Alkaline Phosphatase Activatable Polymeric Cross-Linkers and Their

390

Bioconjugate Chem. 1998, 9, 390−398

Alkaline Phosphatase Activatable Polymeric Cross-Linkers and Their Use in the Stabilization of Proteins Christopher Bieniarz,*,† Michael J. Cornwell,‡ and Douglas F. Young‡ Advanced Drug Delivery, Hospital Products Division, and Diagnostics Division, Abbott Laboratories, Abbott Park, Illinois 60064-3500. Received February 9, 1998

We report the synthesis of polymeric cross-linking agents, poly(glutamic acid) poly(phosphorothioates), and their use in the cross-linking and stabilization of proteins upon treatment with alkaline phosphatase. We have shown that poly(phosphorothioates) are excellent substrates of alkaline phosphatase, yielding thiolated polymers which react covalently with electrophilic groups introduced into the proteins. Three proteins of different structure and function were cross-linked using this method: calf intestinal alkaline phsophatase, glucose oxidase (Aspergillus niger), and (R)-phycoerythrin. The cross-linking of alkaline phosphatase is self-catalyzed since this enzyme catalyzes the hydrolysis of phosphates, unmasking thiolates which react with the maleimide prederivatized alkaline phosphatase. Incubation of buffered solutions of native alkaline phosphatase at 45 °C for 7-14 days resulted in a 35% higher loss of enzymatic activity compared to that of cross-linked enzyme. The effect of cross-linking glucose oxidase is even more notable, ranging from 800% stabilization at 37 °C and pH 9.0 to 3000% at 37 °C and pH 7.4. (R)-Phycoerythrin cross-linked with 1-3 equiv of poly(phosphorothioates) and incubated at 45 °C for 45 days was 20% more fluorescent than the native (R)-phycoerythrin subjected to the same conditions. The stabilizing effect of cross-linking was confirmed by comparing the rate of loss of quaternary structure of the cross-linked (R)-phycoerythrin with that of the native protein.

INTRODUCTION

The stabilization of proteins is an important field of practical and fundamental research. Enhancement of protein stability against denaturing effects of temperature, pH, and chemical agents has great practical applications in the biosensors, biopharmaceuticals, food, and cosmetic industries. Some of the methods used for stabilizing proteins against unfolding consist of molecular modifications, cross-linking, physical entrapment, surface immobilization, and the use of stabilizing carbohydrates and polymer additives. There is a plethora of reports, patents, and symposia dealing with many aspects of protein stabilization consisting of chemical derivatization and inter- or intramolecular cross-linking of the reactive functionalities on the surface of the proteins, presumably leading to increased conformational stability and enhanced resistance to denaturation (1-9). Unfolding of globular proteins generally involves exposure of buried hydrophobic side chains. Chemical cross-linking of proteins appears to be a particularly efficient stabilization method for minimizing unfolding pathways and preventing changes in the quaternary structure of multimeric proteins. Such unfolding often leads to dissociation into inactive protein subunits. Cross-linking approaches were exploited successfully in the stabilization of proteases, endonucleases, and antibodies (6, 7). Similarly, multimeric enzymes have been stabilized by cross-linking with glutaraldehyde (10). Deliberate polymerization of enzymes with the aim of improving their thermal stability * To whom the correspondence should be addressed: Abbott Laboratories, Hospital Products Division D-97D, Abbott Park, North Chicago, IL 60064-3500. Phone: (847) 937-2239. Fax: (847) 938-3645. E-mail: [email protected]. † Hospital Products Division. ‡ Diagnostics Division.

has also been described (11, 12). Several bifunctional cross-linking agents used in chemical cross-linking and stabilization of proteins were reviewed (13). The reactive polymeric cross-linkers described in the above references lead to mostly intermolecular crosslinking of the proteins. The resulting polymeric protein often has a molecular weight well in excess of that of its native state. Moreover, the large adducts often have biological activities quite different from those of the native proteins. This is particularly troublesome when the cross-linked proteins are destined for use in immunoconjugates, biosensors, or other biometric applications because of the problems with nonspecific binding and altered solubilities of the cross-linked proteins. In this paper, we report a novel method of protein cross-linking and stabilization. The cross-linking is based on an alkaline phosphatase activatable linear poly(phosphorothioate). The method is depicted in Scheme 1. Protein (a) is first derivatized with maleimides. To the derivatized protein (b) is added a poly(phosphorothioate) (c), which is then exposed to the action of alkaline phosphatase (ALP).1 ALP very efficiently catalyzes the 1 Abbreviations: ALP, alkaline phosphatase; R-PE, (R)phycoerythrin; GOX, glucose oxidase; DTNB, 5,5′-dithiobis(2nitrobenzoic acid); DMF, dimethylformamide; PGA, poly(Lglutamic acid); EDC, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride; EDTA, ethylenediaminetetraacetic acid; NEM, N-ethylmaleimide; PNPP, p-nitrophenyl phosphate; BSA, bovine serum albumin; SMCC, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate; HRPO, horseradish peroxidase; 4-AAP, 4-aminoantipyrine; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SATA, N-succinimidyl S-acetylthioacetate; SPDP, N-succinimidyl-3-(2-pyridyldithio)propionate; DTT, dithiothreitol.

