Biochemical Characterization of an l-Asparaginase Bioconjugate

Martins, M. B. F., and Cruz, M. E. M. (1994) Characterization of bioconjugates of l-asparaginase and Cu,Zn−superoxide dismutase. Abstracts, Third Eu...
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Bioconjugate Chem. 1996, 7, 430−435

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Biochemical Characterization of an L-Asparaginase Bioconjugate M. Ba´rbara A. F. Martins,* A. Paula V. Gonc¸ alves, and M. Euge´nia M. Cruz Biochemistry Unit, Department of Biotechnology, Instituto Nacional de Engenharia e Tecnologia Industrial, Estrada do Pac¸o do Lumiar, 1699 Lisboa Codex, Portugal. Received August 3, 1995X

In this work is characterized a bioconjugate of L-asparaginase, obtained by linkage of palmitic acid chains to the native enzyme in the presence of substrate as a protein protective molecule. Comparisons between isoelectric points, hydrophobicity, pH, and temperature profiles for the bioconjugate and the native enzyme were performed. A shift of pI from 5.03 to 4.58 was observed after conjugation. The modified enzyme evidences a 10-fold increase of the hydrophobicity. A small shift from 7.5 to 7 of the pH for maximal catalytic activity and a 5 °C increase of temperature for maximal activity were observed with conjugation. Stability studies in human serum and on storage evidence similar behaviors for both bioconjugate and native enzyme. The retention of catalytic activity of the bioconjugate is dependent on the presence of micelles. The bioconjugate evidenced 65% retention of activity when catalytic activity was assayed without a surfactant and 98-100% retention of activity when catalytic activity was assayed in the presence of surfactant micelles. The kinetic characteristics of the bioconjugate and of the native enzyme, in micelles of different hydrophobicities, were compared. The Michaelis constant of native enzyme is 0.030 mM, independent of the surfactant, and the Michaelis constant of the bioconjugate varies with the surfactant, from 0.036 to 0.046 mM.

INTRODUCTION

There is increasing interest in the search for mechanisms to optimize the therapeutic action of enzymes to improve their function and properties (Konrad, 1989). One of the tools to achieve this goal is the construction of bioconjugates, obtained by linkage of different molecules to enzymes (Borman, 1989). This approach, requiring random covalent coupling, may result in modification of the enzyme active site or in conformational alterations of the macromolecule, with a consequent alteration of properties. So, characterization of the bioconjugates needs to be performed. In this paper we report the physicochemical and kinetic characterization of a bioconjugate of L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1), which is an enzyme with antitumor activity against human acute lymphoblastic leukemia (Asselin et al., 1989; Capizzi and Handschumacher, 1982). The bioconjugate (Ac-L-asparaginase) was obtained as described in previous work (Martins et al., 1990), by covalent coupling of palmitic chains to the native enzyme using the conjugation method developed by Torchilin et al. (1980) but including substrate in the reactional medium. Ac-L-asparaginase was developed with the aim of supplying the enzyme surface with hydrophobic chains able to promote the association of the active enzyme with the bilayer of liposomes while preserving the other enzyme characteristics. The association of Ac-L-asparaginase with liposomes was evidenced in previous work in which the liposomal form of the bioconjugate was characterized (Jorge et al., 1994). The physicochemical characteristics of the Ac-L-asparaginase, such as hydrophobicity and charge, could be relevant for the optimization of the liposomal form, as both parameters can help the rational design of a bilayer (Boggs, 1987) to improve the incorporation of a protein. The interaction between proteins and liposomes, other than the covalent bond, can be mediated by electrostatic and/ or hydrophobic interactions between the membrane and * Author to whom correspondence should be addressed (telephone +351+1+7162712; telefax +351+1+7163636; e-mail [email protected]). X Abstract published in Advance ACS Abstracts, June 1, 1996.

