column of uricase-diazo-glass beads (2 g). Samples were withdrawn at the indicated times and assayed for uric acid ( 18) and allantoin (19).
IMMOBILIZED
ENZYME
THERAPY
87
me glass beads added extracorporeal shunt
123456 Time (hrs)
FIG. 6. Alterations in serum and urine acid and urine allantoin levels catalyzed by uricase-glass beads in an extracorporeal shunt on an anesthetized dog (Fig. 4). Blood was allowed to circulate through the extracorporeal shunt for two hours prior to the addition of uricase-glass beads. Uricase-glass beads (6 g) were added to the inner chamber of a cardiotomy reservoir in the extracorporeal shunt at the time indicated. Blood and urine uric acid (18) and urine allantoin (19) levels were determined at the indicated times.
detectable ill effects resulting from the glass bead enzyme therapy. Preand postoperative blood chemistries were normal. The experiment was repeated on a second dog under acute conditions. Again the immobilized uricase was effective in lowering the urate levels and increasing the concentration of allantoin. As in all previous experiments, there was little detectable loss of immobilized uricase activity. Immunological Considerations Ouchterlonies were utilized to demonstrate the formation of antibodies to soluble uricase, as shown in Fig. 7. In addition. when rabbit antiuricase was preincubated with native uricase for 30 minutes a marked inhibition of the uricase activity resulted. This decrease in activity was related to the concentration of antiuricase present. When the rabbit antiuricase was incubated with a similar amount of uricase immobilized on glass beads, there was no detectable inhibition of the immobilized enzyme activity even with concentrations of anituricase which
88
VENTER
ET
AL.
FIG. 7. Ouchterlonies demonstrating the presence of antiuricase in rabbit serum prepared as described in Methods. Uricase (0.02 IU) was placed in the center well and rabbit antiserum in decreasing dilutions in the surrounding wells and incubated at 4°C for five days, then photographed unstained. Normal rabbit serum controls were negative for precipating antibodies to uricase.
caused complete inhibition marized in Fig. 8.
of the native enzyme. These data are sumDISCUSSION
The preliminary data presented here for glass bead immobilized glucose oxidase and uricase demonstrate the feasibility of using glass bead immobilized enzymes in vivo. Glass bead immobilized glucose oxidase
IMMOBILIZED
ENZYME
THERAPY
89
E P a”
Log
of Relative
Antiuricase
Serum
Concentration
FIG. 8. Rabbit antiuricase effects on native uricase (closed circles) and uricase-azo-glass bead (open circles) enzymatic activities. Various dilutions of rabbit antiserum (Methods) were preincubated for 30 minutes at 23°C with equal amounts of native or glass bead immobilized uricase. Uricase activity was determined prior to and subsequent to the antiuricase incubations.
was employed as a model enzyme system because of its stability, availability, rapid turnover and ease of substrate analysis. Chronic studies are planned using this enzyme system. Our findings for immobilized glucose oxidase concur with the recent report (20) on the stability of glass bead immobilized glucose oxidase utilized for an on line blood glucose assay system. The data shown here for glass bead immobilized uricase indicate that while immobilized this enzyme is sufficiently stable for storage and short term enzyme therapy. Long term stability under in viva conditions was not examined. The uricase-glass beads may also provide a continuous source of uricase for uric acid assays (2 1). In agreement with other reports for soluble uricase ( lo- 13), this study demonstrates the hypouricemic activity of uricase immobilized on glass beads. This activity has been shown to be due to a stoichiometric conversion of uric acid to allantoin (Fig. 5). Allantoin, which is slightly more soluble than urate, is cleared rapidly and completely by the kidney, allowing for its rapid removal from the blood. A major limitation to soluble uricase therapy resides in the immunological response to large amounts of intravenously administered foreign protein. Antibody formation rapidly diminished the value of uricase therapy even with increasing enzyme concentration (lo- 13). The use of immobilized uricase, while requiring at present an extracorporeal shunt, offers the distinct advantage of retaining the active enzyme on the glass beads within the shunt. This will permit the use of very high enzyme concentrations and at the same time, limit the possibility of antibody formation. This study has shown that essentially all immobilized enzyme activity is recovered following the various treatments. This suggests that little or no soluble enzyme is directly available for phagocytosis and antibody formation.
90
VENTER
ET AL.