S1043-1802(98)00026-3 CCC: $15.00 © 1998 American Chemical Society Published on Web 04/11/1998

Alkaline Phosphatase Activatable Polymeric Cross-Linkers

Bioconjugate Chem., Vol. 9, No. 3, 1998 391

Scheme 1. ALP-Catalyzed Activation of Poly(phosphorothioate) Polymers and Their Cross-Linking of Proteins

hydrolysis of phosphates, unmasking thiolates on the backbone of the polymer (d). The polymer then reacts with maleimide-derivatized enzyme, resulting in a predominantly intramolecular cross-linking and concomitant stabilization of the protein (e). When the protein (a) is alkaline phosphatase, the process is self-catalyzed in that the very enzyme to be cross-linked catalyzes the hydrolysis of the phosphorothioate bonds and cross-linking of the protein surface. To show the generality of our cross-linking stabilization method, we present results of ALP-induced cross-linking of three proteins of very different structure and function: calf intestinal ALP, glucose oxidase, and (R)phycoerythrin. The proteins also vary in the number of subunits, as R-PE is multimeric, ALP dimeric, and GOX monomeric. EXPERIMENTAL SECTION

Materials. Except as noted, reagents were obtained commercially and used without further purification. All solvents were HPLC grade. Hydrazine monohydrate, ethylenediamine, DTNB, and anhydrous DMF were from Aldrich Chemical Co. PGA, cysteamine S-phosphate, EDC, EDTA, Sephadex G-25, NEM, sodium periodate, sodium cyanoborohydride, glucose, PNPP, BSA, and all buffer components were from Sigma Chemical Co. (St. Louis, MO). SMCC and Traut’s reagent (2-iminothiolane hydrochloride) were from Pierce Chemical Co. (Rockford, IL). Concentration membranes, the Centriprep-30 concentrator, and the Centricon-30 concentrator were from Amicon Co. (Beverly, MD). All dialysis tubing was from Spectrum (Houston, TX). Chromatography Econo and Bio-Sil SEC-400 columns and BIO-REX MSZ(D) resin were from Bio-Rad (Hercules, CA). Bovine intestinal ALP and GOX were from Boehringer Mannheim (Indianapolis, IN). R-PE was from Molecular Probes (Eugene, OR). HRPO, 3,5-dichloro-2-hydroxybenzenesulfonic acid sodium salt, and 4-AAP were from Abbott Laboratories (Abbott Park, IL). General Procedures. Electronic spectra were recorded on a Hewlett-Packard 8452A diode array spectrophotometer. Fluorescence spectra were recorded on a Hitachi F-4010 spectrophotometer. HPLC analyses

were performed on a Spectra-Physics instrument equipped with an SP8490 dual-wavelength detector. All enzyme activity measurements were determined with the initial rate method (14). Synthesis of Poly(glutamic acid) Poly(phosphorothioate) PGA(SPO3)24. PGA (MW ) 50000-100000, 1.00 g, 14 mmol) and cysteamine S-phosphate (0.26 g, 1.4 mmol) were dissolved in 40 mL of deionized water. EDC (1.00 g, 5.2 mmol) was added in 100 mg aliquots every 30 min for 5 h. The product was purified with a Centriprep-30 concentrator against deionized water and then lyophilized. Phosphorothioate Analysis. Quantitation of thiolates on poly(phosphorothioates) was achieved with adaptation of Ellman’s method as described previously (15, 16). To 1.00 mL of a solution of 5.0 mM PGA(SPO3)24 in 0.1 M Tris buffer, 1.0 mM MgCl2, and 0.1 mM ZnCl2 at pH 7.5 (buffer A) was added 30 mL of 10 mM DTNB in buffer A. The solution was incubated for 5 min, and the absorbance at 412 nm was recorded. No free thiol was detected. ALP (10 mL of a 74.1 mM solution) was added and the mixture incubated until no further increase in 412 nm absorbance was detected (30 min). The concentration of thiol (0.12 mM) was calculated from the final 412 nm absorbance (1.54 AU) and the extinction coefficient of 13 000 M-1 cm-1 of DTNB at pH 7.5. We found there to be 24 equiv of phosphorothioate per mole of polymer. PGA(SPO3)24 Cross-Linking of Bovine ALP. To 0.75 mL of an aqueous solution of 74.1 mM ALP was added 1.25 mL of 0.1 M sodium phosphate, 0.1 M NaCl, 1.0 mM MgCl2, and 0.1 mM ZnCl2 at pH 7.0 (buffer B). The enzyme was concentrated to 0.2 mL using a Centricon-30 concentrator. The concentrate was rediluted to 2.0 mL with buffer B and then reconcentrated to 0.2 mL, and the procedure was repeated three times. The volume of the enzyme solution was increased to 1.5 mL with buffer B and the mixture placed in a vial. To 75 mL of DMF was added 0.62 mg (1.87 mmol) of SMCC. This solution was added to 1.46 mL of a 35.5 mM solution of ALP (46.7 nmol) and the mixture allowed to react for 1 h at room temperature while being rotated at 100 rpm on a rotary agitator. Coarse Sephadex G-25 that had