S1043-1802(96)00026-2 CCC: $12.00

the protein, such as the ones occurring with membrane proteins (Jain and Zakim, 1987), but the relative importance of this interaction is difficult to assess (Low, 1987). The electrophoretic mobility and the isoelectric point of the bioconjugate and native enzyme were evaluated by Doppler laser anemometry and compared with gel isoelectric focusing quantification. Due to the resolution of this techniques, some information on the homogeneity of the sample can be obtained (McNeil-Watson, 1988; Dwyer, 1993). The catalytic performances of a bioconjugate are relevant for its usefulness, mainly initial activity and stability and also kinetic constants and pH and temperature profiles. The preservation of catalytic properties of a bioconjugate may be dependent on the use of an appropriate stabilizer of its protein structure. Reported stabilizers include some solvents (Timasheff and Arakawa, 1990) and naturally occurring or synthetic micellar systems able to affect the rates of many chemical reactions either in vivo or in vitro (Fendler and Fendler, 1975). High affinity to L-asparagine, and consequently low Michaelis constant (Km), at physiological pH, are required for the antitumor activity of L-asparaginase (Wirston and Yellin, 1973). With the aim of identifying microenvironment parameters able to maximize the activity of Ac-L-asparaginase, micelles of different hydrophobicities were used. The effect of micellization on enzymatic reactions of the bioconjugate is considered. EXPERIMENTAL PROCEDURES

Materials. Asparaginase Elspar, L-asparagine amidohydrolase (EC 3.5.1.1), type EC-2 from Escherichia coli, was obtained from Merck Sharp & Dohme. The other enzymes or proteins used were from Sigma. 3-[(3Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) was from Sigma. For gel isoelectric focusing were used PhastGel IEF, precast homogeneous polyacrylamide gels, containing Pharmaly as carrier ampholyte, and PhastGel Blue R 350 for Coomassie Blue stain, all from Pharmacia. All other reagents were of analytical grade. The reactional medium for modification of the enzyme was sonicated in a water bath sonicator from Sonorex, © 1996 American Chemical Society

L-Asparaginase

Bioconjugate

Model RK 156. The separations by dialysis were performed using membranes with 12 000 molecular weight cutoff. The absorption spectrophotometry results were obtained in a spectrophotometer from Shimadzu, Model UV-160A. The fluorescence spectroscopy results were obtained in a spectrofluorometer from Hitachi, Model F 3000. The gel isoelectric focusing quantifications were performed in a Phast System from Pharmacia. The Doppler laser light scattering studies were performed in equipment from Malvern, Model Zeta-Sizer-III. Methods. Preparation of Ac-L-asparaginase. The bioconjugate of L-asparaginase was prepared according to the method of Torchilin et al. (1980) with some modifications (Martins et al., 1990). In brief, palmitoyl chloride was added to a 50 mM carbonate buffer, pH 9.4, containing 1% sodium cholate and 8 mM L-asparagine. The mixture was sonicated for 90 s and immediately added to the enzyme and incubated, with gentle stirring, at room temperature. The modified enzyme was recovered from the reactional medium using separation procedures to remove the other compounds of the reactional medium. The palmitic acid was removed by centrifugation (20000g for 30 min) and the modified enzyme recovered in the supernatant. The remaining compounds [surfactant, salts, substrate (L-asparagine), and products from its conversion] were removed by dialysis against water. Native enzyme, used as a reference for all of the experiments, was solubilized in 50 mM carbonate buffer, pH 9.4, containing 1% sodium cholate and 8 mM Lasparagine and the solution dialyzed against water. With this, a similar presence of the different compounds, in the concentrations that remain after dialysis, can be assured. The degree of modification was quantified by a fluorometric assay (Bo¨hlen et al., 1973). Briefly, after the recovery of modified and native enzyme from dialysis, the unblocked -NH2 groups of either acylated or native enzyme were bound to fluorescamine according to the method of Bo¨hlen et al. (1973). The intensity of fluorescence emission of bound fluorescamine at 475 nm was measured for excitation at 390 nm. The degree of modification is [1 - (emission per microgram of modified enzyme/emission per microgram of native enzyme)] × 100. Since fluorescamine can react with trace amines present in the reagents and solvents, a reagent blank was run routinely. For this work were prepared batches of Ac-L-asparaginase with a 30% degree of modification and a 100% retention of activity and of L-asparaginase treated as previously described. Amounts of bioconjugate and native enzyme were lyophilized in individual containers. Assay of Catalytic Activity of the Enzyme. Two procedures were used to quantify the initial rate of conversion of substrate (L-asparagine): the quantification of ammonia, according to the procedure of Chaney and Marbach (1962), and a coupled enzyme system to directly follow the reaction rate as described by Abuchowski et al. (1979). Briefly, in the quantification according to Chaney and Marbach (1962), the reaction was initiated by the addition of the native enzyme or the bioconjugate to a solution of L-asparagine (10 mM) in phosphate buffer (50 mM), pH 7.5, at 37 °C. The enzyme concentration in this mixture was 130 nM. The ammonia was quantified in samples (150 µL) of the reaction medium. To each sample was added 1 mL of a solution containing phenol (10 g L-1) and sodium nitroprusside (0.05 g L-1) plus 1 mL of a solution containing sodium hydroxide (5 g L-1) and sodium hypochloride (0.42 g L-1). The color development was produced after 3 min at 60 °C and the