Phagocytosis of enzymes immobilized on glass beads within an extracorporeal shunt cannot be ruled out at present, although actual engulfment of the 200-700 pm glass beads seems unlikely. This study does demonstrate that if antibodies were formed to the native uricase, this would not be a limiting factor in immobilized enzyme therapy at least in the case of the rabbit antibody. While the requirement for an extracorporeal shunt in immobilized enzyme therapy for hyperuricemia creates a tremendous limitation at present, the combination of this system with kidney dialysis machines could possibly prevent the occurence of hyperuricemia resulting from dialysis. There are also indications that certain patients with chronic hyperuricemia and renal insufficiency could benefit from immobilized uricase therapy (22). We feel, however, that further laboratory investigation is warranted prior to any clinical utilization of glass bead immobilized enzymes for enzyme therapy. Under present investigation in this laboratory are some of the immunological considerations discussed here, along with the evaluation of long term enzyme therapy in the form of implantable immobilized enzyme-blood vessel grafts. SUMMARY
Glass bead immobilized glucose oxidase is shown to be effective in lowering blood glucose levels both in vitro and in viva. While immobilized this enzyme is extremely stable and serves as a good model enzyme for further studies of immobilized enzyme therapy. This study also demonstrates the effectiveness of glass bead immobilized uricase in lowering blood urate levels in vitro and in vivo using dogs with an extracorporeal shunt system. These hypouricemic effects have been shown to be due to a conversion of urate to allantoin. The glass bead immobilized uricase appears to be most stable when coupled to the glass via an azo linkage. Antibodies formed in the rabbit to native uricase do not inhibit the glass bead immobilized uricase activity in concentrations that completely inhibit native uricase activity. ACKNOWLEDGMENTS The authors thank Drs. J. W. Cove11 and J. E. Seegmiller for their helpful discussions concerning this manuscript, and Mr. Richard S. Pavelec for his assistance with the extracorporeal shunt experiments.
REFERENCES 1. DIXON,
J. E., STOLZENBACH,
F. E., BERENSON,J.
A., AND KAPLAN,N. Biophys. Res. Commtm. 52, 905 (1973). 2. BERENSON, J.A., BENEMANN, J.R., HISERODT,J.,KAMEN,M.D.,STOLZENBA~H,F.E., AND KAPLAN, N. O., manuscript in preparation. 3. CHANG. T. M. S., Science 146, 424 (1964).
O.,Biochem.
IMMOBILIZED
ENZYME
THERAPY
91
T. M. S., MACINTOSH, F. C., AND MASON, S. G., Canadian J. oj.Phys. and Phar. 44, 11.5 ( 1965). 5. CHANG, T. M. S., AND POZANSKY, M. J., J. Biomed. Mater. Res. 2, 187 (1968). 6. CHANG, T. M. S., Nature (London) 218, 243 (1968). 7. DIXON, J. E., STOLZENBACH, F. E., LEE, C. T., AND KAPLAN, N. O., Israel .I. Chemistry 12 (1974). 8. WEETALL, H. H., AND HAVEWALA, N. B., in “Enzyme Engineering” p. 141-266. John Wiley, New York, 1972. 9. WYNGAARDEN, J. B., in “The Metabolic Basis of Inherited Disease” (J. B. Stanbury. J. B. Wyngaarden, and D. S. Fredrickson, Eds.), 3rd ed. McGraw-Hill, New York, 4. CHANG,
10. I I. 12. 13.
14. 15. 16.
1972. ALTMAN, K. I., SMULL, K., AND BARRON, E. S. G., Arch. Biochem. 21, 158 (1949). LONDON, M., AND HUDSON, B. P., Science 12.5, 937 (1957). KISSEL, P., LAMARCHE, M., AND ROYER, R., Nafure (London) 217, 72 (1968). BROGARD, J. M., COUMAROS, D., FRANELSHAUSER, J., STAHL, A., AND STAHL, J., Rev. Europ. E’tudes Clin. Biol. XVII, 890 (1972). CHANG, T. M. S., Trans. Amer. Sot. Artif. Int. Organs, XII, 13 (1966). VENTER, B. R., AND KAPLAN, N. O., manuscript in preparation, VENTER, J. C., AND DIXON, J. E., in “Hormones and Cyclic Nucleotides,” in “Colo-
wick’s and Kaplan’s Methods in Enzymology” (B. W. O’Malley and J. G. Hardman, Eds.). Academic Press, New York, 1974. 17. WIDMER, F., DIXON, J. E., AND KAPLAN, N. O., Analytical Biochem. 55, 282 (1973). 18. LIDDLE, L., SEEGMILLER, J. E., AND LASTER, L., .I. Lab. C/in. Med. 54, 903 (1959). 19. STAHL, A., SCHANG, A. M., BROGARD, J. M., AND COUMAROS, D. Ann. Biol. Clin. 28, 377 (1970). 20. DRITISCHILO, W., AND WEIBEL, M. K., Biochemical Medicine 2 1. REHAK, N., EVERSE, J., BERGER, R. L., AND KAPLAN, N. 0.. 22. SEEGMILLER, J. E., personal communication.