392 Bioconjugate Chem., Vol. 9, No. 3, 1998

been previously rehydrated with 0.1 M sodium phosphate, 0.1 M NaCl, and 0.05% NaN3 at pH 7.0 (buffer C) was poured to a bed height of 45 cm in a 1 cm × 50 cm Econo column. The column was equilibrated with buffer B. Following the incubation, SMCC-derivatized ALP was applied to the column and the column eluted with buffer B. Fractions with an A280 of >0.5 AU were pooled, and the A280 of the pool was used to calculate the SMCCderivatized enzyme concentration. To 300 mL of buffer B was added 2.43 mg (34.7 nmol) of PGA(SPO3)24, and this solution was added to 1.86 mL of a 1.40 mg/mL solution of SMCC-derivatized ALP (17.4 nmol) and the mixture allowed to react overnight at 5 °C while being rotated on a rotary agitator. Characterization of PGA-Cross-Linked ALP. PGAcross-linked ALP was evaluated by size exclusion chromatography using a Bio-Sil SEC-400 column and SDSPAGE, as described in the Results. Thermal Stability Evaluation of Cross-Linked ALP. Both the native and the cross-linked enzymes were diluted to 74.1 nM (10 mg/mL) with buffer A. These dilutions were stored at 45 °C in an incubator for the duration of the study. At day 0 and various time points along the course of the study, the activity of the enzymes was evaluated. To separate 1.00 mL volumes of 7 mM PNPP in 0.5 M diethanolamine, 1.0 mM MgCl2, and 0.1 mM ZnCl2 at pH 10.2 (buffer D) was added 20 mL of the 74.1 nM ALP dilutions. The rate change in the 412 nm absorbance was calculated over 14 s intervals. The rate generated by the enzyme preparations at the various points was divided by the rate generated by the same enzyme preparation at day 0 to calculate the percentage of residual enzyme activity for the stressed dilution. PGA(SPO3)19 Cross-Linking, Characterization, and Thermal Stability Evaluation of GOX from Aspergillus niger. GOX experiments were performed in a manner analogous to the ALP procedures except the polymer had an average of 19 phosphorothioate groups. GOX was derivatized with 25, 50, 75, 100, and 150 molar equiv of SMCC as described above for ALP. The stoichiometry of GOX/PGA(SPO3)19 was 1:1. Hydrolysis of phosphate groups on PGA(SPO3)19 was the result of externally added micromolar native ALP. Cross-linked GOX was incubated at 37 °C at pH 7.4 and 9.0 and compared to native GOX under the same conditions. Both the native enzyme and the cross-linked enzyme were diluted to 500 mg/mL with 1.0 mM EDTA and 0.1 M NaCl at pH 7.4 (buffer E). These dilutions were stored in a 37 °C incubator for the duration of the study. At day 0 and various time points along the course of the study, the activity of native and cross-linked enzymes was evaluated. Prior to evaluation, the stressed enzyme preparations were diluted to 2 mg/mL using 0.1 M NaCl and 0.1 M sodium phosphate at pH 7.0 (buffer F). Enzyme activity was monitored in a solution of 2 mM 4-AAP and 8 mM 3,5-dichloro-2-hydroxybenzenesulfonic acid in the presence of 1 unit of HRPO and 100 mM glucose in buffer F. The rate changes were monitored at A550 over 2 min intervals. Thermal stability evaluations of cross-linked GOX at pH 9.0 were carried out like they were for the sample at pH 7.4 except both the native and the cross-linked enzymes were diluted to 500 mg/ mL with 0.1 M sodium phosphate, 1.0 mM EDTA, and 0.1 M NaCl at pH 9.0 (buffer G). Of course, in this application, ALP was added externally to catalyze the hydrolysis of phosphates on PGA(SPO3)19. PGA(SPO3)18 and PGA(SPO3)28 Cross-Linking of R-PE from Porphyra tenera. To 2.5 mL of an aqueous solution of 41.7 mM R-PE was added 2 mL of buffer E.

Bieniarz et al.

This solution was transferred to dialysis tubing with a MW cutoff of 12000-14000 and dialyzed for 24 h each against three 4 L changes of buffer E. To a 1.21 mL aliquot of a 24.1 mM solution of dialyzed R-PE (0.292 nmol) was added a solution of 0.49 mg (1.47 mmol) of SMCC in 200 mL of DMF, and the mixture was allowed to react for 1 h at room temperature while being rotated at 100 rpm on a rotary agitator. A Sephadex G-25 column was prepared as above with buffer E, and following the incubation, the SMCC-derivatized R-PE was applied to a Sephadex G-25 column to remove unreacted SMCC. The column was eluted with buffer E, and 0.75 mL fractions were collected. Fractions with an A566 of >1.0 AU were pooled, and the A566 of the pool was used to calculate the protein concentration of the SMCC-derivatized R-PE. To 1 mL of buffer B was added 10 mg (167 nmol) of PGA(SPO3)18 (MW ) 60 000). To this solution was added 25 mL of 10 mg/mL (1.67 nmol) ALP to deprotect the phosphorothioates. The deprotection reaction was allowed to proceed for 3 h at room temperature while the mixture was being rotated at 100 rpm on a rotary agitator. Following the incubation, a 71 mL (11.5 nmol) aliquot of this solution was added to 1.91 mL of 1.44 mg/mL (11.5 nmol) SMCC-derivatized R-PE and the mixture allowed to react overnight at 5 °C while being rotated at 100 rpm on a rotary agitator. Cross-linking at higher PGA(SPO3)18:R-PE ratios (2:1 and 3:1 molar ratios) was done analogously. Characterization of Cross-Linked R-PE. PGA(SPO3)18 and PGA(SPO3)28 cross-linked R-PE were evaluated by size exclusion chromatography using a Bio-Sil SEC-400 column. Detection was at 280 nm. The mobil phase was buffer E running at a flow rate of 1.0 mL/ min. The primary population generated had a retention time corresponding to a singlet and doublet cross-linked protein with very little polymerization occurring. The residual fluorescence intensity of the cross-linked R-PE was measured and compared to the fluorescence of the native R-PE under the same conditions. The fluorescence generated by the cross-linked R-PE preparations was divided by the value of fluorescence of the native R-PE to calculate the percentage of residual fluorescence for the cross-linked preparations. The result of this evaluation showed that the cross-linked R-PE had retained 92% of the native protein fluorescence intensity. Thermal Stability Evaluation of Cross-Linked R-PE by Fluorescence Measurement and by Size. The thermal stability of PGA(SPO3)18 or PGA(SPO3)28 cross-linked R-PE was evaluated by measuring fluorescence decay of samples thermally stressed at 45 °C and compared to that of native R-PE under the same conditions, using a λexc of 488 nm and a λem of 576 nm. Both the native and the cross-linked proteins were diluted to 100 mg/mL (0.42 mM) with buffer E. These dilutions were stored in a 45 °C incubator for the duration of the study. At day 0 and various time points along the course of the study, the fluorescence intensities of both preparations were evaluated. Prior to evaluation, the stressed protein preparations were diluted to 1 mg/mL (4.2 nM) using buffer E. The fluorescence of the R-PE preparations at various time points was divided by the fluorescence of the same preparation at day 0 to calculate the percentage of residual fluorescence intensity of the stressed protein. The thermal stability of PGA(SPO3)18 and PGA(SPO3)28 cross-linked R-PE was evaluated at 45 °C and compared to that of native R-PE under the same conditions. A BioSil SEC-400 column was used to follow the decomposition of the stressed R-PE into smaller subunits. Detection



