Abstract
The semitelechelic poly[N-(2-hydroxypropyl)methacrylamide] [poly(HPMA)] with a carboxyl end group was prepared with 3-mercaptopropionic acid as a chain transfer agent. Bovine seminal ribonuclease (BSR) and α-chymotrypsin (ChT) were modified with various molecular weights of active poly(HPMA) succinimidyl ester by the reaction with the amino groups of the respective enzyme. The modification of ChT did not significantly change the activity or the substrate specificity of the conjugates towards low-molecular-weight tripeptidic substrates. However, modified ChT activity towards the corresponding poly(ethylene glycol)-based synthetic substrate was significant. The activity decreased as a result of the elevated steric hindrance to the active site of the polymer-modified enzyme. Similarly, the ChT conjugates completely lost their proteolytic activity toward native bovine serum albumin. The autolytic stability of ChT conjugates was improved and the proteolytic stability of the the ChT and BSR conjugates substantially increased compared with the free enzymes. The modification of ChT with poly(HPMA) significantly decreased the immunogenicity of ChT conjugates depending on the molecular weight of the poly(HPMA) and the degree of enzyme substitution.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 213-231 |
| Number of pages | 19 |
| Journal | Journal of Bioactive and Compatible Polymers |
| Volume | 14 |
| Issue number | 3 |
| DOIs | |
| State | Published - May 1999 |
| Externally published | Yes |
ASJC Scopus subject areas
- Bioengineering
- Biomaterials
- Polymers and Plastics
- Materials Chemistry
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In: Journal of Bioactive and Compatible Polymers, Vol. 14, No. 3, 05.1999, p. 213-231.
Research output: Contribution to journal › Article › peer-review
}
TY - JOUR
T1 - Conjugates of semitelechelic poly[N-(2-hydroxypropyl)methacrylamide] with enzymes for protein delivery
AU - Oupický, David
AU - Ulbrich, Karel
AU - Říhová, Blanka
N1 - Funding Information: Oupický David Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, 162 06 Prague, Czech Republic Ulbrich Karel Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, 162 06 Prague, Czech Republic Říhová Blanka Institute of Microbiology, Academy of Sciences of the Czech Republic, 142 20 Prague, Czech Republic 05 1999 14 3 213 231 The semitelechelic poly[N-(2-hydroxypropyl)methacrylamide] [poly(HPMA)] with a carboxyl end group was prepared with 3-mercaptopropionic acid as a chain transfer agent. Bovine seminal ribonuclease (BSR) and α-chymotrypsin (ChT) were modified with various molecular weights of active poly(HPMA) succinimidyl ester by the reaction with the amino groups of the respective enzyme. The modification of ChT did not significantly change the activity or the substrate specificity of the conjugates towards low-molecular-weight tripeptidic substrates. However, modified ChT activity towards the corresponding poly(ethylene glycol)-based synthetic substrate was significant. The activity decreased as a result of the elevated steric hindrance to the active site of the polymer-modified enzyme. Similarly, the ChT conjugates completely lost their proteolytic activity toward native bovine serum albumin. The autolytic stability of ChT conjugates was improved and the proteolytic stability of the ChT and BSR conjugates substantially increased compared with the free enzymes. The modification of ChT with poly(HPMA) significantly decreased the immunogenicity of ChT conjugates depending on the molecular weight of the poly(HPMA) and the degree of enzyme substitution. sagemeta-type Journal Article search-text Conjugates of Semitelechelic Poly[N- (2-hydroxypropyl)meth- acrylamide] with Enzymes for Protein Delivery DAVID OUPICKV AND KAREL ULBRICH* Institute of Macromolecular Chemistry Academy of Sciences of the Czech Republic 162 06 Prague, Czech Republic BLANKA PIHOVA Institute of Microbiology Academy of Sciences of the Czech Republic 142 20 Prague, Czech Republic ABSTRACT: The semitelechelic poly[N-(2-hydroxypropyl)methacrylamideI [poly(HPMA)] with a carboxyl end group was prepared with 3-mercaptopropionic acid as a chain transfer agent. Bovine seminal ribonuclease (BSR) and a-chymotrypsin (ChT) were modified with various molecular weights of active poly(HPMA) succinimidyl ester by the reaction with the amino groups of the re- spective enzyme. The modification of ChT did not significantly change the activ- ity or the substrate specificity of the conjugates towards low-molecular-weight tripeptidic substrates. However, modified ChT activity towards the correspond- ing polyethylene glycol)-based synthetic substrate was significant. The activity decreased as a result of the elevated steric hindrance to the active site of the poly- mer-modified enzyme. Similarly, the ChT conjugates completely lost their proteolytic activity toward native bovine serum albumin. The autolytic stability of ChT conjugates was improved and the proteolytic stability of the ChT and BSR conjugates substantially increased compared with the free enzymes. The modifi- cation of ChT with poly(HPMA) significantly decreased the immunogenicity of ChT conjugates depending on the molecular weight of the poly(HPMA) and the degree of enzyme substitution. *Author to whom correspondence should be addressed. Journal of BIOACTIVE AND COMPATIBLE POLYMERS, Vol. 14 -May 1999 213 0883-9115/99/03 0213-19 $10.00/0 1999 Technomic Publishing Co., Inc. DAVID OUPICKV, KAREL ULBRICH AND BLANKA MIHOVA INTRODUCTION CONJUGATION OF WATER-SOLUBLE polymers with pharmacologically active proteins can be used to reduce the proteolytic degradation of such proteins, improve their biological efficacy and prolong plasma elimi- nation, as well as reduce protein immunogenicity [1,2]. A variety of poly- mers have been used for the covalent modification of enzymes and proteins of therapeutic importance. General requirements for any poly- mer used for this purpose are that it should be water-soluble, biocompatible, non-immunogenic, and devoid of biological activity. The most extensively studied synthetic polymer for the modification of proteins is poly(ethylene glycol) (PEG) [3,4]. An important reason for its attractiveness is the fact that it has been approved by regulatory authori- ties for several pharmaceutical products. Within the last fifteen years, several