9, 32 (1974). Fed. Proc. 33, (1974).
MEDICINE
i2, 79-91 (1975)
A Possible Enzymes
Role
for Glass
as Therapeutic as Enzyme
J. CRAIG VENTER,
Agents
Therapy
for
of Medicine,
Immobilized
(Immobilized
Uricase
Hyperuricemia)
BARBARA R. VENTER, JACK E. DIXON,” ANDNATHAN
Departments
Bead
O.KAPLAN
Biology, and Chemistry, La Jolla, California
University 92037
oj. California,
San Diego.
Received September 25, I974
INTRODUCTION
Glass bead immobilized enzymes have been applied in this laboratory to study enzyme kinetics (1) and to study enzymatic hydrogen production (2). The technique of microencapsulation as developed by Chang and co-workers (3-6) has also been used with success with many enzymes and its potential for use in enzyme therapy established, although with this technique of enzyme insolubilization, enzyme stability is a significant problem (6). The stability of enzymes immobilized on glass beads, however, is tremendously enhanced to the effects of temperature, pH and antibody attack (1,7), and immobilized enzyme columns have been used for extended periods without significant loss of activity (8). We will discuss glass bead immobilized uricase as a possible therapeutic agent for chronic gout with renal insufficiency and immobilized glucose oxidase as a model enzyme system for the extension of studies to the development of semipermanent or permanent implantable shunt systems that will allow for continuous enzyme therapy. Renal failure is the eventual cause of death in 20-250/o of patients with gout. Recent evidence suggests that the incidence of gout is increasing. This is presumably due to the high purine content of modern diets. High urate levels are also produced, for example, as side effects of hemodialysis and of cortisone treatment for acute leukemia (9). The first reported use of uricase to decrease serum urate levels in animals was in 1949 ( 10). Uricase was first tested clinically in humans in 1957 (1 l), and again in 1968 (12) and as recently as 1972 ( 13); nevertheI This work was supported in part by grants from the National Institutes of Health (USPHS CA 1 I683-05), the American Cancer Society (BC-60-P). and Hoffman La Roche Inc. 2 Present Address: Department of Biochemistry, Purdue University, Lafayette, Indiana. 79 (-opyright All right,
Q 1975 by Academic Press, Inc. Printed ol reproduction in any form reserved.
in the United
States.
80
VENTER
ET
AL.
less, in all cases the treatment was eventually limited by antibody formation (1 O-l 3). However, even with this limitation, the effectiveness of uricase in lowering serum urate levels and the consequent reversal of tophi and other gout symptoms was of sufficient proportions for Brogard et al. (13) to recommend utilization of uricase as a possible treatment for acute hyperuricemia, hyperuricemia with uric lithiosis and hyperuricemia with renal insufficiency. Extracorporeal shunt systems are widely used and well characterized for hemodialysis and have been used with microencapsulated enzymes ( 14). Thus we propose such a system could be utilized in conjunction with glass bead immobilized enzymes for applying enzyme therapy to hyperuricemia and as a model system for further study in \vivo of immobilized enzyme systems. MATERIALS Enzyme
Immobilization
AND
and Activity
METHODS
Measurements
Glucose oxidase (EC 1.1.3.4 @D-glucose oxygen oxidoreductase) was coupled to activated aryl amine glass as described in (15) and as follows: One gram of aryl amine glass (16) was added to 10 ml of 2 N HCl and cooled in an ice salt bath to 0°C; 250 mg sodium nitrite were added and the reaction placed under vacuum for 20 min at 0°C. The activated diazonium glass beads were then washed with 80 ml ice cold 1% sulfamic acid followed immediately with 200 ml ice cold distilled water utilizing a sintered glass funnel. The glass beads were then rapidly added to 50 mg of the enzyme in 10 ml 0.1 M phosphate buffer pH 7.4 at 4°C. The reaction was continued for 1 hr. The orange-red glass beads were then filtered and washed slowly with 1 liter of phosphate buffer. The glucose oxidase beads were then allowed to stand overnight in either 1% albumin or 0.1 M pyrophosphate solution to remove noncovalently attached enzyme. Bacterial uricase (urate : oxygen oxidoreductase, EC I .7.3.3) (Novo