Scheme 2. Synthesis of Poly(glutamic acid) Poly(phosphorothioate)

was at 280 nm. The mobile phase was buffer E running at a flow rate of 1.0 mL/min. Both the native protein and the cross-linked protein were diluted to 100 mg/mL with buffer E. These dilutions were stored in a 45 °C incubator for the duration of the study. At day 0 and various time points along the course of the study, the percentage of the total 280 nm absorbance which was due to the small components was evaluated. RESULTS

Phosphorothioates are excellent substrates for alkaline phosphatase (17, 18). We have exploited this feature for an alkaline phosphatase-catalyzed hydrolysis of phosphorothioate-derivatized polymers. The resulting polymeric cross-linkers display many thiols which in a subsequent step may be reacted with the thiol-reactive electrophilic groups on the protein. Thus, the phosphate may be thought of as a protective group for thiolates and alkaline phosphatase as the deprotection reagent. In view of the high reactivity of thiolates toward oxidative coupling, the protective phosphate groups prevent disulfide bond formation. Thus, polymers derivatized with phosphorothioates allow access to a new family of crosslinking and conjugating polymers. Moreover, the high polarity of the phosphates ensures the solubility of even very water insoluble, nonpolar polymer backbones. Although we have prepared several poly(phosphorothioate) polymers, in this work, we focus on crosslinking of three proteins of different structure and function by poly(glutamic acid) poly(phosphorothioate) shown in Scheme 2. Cross-Linking of Bovine ALP. PGA was activated with EDC, and addition of cysteamine S-phosphate at various stoichiometries afforded polymers with various numbers of phosphorothioate groups per polymer. We found that the optimal level of phosphorothioate groups per polymer was between 18 and 24. The synthesis of PGA(SPO3)x polymers is shown in Scheme 2. In preliminary experiments, we ascertained that PGA(SPO3)24 is a good substrate for ALP. Virtually all thiols on the polymer were deprotected after 2 h at 5 °C and pH 7.0 using nanomolar quantities of ALP. This was demonstrated by quantitating the number of thiols as a function of time during the deprotection reaction, using Ellmann’s reagent (DTNB). In previous work, we demonstrated that the derivatization of ALP with the maleimide heterobifunctional agents did not significantly affect the activity of the enzyme; 90-95% of the native enzyme activity remained after functionalization of the enzyme with various extended linkers (16). We confirmed this result in the current work and found that derivatization of the enzyme with 40 equiv of SMCC per enzyme resulted in a loss of only 5% of its activity. PGA(SPO3)24 and ALP were reacted in molar ratios of 1:1, 2:1, 3:1 or 4:1, and 6:1. The ratio of cross-linked multimeric

Figure 1. Dependence of the size of the cross-linked ALP on the stoichiometry of PGA(SPO3)24. Cross-linking ALP with 1 equiv of PGA cross-linker yields 82% of the multimeric aggregate of the enzyme (intermolecular cross-linking), while higher ratios of PGA per enzyme yield more singlet enzyme (intramolecular cross-linking).

structures to cross-linked singlets depends on the PGA(SPO3)24:ALP stoichiometry. Figure 1 shows that at higher PGA(SPO3)24:ALP ratios the cross-linking is prevalently intramolecular, resulting in higher ratios of the surface-cross-linked single enzyme molecule. At low PGA(SPO3)24:ALP ratios, multimeric intermolecular crosslinking predominates. The maleimide-derivatized ALP catalyzes the crosslinking of its own surface in an autocatalytic process which, to the best of our knowledge, is unprecedented. The cross-linked ALP was also examined by SDSPAGE using a Phastgel system. Gradient gels of 7 to 10% polyacrylamide were run under nonreducing and reducing conditions. Results from nonreducing conditions showed that the primary population generated was singlet cross-linked enzyme with very little intermolecular cross-linking occurring. Results from the reducing conditions showed that the cross-linked ALP was not monomerized under conditions that were sufficient to monomerize the native ALP. Gel filtration HPLC analysis of the ALP cross-linked with 3 molar equiv of PGA(SPO3)24 per ALP using the Bio-Sil SEC-400 column in buffer B showed essentially complete replacement of the peak of the SMCC-derivatized ALP at 11.31 min with a


Figure 2. Thermal stability of ALP cross-linked at 2:1 and 4:1 PGA(SPO3)24:ALP molar ratios, with NEM capped and uncapped, at 45 °C in 0.1 M Tris buffer (pH 7.5), 0.1 mM ZnCl2, and 1 mM MgCl2. The data are normalized to 100% activity at day 0 to better visualize the changes.