PEG conjugates have been evaluated clinically [5] including con- jugates with asparaginase [6], adenosine deaminase [7], interleukin-2 [8], superoxide dismutase [9], hemoglobin [10], and uricase [11]. Protein conjugates containing dextran [12] or synthetic polymers other than PEG have also been described, for example, poly[N-(2-hydroxy- propyl)methacrylamide] [poly(HPMA)], poly(N-vinylpyrrolidone) [13], copolymer divinyl ether-maleic acid [14] or poly(N-isopropylacrylamide) [15]. Earlier, poly(HPMA) was used to modify a-chymotrypsin [16] and insu- lin [17] as well as conjugate antibodies as a targetable carrier for drugs [18]. In all these cases, the protein was attached to the poly(HPMA) chain via multiple linkages situated on the side chain of the polymer backbone. This work presents the synthesis and physicochemical and biological characterization of poly(HPMA)-protein conjugates in which the poly- mer chains are attached to the protein molecule via a single polymer end-chain linkage. First, semitelechelic poly(HPMA) was used to modify a-chymotrypsin (ChT), which is a model enzyme used to study poly- mer-enzyme conjugation and physicochemical and biological properties of such conjugates. Then bovine seminal ribonuclease (BSR), an enzyme with potential anti-cancer activity, was conjugated with the semitelechelic poly(HPMA). BSR is a homolog of bovine pancreatic ribo- nuclease A (RNase A). Unlike RNase A, BSR exhibits specific antitumor, aspermatogenic, and immunosuppressive activities [19]. BSR also shows significant anti-cancer activity when administered intratumorally. No activity for BSR has been observed when it was administered i.p., s.c. or i.v. [20,21]. The aim of the poly(HPMA) modification of BSR was to pre- pare conjugates that are stable in the blood stream with the potential as an i.v. anti-cancer drug. 214 Conjugates of Semitelechelic Poly[N-(2-hydroxypropyl)methacrylamide] 215 EXPERIMENTAL Materials and Methods N,N'-Dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (HOSu), 4-dimethylaminopyridine (DMAP), 3-mercaptopropionic acid (MPA), 2,2'-azobisisobutyronitrile (AIBN), phthalaldehyde (OPA), bo- vine pancreatic a-chymotrypsin (ChT) and bovine serum albumin (BSA) were purchased from Fluka Chemie AG, and 2,4,6-trinitrobenzene- sulfonic acid (TNBSA) was purchased from Serva. BSR was a kind gift from Dr. Matousek (Institute of Animal Genetics). N-(2-Hydroxy- propyl)methacrylamide (HPMA) was synthesized as reported [22]. Oligopeptidic substrates HO GlyGlyPheNAp and HOl GlyValPheNAp were prepared as reported [23]. Polymeric substrate PEG-Glu(OH)- NH(CH2)2NH-Glu(GlyValPheNAp)-PEG was prepared using PEG, mo- lecular weight 2,000, as reported [24]. All other reagents were of analyti- cal grade. Synthesis of Semitelechelic Poly(HPMA) Precipitation polymerization of HPMA in acetone was carried out in the presence of the chain transfer agent 3-mercaptopropionic acid (MPA) at 50TC for 24 h. The concentrations were as follows: initiator AIBN 2.7 x 103 M, HPMA 0.798 M and MPA in the range 6 x 104 -9 x 10-2 M. The re- sulting polymers with one carboxylic end-group were purified by double precipitation from methanol solution into a 20-fold excess of mixture ace- tone:diethyl ether (3:1). End-functionalized poly(HPMA)s [poly- (HPMA)-COOH] of different molecular weights were prepared by changing the HPMAIMPA ratio. Synthesis of Hydroxysuccinimide Ester of Semitelechelic Poly(HPMA) The end carboxylic group was converted into active succinimidyl ester by the reaction common in peptide synthesis. Poly(HPMA)-COOH (0.17 mmol) and N-hydroxysuccinimide (HOSu) (1. 70 mmol) were dissolved in 6 mL of DMF (dried with P205, freshly distilled before use) and cooled to -20oC, to this solution, DCC (1.70 mmol) and DMAP (0.33 mmol) in 5 mL of DMF were added. The solution was left overnight at 00C. The NN'-dicyclohexylurea which precipitated during reaction was removed by filtration and the polymer was isolated by precipitation into a mixture DAVID OUPICKY, KAREL ULBRICH AND BLANKA ?UHOVA of dry acetone:diethyl ether (3:1). The succinimidyl ester of poly- (HPMA)-COOH [poly(HPMA)-COOSu] was dried and used directly in the subsequent reaction of enzyme modification. Synthesis of Poly(HPMA) Conjugates a-Chymotrypsin (49 mg, 0.0274 mmol of amino groups) was dissolved in 20 mL of 0.15 M NaCl solution with 0.01 M CaCl2, the solution was cooled to 50C and pH was adjusted to 7.8 with a saturated solution of Na2B407. To this solution, poly(HPMA)-COOSu (0.163 mmol) was added under vigorous stirring. The pH was maintained at 7.8 for 2 h, then brought to 8.0 for 3 h. After 5 h the pH was brought to 8.5 for 0.5 h to hy- drolyze the remaining active ester. The ChT conjugate was isolated from unreacted polymer and low-molecular-weight compounds by preparative size exclusion chromatography using a Sephacryl S300 column (26 x 600 mm, flow rate 12.5 mL/h, 100C and 1 mM HOl as the mobile phase). The fraction containing the conjugate was collected and lyophilized. The conjugates of ChT with two other poly(HPMA)-COOH and the conjugate of BSR were prepared using the same poly(HPMA)-COOSu/ enzyme amino group ratio as described above. Physicochemical Characterization The molecular weights and number-average molecular weights of the semitelechelic poly(HPMA)s were determined by size exclusion chroma- tography (SEC) in 0.05 M TRIS buffer pH 8.1 on a SuperoseTm 12 column (Pharmacia). The column was calibrated usingbroad-range poly(HPMA) standards [25] characterized by Multi-Angle Laser Light Scattering. The content of the end carboxylic groups was determined by titration with 0.05 M NaOH using an automatic titrator (Radiometer). The amount of substituted amino groups on ChT or BSR was determined by the TNBSA assay according to the method of Snyder [26]. The ChT content was evaluated spectrophotometrically (28 = 4.5 X 104 L mol-l cm-t) and by amino acid analysis. The BSR content was evaluated by amino acid analysis. For the amino acid analysis, the protein was hydrolyzed at 1100C for 16 h in 6 M HCl. The hydrolyzed sample was dried over NaOH in vacuo, dissolved in distilled water and then analyzed on an LDC-Analytical amino acid analyzer with a NUCLEOSIL 120-3 C18 (125 x 4 mm) reverse phase column (Macherey Nagel) with precolumn derivatization with OPA using gradient elution with sodium acetate buffer (pH 6.5)-MeOH 0-70%. 