Bioconjugate Chem., Vol. 7, No. 4, 1996 431

absorbance quantified at 625 nm. A calibration curve with standard solutions of 0.02-0.16 µM ammonium chloride was used. In the coupled enzyme system assay the reaction rate of conversion of L-asparagine, at 37 °C, was directly followed by the decrease of absorbance at 340 nm (decrease of NADH concentration) of a solution containing 3 nM of enzyme (native enzyme or bioconjugate) and 10 mM of substrate (L-asparagine). The reaction was initiated by the addition of 10 µL of an enzyme solution to 1000 µL of a reaction mixture with the following composition: 20 mL of glycerol, 11 mg of NADH, 116 units of L-malate dehydrogenase, 180 units of L-glutamic oxaloacetic transaminase, 11 mg of R-ketoglutaric acid, and 1.1 mmol of L-asparagine, made up to 100 mL with 0.1 M Tris-HCl, pH 8.35. Unless specifically noted otherwise, the catalytic assays were performed including 1% cholic acid in reaction medium. Determination of Isoelectric Point. (a) Isoelectric Focusing. Samples of Ac-L-asparaginase and L-asparaginase rehydrated in water solutions of CHAPS (1%) were applied in the isoelectric focusing gel, and the staining procedure was performed using Coomassie Blue; the concentrations and the other procedures of the electrophoresis followed the instructions of the system used. (b) Doppler Laser Anemometry. The microelectrophorese cell of the Doppler laser light scattering equipment was filled using equivalent preparations of bioconjugate or native enzyme, rehydrated to a concentration of 500 µg mL-1, in electrolytes with different pH values. As electrolytes were used buffers of constant and low ionic strength (I ) 0.01). The compositions of each buffer were as follows: for pH 2.5, chloroacetic acid and potassium hydroxide; for pH 3.5, formic acid and potassium hydroxide; for pH 4.5, acetic acid and potassium hydroxide; for pH 5.5, succinic acid and potassium hydroxide; for pH 6.5, phosphoric acid and disodium hydrogen phosphate; for pH 7.5, phosphoric acid and disodium hydrogen phosphate; for pH 8.5, boric acid and sodium tetraborate. The electrode chambers were filled with electrolyte free of enzyme. The electrophoretic mobility was monitored at low ionic strength to promote electrostatic interactions for the bioconjugate and for the native enzyme as a function of the pH. The experiments were performed at 20 °C. The isoelectric point corresponds to the pH of null electrophoretic mobility. Determination of Partition Coefficient. Water and octanol (2.5 mL of each) were mixed in a vortex until formation of an emulsion. The mixture was added to 0.25 mg of protein. The emulsion was stirred during 2 h. The preparation was centrifuged at 5000g during 5 min. The two phases formed after centrifugation were analyzed for protein content using a quantification by intrinsic emission of fluorescence of L-asparaginase or Ac-L-asparaginase. Equivalent preparations without protein were used as reference. The partition coefficient octanol/water was determined as the ratio between the protein present in octanol and water phases. Stability Studies. (a) Storage Stability of Free and Conjugated Enzyme. Several equivalent preparations containing around 600 µg of lyophilized Ac-L-asparaginase or L-asparaginase were stored in a refrigerator (6 °C). After different periods of storage, the preparations were rehydrated with 1 mL of a sodium cholate (1%) water solution and assayed for activity (Chaney and Marbach, 1962) and protein content (Lowry et al., 1951). The retention of activity was determined in relation to initial conditions, either for L-asparaginase or for Ac-Lasparaginase.