new peak at 10.96 min and a small shoulder at a higher molecular weight corresponding to polymerized ALP. The UV-vis detector was set at A280, and the flow rate was 1.0 mL/min. Similar results were observed at 2:1 and 4:1 polymer:enzyme stoichiometries with higher stoichiometries resulting in higher amounts of intramolecular cross-linking and lower multimeric content, as seen in Figure 1. The cross-linked ALP is significantly stabilized against thermal denaturation. Figure 2 shows the extent of stabilization of cross-linked ALP compared to that of the native enzyme. A conspicuous feature of the plot is a steep loss of the enzymatic activity during the first 24 h with the crosslinked and native ALP enzymes. Unlike the native enzyme whose activity decreases throughout the incubation, the PGA-cross-linked ALP recovers activity quite rapidly. This striking behavior is discussed later in the text. The enzyme retains 75-90% of its native activity at day 0. The data in the figure are normalized to better visualize the loss of activity as the result of incubations at 45 °C. We found that N-ethylmaleimide capping of the unreacted thiolates left after the cross-linking was not necessary, and in fact led to 10% lower resistance to thermal denaturation. Figure 3 shows that at 5 °C the number of titratable thiols is essentially constant over time for 2:1 and 4:1 PGA(SPO3)24:ALP ratios. At 45 °C, there is a steady loss of thiolates, probably due to an oxidative dimerization process. Thus, after 5 days at 45 °C, there were only two or three titratable thiols. The PGA(SPO3)24:ALP ratio used was 2:1, 3:1, and 4:1, yielding 32, 55, or 68 free thiolates, respectively, on the cross-linked ALP at the start of the incubations (only 2:1 and 4:1 stoichiometries are shown in Figure 3). Interestingly, size exclusion HPLC analysis of the cross-linked ALP incubated at 45 °C showed no increase in the highermolecular weight material despite the observed decrease of the titratable thiols at this higher temperature. The nature of the maleimide heterobifunctional agent used to derivatize the enzyme is important. Shorter maleimide linkers, i.e. SMCC, resulted in better resis-

Bieniarz et al.

Figure 3. Number of thiols of ALP cross-linked at 2:1 and 4:1 ratios with PGA(SPO3)24 at 5 and 45 °C as a function of time.

Figure 4. Thermal stability of GOX functionalized with 25, 50, 75, 100, and 150 equiv of SMCC, cross-linked at a 1:1 PGA(SPO3)19:GOX molar ratio at 37 °C in phosphate buffer (pH 7.4), compared to that of native GOX. The data are normalized to 100% at day 0.

tance of the cross-linked protein to thermal denaturation than the longer 30-atom maleimide conjugating agents (15). Cross-Linking of GOX. We also applied this crosslinking methodology to GOX (A. niger). Figures 4 and 5 show comparisons of the stabilities at 37 °C of the GOX cross-linked to the native enzyme at two different pHs. The PGA(SPO3)19:GOX ratio was 1:1. At day 0 after cross-linking, GOX derivatized with 50 equiv of maleimide/enzyme had 85% of the native enzyme activity. The figures above show the very high degree of stabilization for GOX. As in previous figures, the data in Figure 4 are normalized to 100% at day 0 to visualize the effect of thermal stress on the various preparations. Figure 4 shows that, at pH 7.4 and at a fixed 1:1 cross-linking polymer:enzyme ratio, stabilization is dependent on the degree of derivatization of GOX by SMCC. Enzyme derivatized with the lowest stoichiometries of SMCC (25 equiv of SMCC per GOX) showed the lowest degree of



Figure 6. Chromatographic changes of R-PE stressed at 45 °C in 0.1 M sodium phosphate and 0.1 M NaCl (pH 7.0): (top) native R-PE and (bottom) R-PE cross-linked at a 1:1 PGA(SPO3)18:R-PE molar ratio. Figure 5. Thermal stability of GOX functionalized with 150 equiv of SMCC, cross-linked at a 1:1 PGA(SPO3)19:GOX molar ratio at 37 °C in phosphate buffer (pH 9.0), compared to that of native GOX. The data are normalized to 100% at day 0.

stabilization. Derivatization of GOX with a progressively higher number of equivalents of SMCC resulted in correspondingly higher degrees of stabilization. GOX that is prefunctionalized with 150 molar equiv of SMCC and then cross-linked with PGA(SPO3)19 in a 1:1 ratio is approximately 8 times more stable than the native enzyme (t1/2 of 4 vs 0.5 day), as shown in Figure 5. The stabilizing effect of cross-linking is even more pronounced at pH 7.4, which is more compatible with GOX. The progress of the cross-linking reaction was followed by HPLC using a Bio-Rad SEC-400 column and 280 nm detection. Thus, before incubation, GOX derivatized with a 50-fold molar excess of SMCC, in the presence of 1 equiv of PGA(SPO3)19 and a micromolar level of alkaline phosphatase, had a GPC signal at 12.10 min, but that signal was completely replaced after 16 h by one at 11.37 min (70%) and at 10.25 min (30%) corresponding to a singlet cross-linked GOX and dimeric and trimeric adducts. When higher PGA(SPO3)19:GOX ratios (3:1 and 5:1) were used, the original signal of GOX was replaced with essentially a single peak at 11.41 min corresponding to a singlet intramolecular cross-linked GOX. Thus, higher ratios of cross-linker per enzyme led to increased content of intramolecular as opposed to intermolecular cross-linking. This trend is consistent with the one observed for ALP shown in Figure 1. Cross-Linking of R-PE. R-PE, a member of the phycobiliprotein system, is a light-harvesting photosynthetic algal pigment, bilin-protein conjugate with a MW of 240 000 (19, 20). It possesses a complex quaternary structure consisting of 13 subunits organized in subunit architecture (Rβ)6γ. It has 34 bilin chromophores covalently conjugated to cystein residues in the subunits. Due to the multiple bilins, the extremely high absorbance coefficient ( ) 2.4 × 106 cm-1 M-1), and the correct mutual orientation of the bilins within the protein domains, R-PE has an exceedingly high fluorescence quantum yield and very large Stokes shift (λex ) 495 nm, λem ) 576 nm). Light absorbed by biliproteins is transferred through a network of pairwise Fo¨rster resonance processes (21, 22). The fluorescence characteristics of the phycobiliproteins depend on the mutual orientation of the chromophores within the protein domains. The top of Figure 6 shows a GPC profile of the chromatographic changes of R-PE stressed at 45 °C with

Figure 7. Thermal stability of R-PE at 45 °C and pH 7.4. Rate of formation of low-molecular weight subunits of R-PE crosslinked at 1:1 and 4:1 PGA(SPO3)18:R-PE molar ratios.