Enzymes) was used without further purification for direct coupling to alkyl and aryl amine glass beads (16) as follows. Gluteraldehyde coupling. To each gram of alkyl amine glass were added 5 ml of a 5% solution of gluteraldehyde in 0.1 M phosphate buffer, pH 7.5. The mixture was placed under vacuum in a dessicator for 60 min at room temperature. The orange gluteraldehyde glass was then washed twice with 100 ml of ice cold phosphate buffer, 0.1 M, pH 7.5 and immediately added to 20 IU of uricase dissolved in 10 ml of a saturated solution of uric acid in ice cold 0.1 M phosphate buffer, pH 7.5. The reaction was continued overnight at 4°C. Uricase and catalase (EC
IMMOBILIZED
ENZYME
THERAPY
81
1.11.1.6 hydrogen peroxide : hydrogen peroxide oxidoreductase) were coupled together to the aryl amine glass by the gluteraldehyde procedure as above, catalase (20 IU/ml) being added to the uricase/uric acid solution prior to the gluteraldehyde glass addition. Diazo coupling. One gram of aryl amine glass was added to IO ml of 2 N HCl and cooled to 0°C: 250 mg of sodium nitrite were added and the reaction placed under vacuum for 20 min at 0°C. The activated diazonium glass beads were then washed with 80 ml ice cold 1% sulfamic acid followed immediately with 200 ml ice cold distilled water, then rapidly added to 20 IU uricase in 10 ml phosphate buffer, pH 7.5, saturated with uric acid and the reaction continued overnight at 4°C. The uricase-glass and the uricase-catalase-glass beads were washed slowly with 2 liters of ice cold phosphate buffer and placed in a 1% albumin solution overnight at 4°C. They were then washed slowly with 2 liters of ice cold phosphate buffer. Enzyme activities were measured on a Perkin-Elmer Model 46 spectrophotometer equipped with an immobilized enzyme reactor system (17). Uricase reactions were run in glycine buffer, 0.1 M, pH 9.4, and the oxidation of uric acid to allantoin followed as a decrease in OD at 292 nm. For storage, uricase-glass beads and glucose oxidase beads were kept at 4°C in 0.1 M glycine buffer, pH 9.4, and phosphate buffer, 0.1 M, pH 7.4, respectively. In vitro blood assays. Canine blood (4 liters) was obtained fresh from dogs anesthetized with 50 mg/kg pentobarbitol and heparinized with sodium heparin. The blood was added to a glass reservoir in a 37°C water bath. The blood was circulated through Tygon tubing into the immobilized enzyme reactor (a cardiotomy reservoir (Model Ql20, Bentley Laboratories, Inc.)) then back to the starting pool with the aid of a peristaltic pump. Ninety-five percent 02, 5% CO, was continuously bubbled into the inner chamber of the cardiotomy reservoir. Samples for substrate analysis were taken from the blood pool. A typical experiment to study the effectiveness of immobilized glucose oxidase or uricase involved circulating the blood for at least 2 hr through the entire system prior to addition of the enzyme-glass beads to the cardiotomy reservoir. An accurate control level of glucose, uric acid or allantoin was thereby established. One to 5 g of enzyme-glass beads were added to the inner chamber of the cardiotomy reservoir and the concentration of the appropriate substrate followed with time. Experiments on human blood were modified from those described for the canine blood because a smaller starting volume was utilized. Two pints of fresh blood (obtained from the San Diego Blood Bank) were warmed to 37°C and pumped over a small column of uricase-glass beads. For
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glucose oxidase experiments, glucose was added to the blood to bring the initial concentration to 400 mg%. For uricase experiments uric acid was added when necessary to bring initial blood levels to 5 mg%. In vivo blood assays. Conditioned dogs were anesthetized with 30 mg/kg pentobarbitol and subjected to artificial respiration. The dogs were heparinized with 10,000 units heparin. The right carotid artery was canulated and connected to a transducer for the continual monitoring of blood pressure. ECG was also continuously monitored. The right femoral vein and artery were canulated to establish an extracorporeal shunt. The femoral artery catheter was connected directly to the cardiotomy reservoir which had been primed with 1 liter of Ringers