432 Bioconjugate Chem., Vol. 7, No. 4, 1996

(b) Stability in Human Serum of Free and Conjugated Enzyme. Equivalent preparations containing 500 µg of L-asparaginase or 450 µg of Ac-L-asparaginase, plus 2 mL of human serum and 2 mL of phosphate saline buffer, were incubated at 37 °C. After different periods of time, a group of three preparations was analyzed for catalytic activity by the direct quantification of product previously described. The retention of activity was determined in relation to initial conditions, either for L-asparaginase or for Ac-L-asparaginase. Determination of Temperature-Activity Profiles. Solutions of L-asparaginase or Ac-L-asparaginase (500 µg mL-1) in water containing 1% of sodium cholate were assayed for catalytic activity by the quantification of ammonia previously described, at temperatures in the range from 25 to 85 °C at pH 7.5. The results were normalized in relation to the maximal activity for each form of the enzyme. Determination of pH-Activity Profiles. Equivalent preparations, containing 500 µg of L-asparaginase or AcL-asparaginase, were rehydrated with 1 mL of buffer with different pH values (5.5-9.0), containing 1% of sodium cholate. In the pH range from 4 to 5.5 was used a 50 mM sodium acetate-acetic acid buffer, in the pH range from 6 to 8 was used a 50 mM phosphate buffer, and in the pH range from 8.5 to 10.5 was used a 50 mM carbonate buffer. Each native enzyme or bioconjugate solution was assayed for protein content (Lowry et al., 1951) and for catalytic activity, at 37 °C, in a reaction medium made of 10 mM L-asparagine in the buffer used for rehydration and using the direct quantification of product previously reported. The results were normalized in relation to the maximal catalytic activity of each form of the enzyme. Determination of the Retention of Catalytic Activity. The retention of catalytic activity (RA) is the ratio

RA (%) ) (initial rate of reaction catalyzed by enzyme under study/initial rate of reaction catalyzed by native enzyme) × 100 The “enzyme under study” in the above equation could be the bioconjugate in a micellar system, the bioconjugate in a nonmicellar system, or the native enzyme in a micellar system. The “native enzyme” is an equal amount of native enzyme without surfactant. All of the quantifications were performed, in conditions of high substrate concentration S . Km, by the coupled enzyme assay with 10 mM L-asparagine. Determination of Kinetic Parameters. The initial reaction rate for L-asparaginase or Ac-L-asparaginase as function of the initial L-asparagine concentration in the range 0.05-2 mM was quantified by the coupled enzyme system previously described, using 1-40 µmol of Lasparagine in the reaction mixture. The kinetic parameters of the bioconjugate and of the native enzyme were determined using the Hanes linearization method (Cornish-Bowden and Wharton, 1988). All of the experiments and quantifications reported were performed in triplicate. The results represent a mean of three independent determinations. RESULTS AND DISCUSSION

Effect of Conjugation on Enzyme Charge and Hydrophobicity. An alteration of charge was observed after conjugation according to the electrophoretic mobility results. A decrease of 0.45 of a pI unit was observed for the conjugated enzyme: the isoelectric point (pI) for L-asparaginase as determined by Doppler laser anemometry (Figure 1) is 5.03 ( 0.07. This result is in accordance

Martins et al.

Figure 1. Electrophoretic mobility of Ac-L-asparaginase (b) and L-asparaginase (9) as function of pH. Table 1. Octanol/Water Partition Coefficients of the Bioconjugate and the Native Enzyme enzyme