0.1 M sodium phosphate and 0.1 M NaCl at pH 7.0. A Bio-Sil SEC-400 column was used to follow the decomposition. Upon incubation, approximately 60% of the protein is fragmented into smaller subunits (R, β, and γ with MWs of 17 000, 18 000, and 30 000, respectively) (20) corresponding to peaks at 14.04 min and an earlier shoulder peak. The bottom of Figure 6 shows a GPC profile of R-PE cross-linked with PGA(SPO3)18 in 1:1 polymer:R-PE ratio and exposed to the same thermal stress as the native protein. The smaller subunit peaks correspond to approximately 40% of the material, with 60% corresponding to the higher-molecular weight crosslinked material. Thus, cross-linked material displayed a lower tendency to decompose into subunits than the native R-PE. The changes in the quaternary structure of R-PE are accompanied by a gradual loss of its fluorescence in solution upon exposure to heat. Figure 7 shows the dependence of the formation of the low-molecular weight fraction in the R-PE solution on the time of incubation at 45 °C and pH 7.4. Clearly, the content of the low-molecular weight component is 30% higher for native R-PE than for R-PE cross-linked with 1 or 4 equiv of PGA(SPO3)18. Concomitant with the loss of the quaternary structure of R-PE was a loss of its fluorescence, as shown in Figure 8.


Figure 8. Thermal stability of R-PE at 45 °C and pH 7.4. Rate of fluorescence loss of R-PE cross-linked at a 3:1 PGA(SPO3)18: R-PE molar ratio. For comparison, native R-PE at 4 and 45 °C is shown. NEM-capped cross-linked R-PE is also shown.

As seen in Figure 8, the fluorescence loss is significantly lower for PGA-cross-linked R-PE than for the native R-PE at 45 °C and pH 7.4. After 18 days at 45 °C, PGA-cross-linked R-PE (open circles) retained 20% more fluorescence than the un-cross-linked, native R-PE (triangles). Capping of the sulfhydryl groups by NEM (solid squares) does not appear to have an effect on the fluorescence. DISCUSSION

Alkaline phosphatase is frequently employed as a label in enzyme immunoassays. Its widespread use may be attributed to the very high second-order rate constant (kcat/Km ) 5 × 107 M-1 s-1), the existence of excellent fluorogenic and chromogenic substrates, a pH optimum 9 to 10.5, which is compatible with an optimal signal from fluorogenic and chromogenic substrates, and reasonably good thermal stability (23-25). In this work, we describe a series of new polymeric substrates of alkaline phosphatase. The poly(phosphorothioates) reported here are excellent enzyme substrates and generate thiolates upon catalysis. The byproduct of the reaction is phosphate ion, an innocuous material which may be left in the reaction medium and does not interfere with the subsequent use of the polymers as cross-linkers and stabilizers of proteins. Thus, the phosphorothioate functionality may be thought of as a protected thiolate nucleophile. While other protected thiolates are widely used in bioconjugations and protein modifications, i.e. SATA and SPDP, these reagents mask thiols as thioacetyl and 2-pyridyl disulfide. The generation of thiols using these reagents is achieved by reductive cleavage of 2-pyridyl disulfide using DTT as a reducing agent which needs to be removed from the reaction medium or by using harsh reaction conditions of 0.2 N NaOH, aqueous NH3 (26), or hydroxylamine (27) which may be incompatible with base-sensitive haptens or proteins. The reaction of the polythiolated polymers with the electrophile-derivatized proteins cross-links the surface of the protein and results in protein stabilization. Figure 2 shows that at suitable stoichiometries of PGA(SPO3)24 the enzyme may be considerably stabilized against thermal denaturation. A striking aspect of this graph is

Bieniarz et al.