216 Conjugates of Semitelechelic Poly[N-(2-hydroxypropyl)methacrylamide] 217 The molecular weights of the conjugates were calculated from the known number of substituted amino groups and the molecular weight of the poly(HPMA) used or, alternatively, from the ChT content. Molecular weights determined by SEC using poly(HPMA) calibration were also cal- culated and were used for comparison of the conjugates. The purity of the purified conjugates was checked by SDS-PAGE elec- trophoresis on the Phastsystem gradient gels 7-15 (Pharmacia LKB). No free ChT or BSR were detected. The apparent hydrodynamic radii (Rhapp) of the conjugates at finite di- lution were determined by dynamic light scattering (DLS). Polarized DLS measurements were made using the apparatus and technique de- scribed in Reference [27]. The angular dependence of Rh,app was not very important due to small sizes of the molecules. Enzymatic Activity of ChT, BSR and Their Conjugates Stock solutions of native ChT and conjugates were prepared in water. The activity of ChT (59% of active protein) was determined by titrating the active site with Z-Tyr-ONp [28]. Stock solutions of low- molecular-weight substrates were prepared in DMSO, the final concen- tration of DMSO in the incubation media not exceeding 10% (vlv). The so- lution of polymeric substrate in the TRIS buffer was prepared and the concentration of 4-nitroanilide groups was determined spectrophotomet- rically (8316 = 12,800 L * mol-1 . cm-'). The measurements of enzymatic ac- tivity were performed in 0.08 M-TRIS buffer (pH 8.0) with 0.1 M CaCl2. The release of 4-nitroaniline was monitored spectrophotometrically at 410 nm (e= 8440 L * mol' . cm-') at 250C and initial rates were calculated. The concentrations in incubation media were as follows: 8.5 x 108 M ChT (HCl GlyValPheNAp and PEG-based substrates); 7.5 x 104 M ChT (HCl * GlyGlyPheNAp); 1 x 10 -5 x 104 M substrates. The kinetic pa- rameters were determined from a Lineweaver-Burk plot of the initial rates of the 4-nitroaniline appearance. The error in the enzymatic activ- ity did not exceed 10% of the values. Enzymatic activity of native and modified BSR was evaluated by the method of Crook [29] using cytidine 2 -:3 '-monophosphate as the sub- strate at two different pH's (7.1 and 5.5). The ability of free ChT and its conjugates to cleave native protein sub- strate was tested on bovine serum albumin at 370C. The incubation mix- ture in 0.1 M TRIS buffer (pH 8.1) contained 10 mg/mL BSA and 2 x 10- M of the appropriate ChT derivative. The samples were withdrawn at specific time intervals, diluted and analyzed by SEC on a Superose 12 col- umn using the conditions mentioned above. The degradation of BSA was DAVID OUPICKV, KAREL ULBRICH AND BLANKA kIHOVA monitored by the decrease in the peak area of BSA with time, using UV and differential refractometric detectors. Autolytic Stability of ChT Conjugates Autolysis of ChT and its conjugates was tested at pH 8.1 in 0.1 M TRIS buffer in the absence of CaCl2. A 0.5 mg protein/mL enzyme solution was prepared and the solution was incubated at 370C. Aliquots were with- drawn from the solution at specific time intervals and the activity deter- mined using HCl * GlyGlyPheNAp as the substrate. Proteolytic Stability of ChT, BSR and Their Conjugates Proteolytic stability of ChT and its conjugates to elastase was tested in 0.1 M TRIS buffer (pH 7.4). The enzymes were dissolved to obtain a final concentration of 0.5 mg protein/mL and were digested at 370C with 0.88 mg/mL of elastase. Proteolytic stability of BSR and its conjugate was esti- mated by incubating 0.5 mg/mL enzyme solution (0.1 M TRIS, pH 7.4) in the presence of 5 mg/mL a-chymotrypsin. Immunogenicity of ChT and BSR Conjugates All experiments were done on 3-month-old female mice of inbred strain A/J from the colony of the Institute of Physiology. Conjugates of N-(2-hydroxypropyl)methacrylamide (P1-ChT - P3-ChT) or nonmodi- fled a-chymotrypsin was used for immunization. Immunization: A control serum taken from the experimental mice be- fore starting the experiment was used. Mice were immunized three times in two-week intervals with 50 ,g of sample, either incorporated in a com- plete Freund's adjuvant (CFA) or applied as a solution. Samples were in- jected subcutaneously (0.2 mL s.c., solution or 0.4 mL s.c., CFA). On day 10, after the last immunization, the mice were exsanguinated and the separated serum stored at -70C. The antibody level in the serum was de- termined by ELISA. Detection of antibodies by ELISA indirect method (double layer): Ab- sorption of antigen on microplates (NUNC Immunoplate maxi Sorp F 96), proceeded overnight at 4C. The wells were filled with 50 ,L of sam- ple (0.5-1 tg/mL). Two separate identical plates were done simulta- neously. The next day, the microplates were rinsed with PBS + 0.1% Tween 20 and incubated at room temperature for 2 h in PBS containing 0.02% gelatin and 1% bovine serum albumin (BSA) to decrease the non-specific binding of immunoglobulins. They were rinsed five more 218 Conjugates of Semitelechelic Poly[N-(2-hydroxypropyl)methacrylamide] 219 times with PBS and PBS with 0.2% Tween 20, filled with 50AL of differ- ent dilutions of the tested sera and the plates were kept overnight at 40C. On the next day, the microplates were rinsed and horse-radish peroxidase-conjugate, affinity purified porcine anti-mouse IgG (Institute of Sera and Vaccines, Prague) was diluted 1:500 and added. After 1 h in- cubation, the conjugate was removed, the microplates rinsed and 0.015% H202 with 1,2-phenylenediamine (Sigma, 5 mg/10 mL substrate) was added. The reaction was stopped after 10 min at room temperature with 20 AL of 2 M H2SO4 and the absorbance of the colored product was mea- sured using a semiautomatic Titertek Multiscan Reader MCC/340 (Flow Laboratories) at a wavelength of 492 nm against a row of wells treated with the substrate. The analysis was repeated using three samples. Every assay included positive and negative control, and a buffer blank to control the specificity of the reaction. Titer of antibodies represents the dilution of sera when absorbance was higher than in the control by 3SD. RESULTS AND DISCUSSION To achieve optimum conditions for the preparation of semitelechelic polymers with a broad range of molecular weights and suitable for en- zyme modification, attention has to be paid to