lactate with 1% albumin. A peristaltic pump was used between the reservoir and the femoral vein. When the lines were opened to blood flow, the pump was set at a speed to match arterial blood flow in order to maintain the arterial pressure and the blood level in the reservoir constant. Volume in the cardiotomy reservoir was 900 ml. Flow rates of 500 to 1,000 ml per minute were used. Blood samples for substrate analysis were taken from the femoral artery proximal to the cardiotomy reservoir. In experiments where urine samples were desired, a Folly catheter was inserted and urine flow rates monitored for each sample. As in the in vitro systems, the extracorporeal shunts were run for l-2 hr prior to the addition of the enzyme glass beads to the system in order to obtain adequate base lines for the substrate to be monitored. Blood glucose levels were measured using glucose Stat Packs (Calbiochem). Blood and urine uric acid were measured by the method of Liddle et al. (18) and blood and urine allantoin by the method of (19). Antibody preparation. A 2 kg New Zealand white rabbit was immunized with intradermal uricase (20 IU) in Freunds complete adjuvant (Dilco). Secondary immunization was carried out after 6 wk with 20 IU uricase in incomplete adjuvant (Dilco). Ten days prior to serum collection, 7.5 IU uricase were given intravenously. The collected rabbit serum was treated with 50% saturated ammonium sulfate overnight at 4”C, centrifuged at 5000 g for 15 min and the supernatant discarded. The pellet was resuspended and then dialyzed against 0.8% sodium chloride for 48 hr with two volume changes. Control serum from an unimmunized rabbit was prepared in an identical manner. RESULTS
Yields and Stability of the Immobilized Uricase When uricase is covalently coupled to porous glass beads by the diazo and gluteraldehyde linkages described (Fig. I) and stored at 4°C in glycine buffer, 0.1 M, pH 9.4, the enzyme is stable for several months.
IMMOBILIZED
ENZYME DIAZO
83
THERAPY
COUPLING
Si- CHz-CH2-CH2-Ntl-EeNH2
-
(~0~0~ + HCI) + Enzyme
glass
Si-CH,-Cn,-,‘,,-N~-e~N=~
-Enzyme
GLUTERALDEHYDE
glass
COUPLING
Si-CH,-CH,-CH,-NH, + CHO WI,), + Enzyme
3
CHO
I I-CH,-CH,-CH,-
ms
N = CH - (CH,),-
CH=N - Enzyme
J I. Diazo and gluteraldehyde enzyme coupling to aryl amine and alkyl amine glass beads. Diazo coupling is presumably to aromatic residues such as tyrosine: gluteraldehyde coupling probably involves lysine residues (I). FIG.
The differences in yields and activities of the immobilized enzymes prepared by the various methods outlined are summarized in Table 1. Due to enzyme inactivation during the coupling process, only IO- 12% of the initial uricase activity in solution was recovered on the glass beads. The uricase coupled to the glass via an azo linkage appears to be the most stable preparation, maintaining 90% of the initial activity over a 50 day period (Fig. 2). The gluteraldehyde coupling yielded a higher TABLE STABILITY
Uricase immobilization procedure Diazo linkage Gluteraldehyde Combined uricase + catalase -both linked through gluteraldehyde
1
OF URICASE-GLASS
BEADS”
Initial activity Activityafter5Odays (I units/g glass) (I units/g glass)
% Activity after 4 hr in blood EL 37°C
2.0 2.1
1.8 1.7
>95%
2.4
1.5
<75%
82%
n The activity and stability of uricase immobilized on glass beads via the indicated linkages: “Initial activity” was assessed immediately following coupling and washing procedures listed in Methods: “Activity after 50 days” was measured following 50 days storage at 4°C in 0.1 M glycine buffer, pH 9.4; “Percent activity in blood” was measured following four hours of incubation in canine blood at 37°C.
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Time (days) FIG. 2. Glass bead immobilized uricase stability at 4°C in glycine buffer, 0.1 M, pH 9.4: Diazo coupled uricase (closed circles); gluteraldehyde coupled uricase (open triangles); gluteraldehyde coupled uricase + catalase (open circles). One hundred milligrams (dry weight) of the appropriate immobilized uricase-glass beads were assayed at the indicated times utilizing an immobilized enzyme reactor (17). Enzyme activities are given as the percentage of initial uricase activity obtained following albumin stripping and washing procedures as described in Methods.