the precipitous loss of enzymatic activity of this homodimeric protein during the first day of incubation at 45 °C. While the native protein does not recover, the cross-linked ALP recovers its activity. After 72 h, the enzyme activity is similar to the one at the beginning of the incubation. This behavior may be explained in terms of a reversible change in the quaternary structure of the cross-linked dimer versus an irreversible loss of the activity of the native enzyme due to monomerization. Alkaline phosphatases are homodimers in their active forms, and monomers have no detectable enzymatic activity (23, 28). The rate of loss of activity after the third day appears to be similar for the cross-linked and native enzymes. This suggests that the initial loss of activity of the native enzyme is due to a monomerization of the enzyme, a process prevented in the cross-linked protein. The quantitation of the number of thiols at 5 and 45 °C incubations of ALP cross-linked at 2:1 and 4:1 ratios with PGA is shown in Figure 3. At 5 °C, the number of thiolates measured by spectrophotometric titration with DTNB is reasonably constant for both 2:1 and 4:1 crosslinked enzymes. However, at 45 °C, the number of thiolates decreases rather rapidly, reaching essentially zero after 5 days. The rate of loss of titratable thiols is much faster than the rate of loss of the enzymatic activity shown in Figure 2. HPLC analysis showed that the process is not accompanied by the formation of larger aggregates. HPLC analysis showed that the ratios of multimers to monomeric cross-linked proteins remain constant over the course of 5 days at 45 °C. This suggests intramolecular oxidative disulfide bond formation between the contiguous thiols on the cross-links. This method of stabilization was also applied to GOX, an oxidoreductase of great importance in biosensor technology and the diagnosis of diabetes (29). As shown in Figure 4, the degree of stabilization of this enzyme is dependent on the level of derivatization of the enzyme with maleimide. The lowest stoichiometries of electrophile resulted in relatively low levels of thermal stabilization, while the highest levels of maleimide resulted in excellent stabilization through reaction with 1:1 PGA(SPO3)19. GOX prederivatized with 150 equiv of maleimide and reacted with 1 equiv of PGA retained 58% of the enzymatic activity after 12 days at pH 7.4 and 37 °C, while 50% of the activity remained after 60 days. Under identical conditions, the native enzyme lost 50% of its activity after 2.5 days. Even at an unfavorable pH of 9.0, cross-linking provided a very significant level of stabilization, as shown in Figure 5. In the past decade, phycobiliproteins such as R-PE have played an increasingly important role as fluorescent labels in single-cell analysis and flow cytofluorimetry. Consequently, there has been interest in conjugates of phycobiliproteins and immunoglobulins directed against specific cellular epitopes (30). The high fluorescence of R-PE depends on the preservation of the native quaternary structure of the protein. R-PE is composed of 13 subunits called phycobilisomes organized in an (Rβ)6γ complex. Each subunit carries one or two bilins for a total of 32 fluorogenic bilins in R-PE. Dissociation of the phycobilisomes occurs as the result of factors which denature the proteins, i.e. low salt, urea, or heat (3133). The hexamers are known to break down to smaller subunits, including Rβ and Rβγ (34). The effect of crosslinking R-PE is seen in Figures 7 and 8. Clearly, the rate of appearance of the low-molecular weight component is lower for the cross-linked protein than for the unmodified protein, as shown in Figure 7. Similarly, cross-linking has an effect in preserving the intrinsic


fluorescence of R-PE as shown in Figure 8. The congruence of the results in Figures 7 and 8 may be explained by the known dependence of the fluorescence energy transfer and pathways on the three-dimensional structure of phycobiliproteins (20, 35). Denaturing conditions lead to disassembly of the subunits and loss of quaternary structure of the proteins. We believe that stabilizing effects of cross-linking may be attributed to the fact that the intramolecular cross-linking of the protein allows less conformational mobility of the subunits and lessens the degree to which the protein disassembles under the denaturing conditions. We found that in all cases the use of higher PGA(SPO3)x:protein molar ratios led to increased content of intramolecular cross-linking. This is demonstrated in Figure 1 for the case of alkaline phosphatase-catalyzed auto-cross-linking but was also observed in the case of R-PE and GOX. This may be explained by entropic considerations. The ability to control the degree of crosslinking and the molecular weight of the cross-linked stabilized proteins is a distinct advantage of this method. The cross-linking process introduces several thiolates into the stabilized protein. These thiolates may serve as functionalities, through which conjugation to other biological molecules or surfaces may be accomplished. We are describing such a bioconjugation in the following paper (36). SUMMARY

We have presented a new, enzyme-catalyzed crosslinking method based on alkaline phosphatase-catalyzed activation of poly(phosphorothioate) polymers. The crosslinked proteins are considerably stabilized against thermal denaturation in solutions. The method is rapid and in most cases leads to quantitative cross-linking of the protein. The byproducts of the polymer activation and cross-linking are phosphate ions which do not interfere in the biological activity of most proteins. Consequently, it may be left in the reaction medium. Poly(phosphorothioate) polymers are very soluble in water which facilitates the cross-linking process and increases the solubility of the stabilized protein. The method allows easy conjugation of the stabilized cross-linked proteins through reactions of thiolates with electrophilic functionalities on other biomolecules. The chemical and biological properties of the cross-linked proteins are retained. The cross-linking process may be adjusted to individual proteins. Excellent control of the molecular weight of the cross-linked species may be exercised by adjusting the stoichiometries of the cross-linked polymer and proteins. The method could be adapted for biomolecules other than proteins. Thus, conjugation of oligonucleotides, carbohydrates, and lipids to proteins may be implemented using alkaline phosphatase activation of phosphorothioate-derivatized species. The conjugation to surfaces, i.e. colloidal gold and microchips, in a spacially controlled manner is an intriguing possibility. ACKNOWLEDGMENT

We thank Dr. Eric Ginsburg of the Abbott Laboratories Advanced Drug Delivery Department for his critical reading of the manuscript. LITERATURE CITED (1) Mer, G., Hietter, H., and Lefevre, J.-F. (1996) Stabilization of proteins by glycosylation examined by NMR analysis of a fucosylated proteinase inhibitor. Nat. Struct. Biol. 3, 45-53.