the proper choice of chain transfer agent with a suitable chain transfer constant CT. The chain transfer constant for a pair of monomer-chain transfer agents is defined as a ratio of the rate constant for the propagation reaction of polymer to the rate constant of the transfer reaction [30]. From a practical stand- point, it is not convenient to use a chain transfer agent with a CT less than unity, because in that case even at moderate conversions, the polymeriza- tion degree rapidly increases due to the preferential consumption of the chain transfer agent. As a result, the polymers with very broad polydispersity are obtained. The broadening of the polymer polydispersity is not rapid if 1 < CT < 2 and the reaction is not pushed over about 60% yield [31]. Semitelechelic poly(HPMA)s of general for- mula shown in Figure 1, were prepared by radical polymerization in the presence of 3-mercaptopropionic acid (MPA). The transfer constant for MPA used in this work was estimated to be close to 2 (data not shown); for the reasons stated above, the polymerization was conducted to maximum 40% conversion. Table 1 shows characteristics of the three semitelechelic poly(HPMA)s used for ChT and BSR modification. By changing the monomer/chain transfer agent ratio from 8 to 133, the resulting num- ber-average molecular weight (MJ) ranged from 2000 to 11,000 as deter- mined by SEC. DAVID OUPICKY, KAREL ULBRICH AND BLANKA kfHOVA CH3 CH2-C -S-CH2CH2-COOH 0;=0 NH -n I CH2 CH-OH OH3 Figure 1. Semitelechelic poly(HPMA). The lower polydispersity (Mw/Mj) with decreasing molecular weight was obtained by removing the low-molecular-weight fractions by reprecipitation of the polymer. The important parameter of semitelechelic polymers designed for protein modification is their func- tionality. The content of the functional carboxyl end group in the poly- mers was determined by the titration with NaOH. The number-average molecular weight was calculated from carboxyl group content, assuming one carboxyl end group per macromolecule. A comparison of this value with the value of molecular weight (Ma) determined by SEC, gives the in- formation about the polymer functionality. The data shown in Table 1 in- dicates that the functionality of the P2 and P3 polymers is practically unity and the amount of unfunctionalized macromolecules is negligible. The disproportion in the case of P1 sample is given by a very low molecu- lar weight of the sample which is almost on the separation limit of the used column. Various functional groups in protein molecule can be used for modifica- tion, but most utilize reagents designed for attachment to the lysine amino groups. The most widely used are active succinimidyl esters (or similar reagents like succinimidyl carbonates) of PEG which react with Table 1. Molecular weights of semitelechelic poly(HPMA) used for enzyme modification. (HPMA)/ Polymer (MPA) (1) Mn (2) Mn(2) P1 8 2940 2690 1710 P2 44 7250 5090 4830 P3 133 19,470 10,920 11,180 O1)Determined by SEC. O2)Calculated from end-group titration. 220 Conjugates of Semitelechelic Poly[N-(2-hydroxypropyl)methacrylamide] 221 amino groups of proteins within a short period of time under mild condi- tions [32] and produce modified proteins with well preserved biological activities [331. Therefore, we used succinimidyl esters of semitelechelic poly(HPMA)s. The active esters, prepared using DCC and HOSu, were used immediately after isolation and drying. In order to avoid hydrolysis, the solid active ester of poly(HPMA) was added directly to a solution of the corresponding enzyme. The data in Table 2 show characteristics of the prepared conjugates. The amount of ChT conjugated was determined by two methods. ChT has a relatively high molar absorption coefficient and its concentration can be well determined spectrophotometrically, although the absorption coef- ficient may change due to the modification. These values were compared with the results obtained by a second method to determine ChT content, amino acid analysis. Amino acid analysis is a direct and reliable method; good agreement with data obtained spectrophotometrically suggests that in this particular case the absorption coefficient of ChT was not signifi- cantly influenced. The amount of BSR in the conjugate was determined only by amino acid analysis due to a low absorption coefficient of this en- zyme. The degree of modification of enzyme amino groups was deter- mined by the TNBSA assay. All the conjugates in this work were prepared using the same ratio be- tween poly(HPMA)-COOSu and accessible amino groups of the appropri- ate enzyme (6:1). ChT contains 17 amino groups (14 Lys and 3 a-amino groups) from which only 14 a-amino groups of lysine are accessible. Ac- cordingly [19], BSR contains 22 accessible amino groups. A large excess of Table 2. Basic characteristics of ChT and BSR conjugates with poly(HPMA). % of ChT ChT Modified Content(1) Content(2) Amino Molecular Molecular Rhapp Sample (%) (%) Groups Weight(3) Weight(4) (nm) ChT 100 100 0 25.000 3.4 0.1 P1-ChT 34.0 38.4 91 65,100 59,300 4.9 0.4 P2-ChT 27.8 27.4 91 91,240 89,900 6.8 0.1 P3-ChT 18.7 17.2 64 145,300 122,800 7.2 0.2 P3-BSR N.D. 18.0 67 7.3 0.2 O1)Determined spectrophotometrically. (2)Determined by amino acid analysis. P31Calculated from the ChT content in the conjugates: M = 100 M(ChT)/(ChT content as determined by amino acid analysis). (4)Calculated from the degree of modification of amino groups [14 amino groups of ChT were used as 100% and Mn obtained from SEC was used for poly(HPMA)]: M = M(ChT) + Mn[poly(HPMA)] x (number of modi- fied amino groups of ChT). DAVID OUPICKV, KAREL ULBRICH AND BLANKA fHOVA poly(HPMA)-COOSu was used in order to achieve as high a degree of amino group modification as possible. Despite using the same molar con- centration, the degree of modification of P1-ChT and P2-ChT (-90%) was significantly higher than in the case of P3-ChT (64%). The reason may be steric hindrance to accessibility to the amino groups after some degree of protein modification for the P3 polymer which has the highest molecular weight, while with the lower molecular weight polymers P1 and P2, the hindrance is not so restricting. Analytical SEC was used for further characterization of the poly- (HPMA)-enzyme conjugates. The elution profiles of ChT and its conju- gates are shown in Figure 2; molecular weights of the conjugates