initial specific activity but was less stable than the diazotized uricase preparation. When uricase and catalase were coupled to the same glass beads, the initial uricase activity was 12.5% greater than the gluteraldehyde uricase alone, however, this preparation was found to be the least stable with only 68% of the starting activity remaining after 50 days. To assess the immobilized enzymes’ stability under in vivo conditions. each type of uricase-glass complex was incubated in oxygenated human or canine blood at 37°C in a continuously flowing system. It was found that greater than 95% of the diazo-uricase-glass enzymatic activity was recoverable, whereas for the uricase-gluteraldehyde-glass, only 82% of the initial activity was recoverable following four hours of incubation. The glucose oxidase-gluteraldehyde-glass was stable under these conditions with more than 95% of the starting enzymatic activity recoverable after four hours of incubation. Immobilized-Glucose
Oxidase (In Vitro and In Vivo Assays)
To assess the ability of the glass bead immobilized enzymes to remove substrates from blood, 1 g of the glucose oxidase-glass beads, representing 50 IU of glucose oxidase activity, was added to the inner chamber of the cardiotomy reservoir and 4 liters of canine blood initially containing 400 mg% glucose were pumped continuously through the chamber for three hours. This resulted in the removal of 100% of the glucose, as shown in Fig. 3. This experiment was repeated three times with the same gram of glucose oxidase-glass beads. Each experiment
IMMOBILIZED
ENZYME
8.5
THERAPY
FIG. 3. Four liters of canine blood with 400 mg% blood glucose were continuously circulated through a cardiotomy reservoir at 37°C for three hours. The inner chamber of the cardiotomy reservoir contained 1 g of glucose oxidase glass beads, representing 50 IU of glucose oxidase. Blood samples were removed at the indicated times and assayed for glucose levels (Methods). These data represent the oxidation of 16 g of glucose in the three hour period.
represents the oxidation of 16 g of glucose in a three hour period, When the glucose oxidase glass beads were recovered after the three experiments, more than 95% of the activity on the glass remained. The glucose oxidase-glass beads were then tested in vivo for their ability to oxidize blood glucose. One gram of glucose oxidase glass beads was added to a cardiotomy reservoir in an extracorporeal shunt system established on an anesthetized dog. As Fig. 4 shows, there was a significant and rapid drop in the blood glucose level, resulting in the death of
I
IO
4
20 40 50 Tirne3?min~ FIG. 4. Glass bead immobilized glucose oxidase lowering of blood glucose levels in an anesthetized dog. Glucose-oxidase-glass beads were added to the inner chamber of a cardiotomy reservoir on line in an extracorporeal shunt at time 0. Blood glucose samples were withdrawn from the femoral artery proximal to the cardiotomy reservoir.
0
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VENTER
the dog within 40 minutes. almost full activity. Immobilized
Uricase-
ET AL.
Again the recovered glass bead enzyme had
In Vitro and In Vivo Assays
Due to the low uric acid levels and the presence of allantoin in canine blood, human blood was chosen to test the effectiveness of the uricaseglass beads in lowering serum urate levels. One liter of fresh whole human blood was continuously circulated over a column of 2 g of uricase-diazo-glass beads at 37°C. Samples were removed throughout the four hours of incubation and assayed for uric acid and allantoin levels. As Fig. 5 shows, the serum urate was stoichiometrically converted to allantoin, the conversion rate being 5 mg urate/hr/liter of blood/g of uricase glass beads. This represents the oxidation of 40 mg urate in the four hours of incubation. Full uricase activity was recovered on the glass beads from these experiments. The glass bead immobilized uricase was tested on a Dalmatian dog for its ability to lower serum urate levels under in vivo conditions. The Dalmatian was placed on a high meat diet and probenecid (500 mg three times a day) for one week prior to the experiment. This combination resulted in an increase in the urate level from l/2 mg% to 2.4 mg% and it was maintained at this level. An extracorporeal shunt was established as previously described. Following the addition of 6 g of uricase-glass beads to the extracorporeal shunt reaction chamber, there was a substantial decrease in both serum and urinary uric acid levels and a corresponding increase in the urinary allantoin levels. These results are summarized in Fig. 6. The postoperative period was unremarkable with no
FIG. 5. Stoichiometric conversion of blood uric acid (open circles) to blood allantoin (closed circles) catalyzed by uricase-glass beads. One liter of fresh whole human blood at 37°C was continuously pumped over a small column of uricase-diazo-glass beads (2 g). Samples were withdrawn at the indicated times and assayed for uric acid ( 18) and allantoin (19).