partition coefficient

Ac-L-asparaginase L-asparaginase

1.78 0.13

with the pI we obtain by isoelectric focusing, which is 5.03 ( 0.3. Results reported in the literature for the pI of native E. coli L-asparaginase are in the range 4.65.35 (Wriston and Yellin, 1973) and 4.9-5.6 (Capizzi and Handschumacher, 1982). The pI of Ac-L-asparaginase determined by Doppler laser anemometry is 4.58 ( 0.05 (Figure 1), while by isoelectric focusing a discrepancy between the results of pI for the bioconjugate was observed (results not shown). As previously reported (Martins and Cruz, 1994), the Doppler laser anemometry technique evidences the advantage of quantification of the pI of hydrophobic macromolecules which have low mobility in a gel matrix. The decrease of pI was expected due to the blockage, by conjugation with palmitic chains, of 30% of the accessible -NH2 [pKa values of -NH2 are in the range 9.3-9.5 (Means and Feeney, 1971)]. This degree of modification, quantified by the fluorometric assay described under Experimental Procedures, is consistent with previous results (Martins et al., 1990). Figure 1 also evidences a deviation between the electrophoretic mobilities of both forms of the enzyme, which must be related to the different protonation of the two forms. This alteration of charge points to a different interaction of Ac-L-asparaginase with charged liposomal bilayers and could be an indicative factor to the selection of lipid compositions. Table 1 shows the octanol/water partition coefficient for the native enzyme and the bioconjugate, quantified as a measure of hydrophobicity. To avoid perturbation of protein partitioning in the water/organic solvent systems by the presence of other compounds, the bioconjugate was separated from the reaction medium as described under Experimental Procedures. An increase of the affinity of the bioconjugate to octanol of more than 10-fold was observed without evidence for accumulation of Ac-L-asparaginase at the interface. The amount of bioconjugate solubilized in octanol is in accordance with results previously published (Martins et al., 1990). In these results a solubilization of bioconjugate in octanol, at concentrations as much as 0.46 µM, was correlated with the solutions turbidity. These results evidence a significative increase of the affinity of the bioconjugate to hydrophobic mediums, as expected after introduction of hydrophobic palmitoyl groups in the enzyme molecule. Evaluation of the Homogeneity of the Bioconjugate. The unimodal zeta potential distribution of either Ac-L-asparaginase or L-asparaginase (Figure 2) can be correlated with a homogeneous charge distribution of

L-Asparaginase

Bioconjugate

Bioconjugate Chem., Vol. 7, No. 4, 1996 433

Figure 2. Zeta potential distribution of Ac-L-asparaginase and L-asparaginase at pH 6.5, quantified by microelectrophorese in electrolyte solution. The concentration of native enzyme or bioconjugate was 500 µg mL-1.

Figure 5. Stability of Ac-L-asparaginase (b) and L-asparaginase (9) in human serum at 37 °C. Half-lives were calculated using the inverted decay model.

Figure 3. Effect of pH on the activity of Ac-L-asparaginase (b) and L-asparaginase (9).

Figure 6. Stability of Ac-L-asparaginase (b) and L-asparaginase (9) on storage a 6 °C in lyophilized form.

Figure 4. Effect of temperature on the activity of Ac-Lasparaginase (b) and L-asparaginase (9).

both samples (McNeil-Watson, 1988) and points to the absence of dissociation or aggregation either of native enzyme or of bioconjugate molecules for a concentration of 500 µg mL-1 at pH 6.5. In this study we analyze the homogeneity of only those samples having electrophoretically similar populations. To perform a homogeneity evaluation, based on other characteristics, for example, the hydrophobic affinity, reversed phase chromatography could be used. Effect of Conjugation on Catalytic Activity Dependence on pH and Temperature. Results of catalytic activity of the bioconjugate normalized in relation to the maximal catalytic activity are shown. Figure 3 evidences a similar catalytic activity for both bioconjugate and native enzyme over all of the pH range used. A similar pH dependence of the reaction catalyzed by the native enzyme in the pH range from 4 to 8 is reported in the literature (Ehrman, 1971). Figure 4 shows a similar dependence of the catalytic activity on temperature both for bioconjugate and for