increased with molecular weight of the modifying poly(HPMA). The broadening of the conjugate peaks compared with the peak of unmodified ChT demonstrates the increasing heterogeneity of the conjugates (the same effect was demonstrated by electrophoresis; not shown). The heter- ogeneity is caused by the different degree of modification of the protein amino groups and the polydispersity of the poly(HPMA). The first type of heterogeneity can be suppressed by a high degree of protein modification because at lower modification, there is a higher possibility of different de- grees of modification. This can be assumed for P1-ChT and P2-ChT, where almost all the available protein amino groups are modified. P3-ChT is the most polydisperse of the three conjugates because of the higher polydispersity of P3 and because its lower degree of amino group modification. Shown in Figure 2 are the elution volumes of three protein standards which demonstrated the large change in hydrodynamic volume caused by the ChT modification with relatively low-molecular-weight poly(HPMA). The dramatic change in the hydrodynamic volume of the conjugates is due to the different solution behavior of the two components of the conju- gates. Whereas protein structures are very compact, the poly(HPMA) coils are extended. This fact causes difficulties in column calibration when trying to determine the molecular weight of the conjugates by SEC. The difficulties were demonstrated with P2-ChT. From Figure 2, the mo- lecular weight of P2-ChT can be estimated using protein standards close to that of aldolase (molecular weight 158,000), while using poly(HPMA) calibration, the molecular weight would be 35,900. The conjugate mole- cule is composed of the two different components and so the actual molec- ular weight should be between these limits. An easy way to calculate the actual molecular weight of the conjugates is from the ChT content or from the degree of modification of ChT amino groups. The results of both calculations are listed in the fourth and fifth columns of Table 2. The first method of calculation relies on the accuracy of ChT determination, while 222 Conjugates of Semitelechelic Poly[N-(2-hydroxypropyl)methacrylamide] 223 1.2 1 2 3 ChT 1.0 ---P1-ChT P2-ChT / l l --P3-ChT 0.4 1 0.2 a i 0.0 - 0 5 10 15 20 25 Elution volume [mL] Figure 2. SEC elution profiles of ChT and its conjugates (elution volumes of protein stan- dards 1-ferritin, 2-aldolase, 3-albumin). the second one relies on the determination accuracy of the degree of amino group modification. Amino acid analysis used in this study for the determination of protein content is a direct and reliable method. The ob- served good agreement between values of both molecular weights in the fifth and sixth columns of Table 2 serves as support that the TNBSA method gives reliable results, even though this method is considered to be subject to large errors [34]. It has been shown that the blood clearance time of PEG conjugates was related more to the effective size rather than to the molecular weight. An abrupt reduction in clearance was seen when the conjugate size exceeded the permeability threshold of the kidney [35], around 70,000 for proteins, which corresponds to the molecular weight of serum albumin. This means that increasing the hydrodynamic volume of the conjugates above that of albumin should decrease the systemic clearance rate. Shown in Figure 2 poly(HPMA) with a molecular weight of 5000 (P2), increased the hydrody- namic volume of the conjugate P2-ChT above that of albumin. The hydro- dynamic size can be more accurately determined by dynamic light scattering (DLS) rather than by size exclusion chromatography (SEC). The apparent hydrodynamic radii determined by DLS are also shown in Table 2. The last column of the table shows a clear increase in the size of the conju- gates with increasing molecular weight of the used polymer. The apparent DAVID OUPICKV, KAREL ULBRICH AND BLANKA MIHOVA hydrodynamic radius of ChT was more than doubled after conjugation with the three poly(HPMA)s. In many cases, enzyme modification leads to substantial loss of the en- zymatic activity which is explained by denaturation of the protein struc- ture during modification, modification of essential functional groups, refolding of the protein chain upon attachment of polymer, altering the active site making it less effective, or by steric hindrance preventing ac- cess of substrates to the active site. Table 3 shows the relative activities of the modified ChT and BSR. The enzymatic activity decreased both the ChT and BSR conjugates. However, the relative activity of the ChT conju- gates increased with increasing poly(HPMA) molecular weight with P3 in both cases giving the best activity toward small synthetic substrates. The significant decrease in enzymatic activity of ChT conjugates to- ward a PEG substrate observed for P1-ChT and P3-ChT is probably due to steric hindrance to the active site by the polymeric substrate [36]. The ability of the PEG substrate to access the active site of ChT was inversely proportional to the thickness of the protecting poly(HPMA) shell. The proteolytic activity of ChT conjugates toward albumin (native protein substrate) is shown in Figure 3. Albumin was readily degraded by the un- modified ChT, while all three conjugates showed no activity towards this substrate. The loss of the proteolytic activity towards native proteins is probably due to the protecting poly(HPMA) shell as in the case of PEG. Unlike the PEG, BSA is not able to penetrate the poly(HPMA) shell of the conjugates. A similar loss of activity of ChT modified with PEG (5000) to- ward BSA was reported by Chiu [37]. The P3-BSR conjugate with 67% of the protein amino groups modified with P3 retained 84% activity (Table 3) with respect to the native en- zyme. Changes in the relative activity of conjugate with change of pH from 7.1 to 5.5 were observed. The activity of unmodified BSR was 1820 Table 3. Relative activities of ChT conjugates. Activity(1) Activity(2) Activity(3) Conjugate (%/0) (%) (%) P1-ChT 74 72 64 P2-ChT 88 88 N.D. P3-ChT 109 112 46 P3-BSR 84(1) - (1)HCI GlyVaiPheNAp. (2)CH0 GlyGlyPheNAp. (3) PEG2-GlyValPheNAp. (4)Cytidine 2':3' cyclic monophosphate. 224 Conjugates of Semitelechelic Poly[N-(2-hydroxypropyl)methacrylamide] 225 100 * * * 80 \ ChT -.