IMMOBILIZED
ENZYME
THERAPY
87
me glass beads added extracorporeal shunt
123456 Time (hrs)
FIG. 6. Alterations in serum and urine acid and urine allantoin levels catalyzed by uricase-glass beads in an extracorporeal shunt on an anesthetized dog (Fig. 4). Blood was allowed to circulate through the extracorporeal shunt for two hours prior to the addition of uricase-glass beads. Uricase-glass beads (6 g) were added to the inner chamber of a cardiotomy reservoir in the extracorporeal shunt at the time indicated. Blood and urine uric acid (18) and urine allantoin (19) levels were determined at the indicated times.
detectable ill effects resulting from the glass bead enzyme therapy. Preand postoperative blood chemistries were normal. The experiment was repeated on a second dog under acute conditions. Again the immobilized uricase was effective in lowering the urate levels and increasing the concentration of allantoin. As in all previous experiments, there was little detectable loss of immobilized uricase activity. Immunological Considerations Ouchterlonies were utilized to demonstrate the formation of antibodies to soluble uricase, as shown in Fig. 7. In addition. when rabbit antiuricase was preincubated with native uricase for 30 minutes a marked inhibition of the uricase activity resulted. This decrease in activity was related to the concentration of antiuricase present. When the rabbit antiuricase was incubated with a similar amount of uricase immobilized on glass beads, there was no detectable inhibition of the immobilized enzyme activity even with concentrations of anituricase which
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FIG. 7. Ouchterlonies demonstrating the presence of antiuricase in rabbit serum prepared as described in Methods. Uricase (0.02 IU) was placed in the center well and rabbit antiserum in decreasing dilutions in the surrounding wells and incubated at 4°C for five days, then photographed unstained. Normal rabbit serum controls were negative for precipating antibodies to uricase.
caused complete inhibition marized in Fig. 8.
of the native enzyme. These data are sumDISCUSSION
The preliminary data presented here for glass bead immobilized glucose oxidase and uricase demonstrate the feasibility of using glass bead immobilized enzymes in vivo. Glass bead immobilized glucose oxidase
IMMOBILIZED
ENZYME
THERAPY
89
E P a”
Log
of Relative
Antiuricase
Serum
Concentration
FIG. 8. Rabbit antiuricase effects on native uricase (closed circles) and uricase-azo-glass bead (open circles) enzymatic activities. Various dilutions of rabbit antiserum (Methods) were preincubated for 30 minutes at 23°C with equal amounts of native or glass bead immobilized uricase. Uricase activity was determined prior to and subsequent to the antiuricase incubations.
was employed as a model enzyme system because of its stability, availability, rapid turnover and ease of substrate analysis. Chronic studies are planned using this enzyme system. Our findings for immobilized glucose oxidase concur with the recent report (20) on the stability of glass bead immobilized glucose oxidase utilized for an on line blood glucose assay system. The data shown here for glass bead immobilized uricase indicate that while immobilized this enzyme is sufficiently stable for storage and short term enzyme therapy. Long term stability under in viva conditions was not examined. The uricase-glass beads may also provide a continuous source of uricase for uric acid assays (2 1). In agreement with other reports for soluble uricase ( lo- 13), this study demonstrates the hypouricemic activity of uricase immobilized on glass beads. This activity has been shown to be due to a stoichiometric conversion of uric acid to allantoin (Fig. 5). Allantoin, which is slightly more soluble than urate, is cleared rapidly and completely by the kidney, allowing for its rapid removal from the blood. A major limitation to soluble uricase therapy resides in the immunological response to large amounts of intravenously administered foreign protein. Antibody formation rapidly diminished the value of uricase therapy even with increasing enzyme concentration (lo- 13). The use of immobilized uricase, while requiring at present an extracorporeal shunt, offers the distinct advantage of retaining the active enzyme on the glass beads within the shunt. This will permit the use of very high enzyme concentrations and at the same time, limit the possibility of antibody formation. This study has shown that essentially all immobilized enzyme activity is recovered following the various treatments. This suggests that little or no soluble enzyme is directly available for phagocytosis and antibody formation.
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ET AL.