native enzyme; maximal activity was observed at 65 and 60 °C for Ac-L-asparaginase and L-asparaginase, respectively. Stability of Free and Conjugated Enzyme. A sharp decrease in the retention of activity of L-asparaginase and the conjugated form was observed in the first 2 h of incubation in human serum (Figure 5). This behavior is evidenced by the respective half-lives: 7.9 h for native enzyme and 6.9 h for bioconjugate, calculated using the inverted decay model. During the following 2-48 h the rate of activity variation is reduced. No significant differences between the retention of activity for both the bioconjugate and the native enzyme after 360 days of storage at 6 °C in lyophilized form were found (Figure 6). Effect of Surfactant Micelles on Kinetic Characteristics of the Bioconjugate. The effect of micellar systems of natural and synthetic surfactants on the retention of catalytic activity and on the kinetic parameters (Michaelis constant, Km, and maximal reaction rate, V) of the bioconjugate was evaluated and compared with the performances of native enzyme (Table 2). Surfactants with different charge and hydrophilic-lipophilic balance (HLB) were used: two anionic surfactants, cholic acid (HLB ) 18) and deoxycholic acid (HLB ) 16), and two nonionic surfactants, Brij 76 (HLB ) 12) and Synperonic F-68 (HLB ) 29). The results of retention of catalytic activity evidence a decrease of activity of bioconjugate in the absence of micellar medium. No decrease of catalytic activity of the bioconjugate was observed in micellar medium. This result points to the stabilization of the bioconjugate molecule by the presence of micelles independent of its charge or hydrophobicity. A comparison between the Michaelis constants of L-asparaginase and Ac-L-asparaginase, in micellar medium (Table 2), evidences a slight decrease of the Km of the bioconjugate. In contrast with a constant Km for L-asparaginase, independent of the composition of mi-

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Table 2. Effect of Surfactant Micelles on Kinetic Characteristics of Conjugated and Native Enzymesa surfactantb

enzyme

cholic acid Ac-Lasparaginase deoxycholic acid Brij 76 Synperonic F-68 L-asparaginase

cholic acid deoxycholic acid Brij 76 Synperonic F-68

retention Km (M) V of act. (%) (× 10-5) (M s-1) >98 >98 >98 >96 65 100 100 100 100 100

4.1 ndc 3.6 4.6 nd 3.0 nd 3.1 3.1 3.0

133 nd 125 125 nd 129 nd 125 118 101

a Linearization performed according to the Hanes method. Concentrations of surfactants: cholic acid, 1%; deoxycholic acid, 0.32%; Brij 76, 0.003%; Synperonic F-68, 0.053%. c nd, not done.

b

celles, a slight increase of this parameter was observed as function of the HLB of the surfactant. The maximal reaction (V) for the bioconjugate and the native enzyme also evidences slight differences. The effect of modification on these parameters can be discussed in terms of V/Km, which is considered (Creighton, 1984) to be the most critical parameter in determining the specificity of an enzyme for a substrate. For the native enzyme in the absence of surfactant this ratio is 3.4 × 106, while in the presence of micelles of surfactants it is as follows: cholic acid, 4.3 × 106; Brij 76, 4.0 × 106; Synperonic F-68, 3.9 × 106. For the bioconjugate in micelles of cholic acid this ratio is 3.2 × 106, in micelles of Brij 76 it is 3.5 × 106, or in micelles of Synperonic F-68 it is 2.7 × 106. The slight difference between the V/Km of the bioconjugate and that of the native L-asparaginase, which can correspond to a small alteration of the affinity of the enzyme to substrate, can be due to hydrophobic interactions of the bioconjugate with micelles. Therefore, the localization of the bioconjugate and the native enzyme in a micellar medium may be different. If the bioconjugate becomes entrapped in the micelle, some conformational or microenvironmental alterations can occur and the accessibility of substrate to the bioconjugate can be perturbed, namely by steric hindrances. This may have consequences on the kinetic parameters of each enzyme form. These alterations, based on hydrophobic interactions, can depend on the surfactant hydrophilic-lipophilic characteristics. The slight increase in alteration of affinity with conjugation which leads to a Km for the bioconjugate in the range (3.6-4.6) × 10-5 M does not limit its clinical usefulness; the Km range for clinically useful L-asparaginase is (1-5) × 10-5 M (Capizzi and Cheng, 1981). CONCLUSIONS