\-P1-ChT 60 .SX X 40 20 0 0 2 4 6 8 10 12 14 16 Time of proteolysis [hJ Figure 3. Time course of proteolysis of albumin by ChT and its conjugate (at 370C in 0.1 M TRIS buffer, pH 8.1, 10 mg/mL BSA, 2 x 105 M ChT or the conjugate). U/g (units per g of enzyme) at pH 7.1 and 1020 U/g at pH 5.5. The activity of P3-BSR conjugate was 1530 U/g at pH 7.1 and 1500 U/g at pH 5.0. Al- though the activity of the conjugate did not significantly change with pH the activity of the unmodified BSR was decreased (to 56% of that at pH 7.1). The reason for the difference in the behavior of BSR and its conju- gate could be due to the change in the available amino groups on BSR af- ter modification. In order to get a better understanding of the changes in the ChT activity induced by poly(HPMA) modification, kinetic study was performed using three different substrates. These differed in their suitability as a substrate for ChT (GlyGlyPheNAp, GlyValPheNAp) and their molecular weight [GlyValPheNAp, PEG-Glu(OH)-NH(CH2)2NH-Glu(GlyValPheNAp)PEG], as shown in Table 4. The native ChT and its conjugates exhibited activity toward all the synthetic 4-nitroanilide substrates examined. The relative activity ratios for low-molecular-weight substrates remained approxi- mately the same for all the conjugates and the modification did not appar- ently cause a dramatic change in specificity. A comparison of P1-ChT with P2-ChT makes it possible to see the effect of the molecular weight of poly(HPMA) on the enzymatic activity of the conjugates. The higher ac- tivity of P2-ChT toward both low-molecular-weight substrates, due to DAVID OUPICKY, KAREL ULBRICH AND BLANKA 1MHOVA Table 4. Michaelis-Menten parameters for the kinetics of hydrolysis of some substrates with ChT conjugates. GlyGlyPheNAp GlyValPheNAp PEG2GlyValPheNAp k2 KM k2 KM k2 KM Sample (so1) (mM) k2WKM (s1) (mM) k2lKM (s1) (mM) k2IKM ChT 0.353 1.052 335 12.97 1.565 8290 6.05 0.673 8990 P1 -ChT 0.348 1.807 193 11.15 1.770 6300 4.46 1.247 3570 P2-ChT 0.372 1.612 231 11.45 1.458 7850 N.D. N.D. N.D. P3-ChT 0.424 1.364 311 16.29 1.693 9620 4.24 2.152 1970 lower Km, indicate better affinity of P2-ChT conjugate toward these sub- strates. The difference could be given by the very low molecular weight of P1 which can react with amino groups that are not accessible for the poly(HPMA)s with higher molecular weights and this can cause damage of the active site. The most active conjugate toward the low- molecular-weight substrates was P3-ChT; in the case of HCl GlyGly- PheNAp, it was even more active than unmodified ChT. The high activity is given in both cases by a high k2 and in the case of HC1 * GlyGlyPheNAp, also by a low KM. The increasing activity of the conjugates (P1-ChT -* P3-ChT) may also be due to increases in the hydrophilic poly(HPMA) which could have a solubilizing effect and thus "open" the access to the ac- tive site of the enzyme for low-molecular-weight substrates. The activity toward PEG substrates shows an opposite dependence on the poly(HPMA) content than in the case of low-molecular-weight substrates. With this substrate, P3-ChT was less active, mainly due to high KM. As already mentioned, one of the most important goals in the modifica- tion of enzymes is to increase their stability. It is well known that ChT readily undergoes autolysis, which could be slowed down by the presence of Ca2+ ions stabilizing the enzyme molecules in solution. Shown in Fig- ure 4 the autolysis of the conjugates is almost entirely suppressed com- pared with the unmodified ChT. These results are consistent with the loss of activity toward native protein substrates, although there is some mini- mal decrease in the activity of the conjugates (5-10%). The differences among the conjugates were more pronounced after a 24 hour autolysis. Unmodified native ChT lost over 90% of its original activity, whereas P1-ChT 25%, P2-ChT 20% and P3-ChT 10% of their original activity. It seems that the highest molecular weight poly(HPMA) gives the conju- gate the highest autolytic stability. Although the conjugates did not show any proteolytic activity toward albumin over the time range studied, it is possible that they could exhibit some activity over a longer period of time. The results obtained after a 24 hour incubation would then be in good 226 Conjugates of Semitelechelic Poly[N-(2-hydroxypropyl)methacrylamide] 227 120 100I S 80 _ \ + ~~~~~~~~ChT ^~~~~~~~~~~~~~~~ 60 P-ChT "60 B \ - ~~~~~~~~~P2-ChT 40 A: 40 b ~~~~~~~~P3-ChT 20 0 0 1 2 3 4 5 6 Time [hi Figure 4. Autolysis of ChT and its conjugates (at 3700 in 0.1 M TRIS buffer, pH 8.1, 0.5 mg/mL protein). agreement with the data on the enzymatic activity towards PEG sub- strate, where the P3-ChT showed the lowest activity of all the conjugates. Stability to proteolytic enzymes is one of the most important require- ments for the conjugates used for therapeutic purposes. The proteolytic stability of ChT and its conjugates was examined by incubation with elastase. It was necessary to choose concentrations that would minimize the effect of ChT autolysis and the proteolysis of elastase by ChT, espe- cially for the digestion of unmodified native ChT. The results of proteoly- sis of ChT and its conjugates are presented in Figure 5. The rate of proteolysis of unmodified ChT is very fast; after 60 minutes the activity decreased to only 5% of its original value. The proteolytic stability of the ChT was significantly enhanced after modification with poly(HPMA). The most stable was the P3-ChT conjugate which decreased access of elastase to ChT through the protecting shell of poly(HPMA). The results of the proteolytic stability obtained for BSR and its conju- gate are shown in Figure 6. In this case, the enzyme was incubated with ChT, since the digestion with elastase was relatively slow. Similar to the ChT conjugates, BSR proteolysis was significantly reduced after modifi- cation with poly(HPMA). In addition to preserving activity, good proteolytic stability and pro- longed persistence in circulation, the most important factor limiting the 228 DAVID OuPIcKV, KAREL ULBRICH AND BLANKA IfHOVA 1001 80 \ ~60 40- A \ P2-ChT -*P3-ChT 20 0 10 20 30 40 50 60 Time [min] Figure 5. Proteolysis of ChT and its conjugates by elastase (at 370C in 0.1 M TRIS buffer, pH 7.4, 0.88 mg/mL elastase, 0.5 mg/mL ChT). -70 60 ,! 