Phagocytosis of enzymes immobilized on glass beads within an extracorporeal shunt cannot be ruled out at present, although actual engulfment of the 200-700 pm glass beads seems unlikely. This study does demonstrate that if antibodies were formed to the native uricase, this would not be a limiting factor in immobilized enzyme therapy at least in the case of the rabbit antibody. While the requirement for an extracorporeal shunt in immobilized enzyme therapy for hyperuricemia creates a tremendous limitation at present, the combination of this system with kidney dialysis machines could possibly prevent the occurence of hyperuricemia resulting from dialysis. There are also indications that certain patients with chronic hyperuricemia and renal insufficiency could benefit from immobilized uricase therapy (22). We feel, however, that further laboratory investigation is warranted prior to any clinical utilization of glass bead immobilized enzymes for enzyme therapy. Under present investigation in this laboratory are some of the immunological considerations discussed here, along with the evaluation of long term enzyme therapy in the form of implantable immobilized enzyme-blood vessel grafts. SUMMARY
Glass bead immobilized glucose oxidase is shown to be effective in lowering blood glucose levels both in vitro and in viva. While immobilized this enzyme is extremely stable and serves as a good model enzyme for further studies of immobilized enzyme therapy. This study also demonstrates the effectiveness of glass bead immobilized uricase in lowering blood urate levels in vitro and in vivo using dogs with an extracorporeal shunt system. These hypouricemic effects have been shown to be due to a conversion of urate to allantoin. The glass bead immobilized uricase appears to be most stable when coupled to the glass via an azo linkage. Antibodies formed in the rabbit to native uricase do not inhibit the glass bead immobilized uricase activity in concentrations that completely inhibit native uricase activity. ACKNOWLEDGMENTS The authors thank Drs. J. W. Cove11 and J. E. Seegmiller for their helpful discussions concerning this manuscript, and Mr. Richard S. Pavelec for his assistance with the extracorporeal shunt experiments.
REFERENCES 1. DIXON,
J. E., STOLZENBACH,
F. E., BERENSON,J.
A., AND KAPLAN,N. Biophys. Res. Commtm. 52, 905 (1973). 2. BERENSON, J.A., BENEMANN, J.R., HISERODT,J.,KAMEN,M.D.,STOLZENBA~H,F.E., AND KAPLAN, N. O., manuscript in preparation. 3. CHANG. T. M. S., Science 146, 424 (1964).
O.,Biochem.
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ENZYME
THERAPY
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T. M. S., MACINTOSH, F. C., AND MASON, S. G., Canadian J. oj.Phys. and Phar. 44, 11.5 ( 1965). 5. CHANG, T. M. S., AND POZANSKY, M. J., J. Biomed. Mater. Res. 2, 187 (1968). 6. CHANG, T. M. S., Nature (London) 218, 243 (1968). 7. DIXON, J. E., STOLZENBACH, F. E., LEE, C. T., AND KAPLAN, N. O., Israel .I. Chemistry 12 (1974). 8. WEETALL, H. H., AND HAVEWALA, N. B., in “Enzyme Engineering” p. 141-266. John Wiley, New York, 1972. 9. WYNGAARDEN, J. B., in “The Metabolic Basis of Inherited Disease” (J. B. Stanbury. J. B. Wyngaarden, and D. S. Fredrickson, Eds.), 3rd ed. McGraw-Hill, New York, 4. CHANG,
10. I I. 12. 13.
14. 15. 16.
1972. ALTMAN, K. I., SMULL, K., AND BARRON, E. S. G., Arch. Biochem. 21, 158 (1949). LONDON, M., AND HUDSON, B. P., Science 12.5, 937 (1957). KISSEL, P., LAMARCHE, M., AND ROYER, R., Nafure (London) 217, 72 (1968). BROGARD, J. M., COUMAROS, D., FRANELSHAUSER, J., STAHL, A., AND STAHL, J., Rev. Europ. E’tudes Clin. Biol. XVII, 890 (1972). CHANG, T. M. S., Trans. Amer. Sot. Artif. Int. Organs, XII, 13 (1966). VENTER, B. R., AND KAPLAN, N. O., manuscript in preparation, VENTER, J. C., AND DIXON, J. E., in “Hormones and Cyclic Nucleotides,” in “Colo-
wick’s and Kaplan’s Methods in Enzymology” (B. W. O’Malley and J. G. Hardman, Eds.). Academic Press, New York, 1974. 17. WIDMER, F., DIXON, J. E., AND KAPLAN, N. O., Analytical Biochem. 55, 282 (1973). 18. LIDDLE, L., SEEGMILLER, J. E., AND LASTER, L., .I. Lab. C/in. Med. 54, 903 (1959). 19. STAHL, A., SCHANG, A. M., BROGARD, J. M., AND COUMAROS, D. Ann. Biol. Clin. 28, 377 (1970). 20. DRITISCHILO, W., AND WEIBEL, M. K., Biochemical Medicine 2 1. REHAK, N., EVERSE, J., BERGER, R. L., AND KAPLAN, N. 0.. 22. SEEGMILLER, J. E., personal communication.
9, 32 (1974). Fed. Proc. 33, (1974).