Bioconjugate Chem., Vol. 9, No. 3, 1998 397 (2) Sobolov, S. B., Bartoszko-Malik, A., Oeschger, T. R., and Montelbano, M. M. (1994) Cross-linked enzyme crystals of fructose diphosphate aldolase: development as a biocatalyst for synthesis. Tetrahedron Lett. 35, 7751-7754. (3) St. Clair, N., and Navia, M. A. (1992) Cross-linked enzyme crystals as robust biocatalysts. J. Am. Chem. Soc. 114, 73147316. (4) Suh, J., and Hwang, B. K. (1992) Enhancement of durability of chymotripsin through cross-linking with poly(allylamine). Bioorg. Chem. 20, 223-235. (5) Torchilin, V. P., and Martinek, K. (1979) Enzyme stabilization without carriers. Enzyme Microb. Technol. 1, 74-82. (6) Wang, P., Hill, T. G., Bednarski, M. D., and Callstrom, M. R. (1991) The catalytic formation of peptide bonds with carbohydrate protein conjugates of proteases [CPC(Proteases)]. Tetrahedron Lett. 32, 6827-6830. (7) Wang, P., Hill, T. G., Wartchow, C. A., Huston, M. E., Oehler, L. M., Bradley Smith, M., Bednarski, M. D., and Callstrom, M. R. (1992) New carbohydrate-based materials for the stabilization of proteins. J. Am. Chem. Soc. 114, 378380. (8) Schellman, J. A. (1987) The thermodynamic stability of proteins. Annu. Rev. Biophys. Chem. 16, 115-137. (9) Pollak, A., Blumenfeld, H., Wax, M., Baoughn, R. L., and Whitesides, G. M. (1980) Enzyme immobilization by condensation copolymerization into cross-linked polyacrylamide gels. J. Am. Chem. Soc. 102, 6324-6336. (10) Gottschalk, N., and Jaenicke, R. (1987) Chemically crosslinked lactate dehydrogenase: stability and reconstruction after glutaraldehyde fixation. Biotechnol. Appl. Biochem. 9, 389-400. (11) Freytag, J. W., and Ishikawa, E. (1987) Immunoassays utilizing covalent conjugates of polymerized enzyme and antibody. U.S. Patent 4,657,853. (12) Pedersen, S., and Jørgensen, O. B. (1991) Immobilization of enzymes by cross-linking with a cross-linking agent and a polymer containing 1-aminoethylene moiety. PCT WO 91/ 08287. (13) Wong, S. S., and Wong, L.-J. C. (1992) Chemical crosslinking and the stabilization of proteins and enzymes. Enzyme Microb. Technol. 14, 866-874. (14) Segel, I. H. (1976) Biochemical Calculations, 2nd ed., p 216, John Wiley & Sons, New York. (15) Bieniarz, C., Husain, M., Barnes, G., King, C. A., and Welch, C. J. (1996) Extended length heterobifunctional coupling agents for protein conjugation. Bioconjugate Chem. 7, 88-95. (16) Husain, M., and Bieniarz, C. (1994) Fc site-specific labeling of immunoglobulins with calf intestinal alkaline phosphatase. Bioconjugate Chem. 5, 482-490. (17) Neumann, H., Boross, L., and Katchalski, E. (1967) Hydrolysis of S-substituted monoesters of phosphorothioic acid by alkaline phosphatase from Escherichia coli. J. Biol. Chem. 242, 3142-3147. (18) Neumann, H. (1968) Substrate selectivity in the action of alkaline and acid phosphatases. J. Biol. Chem. 243, 46714676. (19) Glazer, A. N., and Stryer, L. (1984) Phycofluor probes. Trends Biochem. Sci., 423-427. (20) Glazer, A. N. (1989) Light guides. Directional energy transfer in a photosynthetic antenna. J. Biol. Chem. 264, 1-4. (21) Lakowicz, J. R. (1983) Principles of Fluorescence Spectroscopy, pp 303-339, Plenum Press, New York. (22) MacColl, R., and Guard-Rriar, D. (1987) Phycobiliproteins, CRC Press, Boca Raton, FL. (23) Butterworth, P. J. (1983) Alkaline phosphatase. Cell Biochem. Funct. 1, 66-70. (24) Maggio, E. T. (1980) Enzyme-Immunoassay, pp 64-65, CRC Press, Boca Raton, FL. (25) McComb, R. B., Bowers, G. N., and Posen, S. (1979) Alkaline Phosphatase, pp 289-372, Plenum Press, New York. (26) Greene, T. W., and Wuts, P. G. M. (1991) Protective Groups in Organic Synthesis, p 298, John Wiley & Sons, New York. (27) Duncan, R. J. S., Weston, P. D., and Wrigglesworth, R. (1983) A new reagent which may be used to introduce

398 Bioconjugate Chem., Vol. 9, No. 3, 1998 sulfhydryl groups into proteins, and its use in preparation of conjugates for immunoassay. Anal. Biochem. 132, 68-73. (28) Walsh, C. (1979) Enzymatic Reaction Mechanisms, p 192, W. H. Freeman and Co., San Francisco. (29) Turner, A. P. F., Karube, I., and Wilson, G. S. (1987) Biosensors. Fundamentals and Applications, Oxford Science Applications, Oxford. (30) Oi, V. T., Glazer, A. N., and Stryer, L. (1982) Fluorescent phycobiliprotein conjugates for analyses of cells and molecules. J. Cell Biol. 93, 981-986. (31) MacColl, R., Malak, H., Cipollo, J., Label, B., Ricci, G., MacColl, D., and Eisele, L. E. (1995) Studies on the dissociation of cryptomonad biliproteins. J. Biol. Chem. 270, 2755527561. (32) MacColl, R., and Eisele L. E. (1996) R-Phycoerythrins having two conformations for the same aggregate. Biophys. Chem. 61, 161-167.

Bieniarz et al. (33) Wilbanks, S. M., and Glazer, A. N. (1993) Rod structure of a Phycoerythrin II-containing phycobilisome. J. Biol. Chem. 268, 1236-1241. (34) Gingrich, J. C., Williams, R. C., and Glazer, A. N. (1982) Rod substructure in cyanobacterial phycobilisomes: phycoerythrin assembly in Synechocystis 6701 phycobilisomes. J. Cell Biol. 95, 170-178. (35) Glazer, A. N., Yeh, S. W., Webb, S. P., and Clark, J. H. (1985) Disk-to-disk transfer as the rate-limiting step for energy flow in phycobilisomes. Science 227, 419-423. (36) Bieniarz, C., Young, D. F., and Cornwell, M. J. (1997) Thermally stabilized immunoconjugates: conjugation of antibodies to alkaline phosphatase stabilized with polymeric cross-linkers. Bioconjugate Chem. 9, 399-402.

BC9800264

Alkaline Phosphatase Activatable Polymeric Cross-Linkers and Their

Recommend Documents