50 ,40 P: 0 1 2 3 4 5 6 Time [hj Figure 6. Proteolysis of BSR and its conjugate by ChT (at 37TC in 0.1 M TRIS buffer, pH 7.4,5 mg/mL ChT, 0.5 mg/mL BSR). Conjugates of Semitelechelic Poly[N-(2-hydroxypropyl)methacrylamide] 229 Table 5. Immunogenicity of ChTl BSR and its conjugates. Sample Antibody Titer ChT 250,000 P1-ChT 144,000 P2-ChT 13,000 P3-ChT 9000 BSR 10,486,000 P3-BSR 621,000 use of enzymes for therapy is immunogenicity of conjugates. The data on the immunogenicity of poly(HPMA)-enzyme conjugates are shown in Table 5. Significant decreases in immunogenicity of chymotrypsin after modification with poly(HPMA) were observed. This decrease is more pro- nounced with increasing poly(HPMA) molecular weight. The decrease in immunogenicity of the conjugate Pl-ChT is very low, indicating that the poly(HPMA) shell on the protein surface is not thick enough to prevent the enzyme from being recognized by the immune system. The dramatic decrease with the conjugates P2-ChT and P3-ChT is in accordance with the results of several authors, who showed that the attachment of high-molecular-weight PEGs was more efficient in reducing or eliminat- ing antigenic or immunogenic properties of proteins than the use of their low-molecular-weight homolognes [38,39]. The immunogenicity of BSR was also significantly decreased after modification with poly(HPMA) as shown in Table 5. The decrease is relatively lower compared to the chymotrypsin conjugates. The reason for the dramatic decrease in the immunogenicity of P3-BSR compared to the chymotrypsin conjugates re- quires more detailed study, which is under way. CONCLUSIONS Poly(HPMA) conjugates of chymotrypsin or bovine seminal ribonuclease, an enzyme with anti-cancer activity, have been synthe- sized. The conjugation of poly(HPMA) with chymotrypsin does not influ- ence the substrate specificity and enzymatic activity toward low-molecular-weight substrates. In contrast, the enzymatic activity to- ward protein substrate BSA was totally suppressed. The ChT conjugates showed significantly improved autolytic and proteolytic stability com- pared with unmodified ChT. The immunogenicity of ChT conjugates was dramatically decreased with increasing molecular weight of poly(HPMA). The activity of the BSR conjugate was well preserved and DAVID OUPICKV, KAREL ULBRICH AND BLANKA MIHOVA the proteolytic stability was improved similarly to that of ChT conju- gates. The immunogenicity of BSR conjugate was also decreased compare to free BSR. ACKNOWLEDGMENT The authors thank the Grant Agency of the Czech Republic for sup- porting the project by grant no. 307/96/K226. REFERENCES 1. R. Duncan and F. Spreafico. 1994. Clin. Pharmacokinet., 27, 290. 2. A. H. Sehon, 1992. Poly(Ethylene Glycol) Chemistry: Biotechnical and Bio- medical Applications. Harris, J. M., ed. New York: Plenum Press, p. 139. 3. N. V Katre. 1993. Adv. Drug Delivery Rev., 10, 91. 4. F. Fuertges and A. Abuchowski. 1990. J. Controlled Release, 11, 139. 5. D. Putnam and J. Kopecek. 1995. Adv. Polym. Sci., 122, 57. 6. D. H. Ho, N. S. Brown, and A. Yen. 1986. Drug Metab. Dispos., 14, 349. 7. M. S. Hershfield, R. H. Buckley, and M. L. Greenberg. 1987. New Engl. J. Med., 316, 589. 8. F. J. Meyers, C. Paradise, and S. A. Scudder. 1991. Clin. Pharmacol. Ther., 49, 307. 9. P. D. Thomson, G. Till, and J. 0. Wolliscroft. 1990. Burns, 16, 406. 10. M. L. Nucci, R. Shorr, and A. Abuchowski. 1991. Adv. Drug Delivery Rev., 6, 133. 11. S. Davis, Y K. Park, and F F. Davis. 1981. Lancet, 2, 281. 12. R. Fagnani, M. S. Hagan, and R. Bartholomew. 1990. Cancer Res., 50,3638. 13. P Caliceti, 0. Schiavon, M. Morpurgo, and F. M. Veronese. 1995. J. Bioact. Compat. Polym., 10, 103. 14. H. Maeda, T. Oda, and Y Matsumura. 1988. J. Bioact. Compat. Polym., 3,27. 15. M. Matsukata, Y. Takei, T. Aoki, K. Sanui, N. Ogata, Y Sakurai, and T. Okano. 1994. J. Biochem., 116, 682. 16. A. Laane, V Chytry, and M. Haga. 1981. Collect. Czech. Chem. Commun., 46, 1466. 17. V Chytry, J. Vrana, and J. Kopedek. 1978. Makromol. Chem., 179, 329. 18. B. MihovA, J. Strohalm, D. Plocova', V Subr, M. Jelinkov4, M. Sirovd, and K. Ulbrich. 1996. J. Controlled Release, 40, 303. 19. J. Dostdl and J. Matousek. 1973. J. Reprod. Fertil., 33, 263. 20. J. S. Kim, J. Soudek, J. Matousek, and R. T. Rainest. 1995. J. Biol. Chem., 270, 10525. 21. J. S. Kim, J. Soudek, J. Matousek, and R. T. Rainest. 1995. J. Biol. Chem., 270, 31097. 230 Conjugates of Semitelechelic Poly[N-(2-hydroxypropyl)methacrylamide] 231 22. K. Ulbrich. 1998. J. Controlled Release., in press. 23. J. Pato, M. Azori, K. Ulbrich, and J. Kopedek. 1984. Makromol. Chem., 185, 231. 24. M. Pechar, J. Strohalm, and K. Ulbrich. 1995. Collect. Czech. Chem. Commun., 60, 1765. 25. E. 0. Nwankwo and S. D. Abbott. 1995. J. Apple. Polym. Sci., 58, 191. 26. S. L. Snyder and P Z. Sobocinski. 1975. Anal. Biochem., 64, 284. 27. t. Kofidk, P St~panek, and B. Sedl6dek. 1984. Czech. J. Phys. (Engl. Transl.), A 34, 497. 28. F. J. Kezdy and E. T. Kaiser, 1970. Proteolytic Enzymes. Perlmann, G. E. and Lorand, L., eds. New York: Academic Press, p. 3. 29. E. M. Crook, A. P Mathias, and B. R. Rabin. 1960. Biochem. J., 74, 234. 30. P J. Flory. 1971. Polymer Chemistry. London: Cornell University Press. 31. E. Ranucci, G. Spagnoli, L. Sartore, and P Ferruti. 1994. Macromol. Chem. Phys., 195, 3469. 32. S. Zalipsky and Ch Lee, 1992. Poly(Ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications. Harris, J. M., ed. New York: Plenum Press, p. 347. 33. C. Monfardini, 0. Schiavon, P Caliceti, M. Morpurgo, J. M. Harris, and F. M. Veronese. 1996. Bioconjugate Chem., 6, 62. 34. F. M. Veronese, 1992. Poly(Ethylene Glycol) Chemistry: Biotechnical and Bio- medical Applications. Harris, J. M., ed. New York: Plenum Press, p. 127. 35. M. J. Knauf, D. P Bell, P Hirtzer, Z. P Luo, J. D. Young, and N. V Katre. 1988. J. Biol. Chem., 263, 15064. 36. I. Fuke, T. Hayashi, Y Tabata, and Y Ikada. 1994. J. Controlled Release, 30, 27. 37. H. Chiu, S. Zalipsky, P Kope~kova, and J. Kopedek. 1993. Bioconjugate Chem., 4, 290. 38. K. A. Sharp, M. Yalpani, S. J. Howard, and D. E. Brooks. 1986. Anal. Biochem., 154, 110. 39. T. Suzuki, K. Ikeda, and T. Tomono. 1989. J. Biomater. Sci., Polym. Ed., 1,71. 1. R. Duncan and F. Spreafico . 1994 . Clin. Pharmacokinet. , 27 , 290 . 2. A. H. Sehon , 1992 . Poly(Ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications . Harris, J. M. , ed. New York : Plenum Press , p. 139 . 3. N. V. Katre . 