Consistent with the purpose of the construction of a bioconjugate with affinity to the matrix lipid of liposomes, a significant increase in the hydrophobicity of the enzyme was observed after conjugation. Also, an alteration of surface charge was evidenced by the electrophoretic mobility curves. Considering these results, both hydrophobic and electrostatic interactions can explain the association between Ac-L-asparaginase and the bilayers of liposomes previously reported (Martins et al., 1990; Jorge, 1994). The solubilization of Ac-L-asparaginase in octanol, up to a concentration of 0.46 µM, points to the potentiality of this bioconjugation to promote the solubilization of hydrophilic enzymes in organic solvents. The promotion of solubilization of hydrophilic enzymes in nonpolar organic solvents, by the development of specific methods of dissolution, was reported by Mozhaev et al. (1991). A minimized perturbation must be achieved for

the structure of this bioconjugate, considering the results of the effect of the chemical modification on the pH and temperature activity profiles, on the storage stability, on the stability in human serum, on the catalytic activity, and on the substrate affinity. The high retention of catalytic properties of this bioconjugate overcame the limitations reported in the literature (Claassen and Rooijen, 1983) to the modification of L-asparaginase by the palmitoyl chloride method. This must be the result of the following: (1) the mild conditions of this conjugation process (Torchilin et al., 1980); (2) the protective effect of substrate during conjugation (Martins et al., 1990); and (3) the stabilizing effect of the hydrophobic portions of naturally occurring or synthetic micellar systems. In conclusion, the preservation of catalytic properties following conjugation of acyl chains to the native enzyme, as reported, evidences the potential of Ac-L-asparaginase as a candidate to substitute for the native enzyme in processes requiring a hydrophobic interaction with a microenvironment. ACKNOWLEDGMENT

We thank Prof. Dr. Joaquim M. S. Cabral for the fruitful discussion during the preparation of the manuscript. LITERATURE CITED Abuchowski, A., Es, T. E., Palczuk, N. C., McCoy, J. R., and Davis, F. F. (1979) Treatment of L5178Y tumor-bearing BDF1 mice with a nonimmunogenic L-glutaminase-L-asparaginase. Cancer Treatment Rep. 63, 1127-1132. Asselin, B. L., Ryan, D., Frantz, N., Bernal, S. D., Leavitt, P., Sallan, S. E., and Cohen, H. J. (1989) In vitro and in vivo killing of acute lymphoblastic leukemia cells by L-asparaginase. Cancer Res. 49, 4363-4368. Boggs, J. M. (1987) Lipid intermolecular hydrogen bonding: influence on structural organization and membrane function. Biochim. Biophys. Acta 906, 353-404. Bo¨hlen, P., Stein, S., Dairman, W., and Undenfriend, S. (1973) Fluorimetric assay of proteins in the nanogram range. Arch. Biochem. Biophys. 155, 213-220. Borman, S. (1989) Bioconjugate chemistry attracts growing interest. Chem. Eng. News 67 (May 8), 25-28. Capizzi, R. L., and Cheng, Y. C. (1981) Therapy of neoplasia with asparaginase. Enzymes as Drugs (J. C. Holcenberg, Ed.) pp 2-24, Wiley, New York. Capizzi, R. L., and Handschumacher, R. E. (1982) Asparaginase. Cancer Medicine (J. F. Holland and E. Frei III, Eds.) pp 920933, Lea and Febiger, Philadelphia. Chaney A. L., and Marbach E. P. (1962) Modified reagents for determination of urea and ammonia. Clin. Chem. 8, 130132. Claassen, E., and Rooidjen, N. (1983) A comparative study on the effectiveness of various procedures for attachment of two proteins (L-asparaginase and horseradish peroxidase) to the surface of liposomes. Prep. Biochem. 13, 167-174. Cornish-Bowden, A., and Wharton, C. W. (1988) Simple enzyme kinetics. Enzyme Kinetics (D. Rickwood, Ed.) pp 1-18, IRL Press, Oxford, U.K. Creighton, T. E. (1984) Proteins: Structure and Molecular Principles, pp 405-409, W. H. Freeman, New York. Dwyer, D. L. (1993) Electrophoretic techniques of analysis and isolation. Protein Biotechnology (F. Franks, Ed.) pp 313-363, Humana Press, Totowa, NJ. Ehrman, M., Cedar, H., and Scwartz, J. H. (1971) L-Asparaginase of Escherichia coli: studies on the enzymatic mechanism of action. J. Biol. Chem. 246, 88-94. Fendler, J. H., and Fendler, E. J. (1975) Micellar effects on hydrophobic interactions and protein Structure. Catalysis in

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Bioconjugate

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