1993 . Adv. Drug Delivery Rev. , 10 , 91 . 4. F. Fuertges and A. Abuchowski . 1990 . J. Controlled Release , 11 , 139 . 5. D. Putnam and J. Kopeček . 1995 . Adv. Polym. Sci. , 122 , 57 . 6. D. H. Ho , N. S. Brown , and A. Yen . 1986 . Drug Metab. Dispos. , 14 , 349 . 7. M. S. Hershfield , R. H. Buckley , and M. L. Greenberg . 1987 . New Engl. J. Med. , 316 , 589 . 8. F. J. Meyers , C. Paradise , and S. A. Scudder . 1991 . Clin. Pharmacol. Ther. , 49 , 307 . 9. P. D. Thomson , G. Till , and J. O. Wolliscroft . 1990 . Burns , 16 , 406 . 10. M. L. Nucci , R. Shorr , and A. Abuchowski . 1991 . Adv. Drug Delivery Rev. , 6 , 133 . 11. S. Davis , Y. K. Park , and F. F. Davis . 1981 . Lancet , 2 , 281 . 12. R. Fagnani , M. S. Hagan , and R. Bartholomew . 1990 . Cancer Res. , 50 , 3638 . 13. P. Caliceti , O. Schiavon , M. Morpurgo , and F. M. Veronese . 1995 . J. Bioact. Compat. Polym. , 10 , 103 . 14. H. Maeda , T. Oda , and Y. Matsumura . 1988 . J. Bioact. Compat. Polym. , 3 , 27 . 15. M. Matsukata , Y. Takei , T. Aoki , K. Sanui , N. Ogata , Y. Sakurai , and T. Okano . 1994 . J. Biochem. , 116 , 682 . 16. A. Laane , V. Chytrý , and M. Haga . 1981 . Collect. Czech. Chem. Commun. , 46 , 1466 . 17. V. Chytrý , J. Vrána , and J. Kopeček . 1978 . Makromol. Chem. , 179 , 329 . 18. B. Říhová , J. Strohalm , D. Plocová , V. Šubr , M. Jelínková , M. Šírová , and K. Ulbrich . 1996 . J. Controlled Release , 40 , 303 . 19. J. Dostál and J. Matoušek . 1973 . J. Reprod. Fertil. , 33 , 263 . 20. J. S. Kim , J. Souček , J. Matoušek , and R. T. Rainest . 1995 . J. Biol. Chem. , 270 , 10525 . 21. J. S. Kim , J. Souček , J. Matoušek , and R. T. Rainest . 1995 . J. Biol. Chem. , 270 , 31097 . 22. K. Ulbrich . 1998 . J. Controlled Release ., in press. 23. J. Pató , M. Azori , K. Ulbrich , and J. Kopeček . 1984 . Makromol. Chem. , 185 , 231 . 24. M. Pechar , J. Strohalm , and K. Ulbrich . 1995 . Collect. Czech. Chem. Commun. , 60 , 1765 . 25. E. O. Nwankwo and S. D. Abbott . 1995 . J. Appl. Polym. Sci. , 58 , 191 . 26. S. L. Snyder and P. Z. Sobocinski . 1975 . Anal. Biochem. , 64 , 284 . 27. Č. Koňák , P. Štěpánek , and B. Sedláček . 1984 . Czech. J. Phys. (Engl. Transl.), A34 , 497 . 28. F. J. Kezdy and E. T. Kaiser , 1970 . Proteolytic Enzymes . Perlmann, G. E. and Lorand, L. , eds. New York : Academic Press , p. 3 . 29. E. M. Crook , A. P. Mathias , and B. R. Rabin . 1960 . Biochem. J. , 74 , 234 . 30. P. J. Flory . 1971 . Polymer Chemistry . London : Cornell University Press . 31. E. Ranucci , G. Spagnoli , L. Sartore , and P. Ferruti . 1994 . Macromol. Chem. Phys. , 195 , 3469 . 32. S. Zalipsky and Ch Lee , 1992 . Poly(Ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications . Harris, J. M. , ed. New York : Plenum Press , p. 347 . 33. C. Monfardini , O. Schiavon , P. Caliceti , M. Morpurgo , J. M. Harris , and F. M. Veronese . 1996 . Bioconjugate Chem. , 6 , 62 . 34. F. M. Veronese , 1992 . Poly(Ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications . Harris, J. M. , ed. New York : Plenum Press , p. 127 . 35. M. J. Knauf , D. P. Bell , P. Hirtzer , Z. P. Luo , J. D. Young , and N. V. Katre . 1988 . J. Biol. Chem. , 263 , 15064 . 36. I. Fuke , T. Hayashi , Y. Tabata , and Y. Ikada . 1994 . J. Controlled Release , 30 , 27 . 37. H. Chiu , S. Zalipsky , P. Kopečková , and J. Kopeček . 1993 . Bioconjugate Chem. , 4 , 290 . 38. K. A. Sharp , M. Yalpani , S. J. Howard , and D. E. Brooks . 1986 . Anal. Biochem. , 154 , 110 . 39. T. Suzuki , K. Ikeda , and T. Tomono . 1989 . J. Biomater. Sci., Polym. Ed. , 1 , 71 .
PY - 1999/5
Y1 - 1999/5
N2 - The semitelechelic poly[N-(2-hydroxypropyl)methacrylamide] [poly(HPMA)] with a carboxyl end group was prepared with 3-mercaptopropionic acid as a chain transfer agent. Bovine seminal ribonuclease (BSR) and α-chymotrypsin (ChT) were modified with various molecular weights of active poly(HPMA) succinimidyl ester by the reaction with the amino groups of the respective enzyme. The modification of ChT did not significantly change the activity or the substrate specificity of the conjugates towards low-molecular-weight tripeptidic substrates. However, modified ChT activity towards the corresponding poly(ethylene glycol)-based synthetic substrate was significant. The activity decreased as a result of the elevated steric hindrance to the active site of the polymer-modified enzyme. Similarly, the ChT conjugates completely lost their proteolytic activity toward native bovine serum albumin. The autolytic stability of ChT conjugates was improved and the proteolytic stability of the the ChT and BSR conjugates substantially increased compared with the free enzymes. The modification of ChT with poly(HPMA) significantly decreased the immunogenicity of ChT conjugates depending on the molecular weight of the poly(HPMA) and the degree of enzyme substitution.
AB - The semitelechelic poly[N-(2-hydroxypropyl)methacrylamide] [poly(HPMA)] with a carboxyl end group was prepared with 3-mercaptopropionic acid as a chain transfer agent. Bovine seminal ribonuclease (BSR) and α-chymotrypsin (ChT) were modified with various molecular weights of active poly(HPMA) succinimidyl ester by the reaction with the amino groups of the respective enzyme. The modification of ChT did not significantly change the activity or the substrate specificity of the conjugates towards low-molecular-weight tripeptidic substrates. However, modified ChT activity towards the corresponding poly(ethylene glycol)-based synthetic substrate was significant. The activity decreased as a result of the elevated steric hindrance to the active site of the polymer-modified enzyme. Similarly, the ChT conjugates completely lost their proteolytic activity toward native bovine serum albumin. The autolytic stability of ChT conjugates was improved and the proteolytic stability of the the ChT and BSR conjugates substantially increased compared with the free enzymes. The modification of ChT with poly(HPMA) significantly decreased the immunogenicity of ChT conjugates depending on the molecular weight of the poly(HPMA) and the degree of enzyme substitution.
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U2 - 10.1177/088391159901400302
DO - 10.1177/088391159901400302
M3 - Article
AN - SCOPUS:0033041990
SN - 0883-9115
VL - 14
SP - 213
EP - 231
JO - Journal of Bioactive and Compatible Polymers
JF - Journal of Bioactive and Compatible Polymers
IS - 3
ER -