Affinity Capture of a Mammalian DNA Polymerase β by Inhibitors

Bioconjugate Chem. 2005, 16, 97−104

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Affinity Capture of a Mammalian DNA Polymerase β by Inhibitors Immobilized to Resins Used in Solid-Phase Organic Synthesis Kouji Kuramochi,† Tetsuya Haruyama,‡ Ryo Takeuchi,‡ Takashi Sunoki,# Madoka Watanabe,‡ Masahiko Oshige,† Susumu Kobayashi,†,# Kengo Sakaguchi,†,‡ and Fumio Sugawara*,†,‡ Genome and Drug Research Center, Department of Applied Biological Science, and Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda, Chiba 278-8510, Japan. Received August 24, 2004; Revised Manuscript Received November 28, 2004

The application of resins normally used in solid-phase organic synthesis to the affinity capture of a mammalian DNA polymerase β (pol β) is reported. Lithocholic acid (LCA), an inhibitor of pol β, was immobilized on various solid supports, and the batch affinity purification of pol β from a mixture of proteins using these LCA-immobilized resins was examined. Of the resins tested, TentaGel was the most effective at purifying pol β and at resisting nonspecific absorption of proteins. The immobilized LCA recognized pol β specifically, which resulted in pol β binding to the resin. Using the LCA-immobilized resin, it was possible to purify pol β from a mixture of proteins. Furthermore, it was possible to concentrate pol β from a crude nuclear extract of human T lymphoma Molt4 cells. To facilitate the immobilization of compounds on TentaGel resins, we also designed and prepared photoaffinity beads containing a photoreactive group at the free termini of the TentaGel resin. The pol β inhibitors LCA, C18-β-SQDG, and epolactaene were immobilized on the photoaffinity beads by photoreaction. The batch affinity purification of pol β from a protein mixture could be also achieved with these beads.

INTRODUCTION

DNA replication, repair, and recombination are important processes for all organisms. Many classes of DNA polymerases (pols) have been identified in multicellular eukaryotes, and recent investigations have revealed that eukaryotic cells contain at least 14 types of DNA pol (1). The purification of DNA pols is still a laborious and timeconsuming endeavor. For example, a combination of many types of column chromatography (for example, phosphocellulose, human serum conjugated sepharose, rabbit IgG conjugated sepharose, and monoclonal antibody conjugated sepharose column chromatography) has been often used for the purification of DNA polymerase β. One approach to studying the various roles of each of the DNA pols is to use inhibitors as tools and molecular probes to distinguish among the various DNA pols and to clarify their biological and in vivo functions. We previously identified a number of such inhibitors against DNA pols by screening both natural and synthetic products (2-5). Use of these inhibitors against DNA pols as molecular probes is also important for purifying DNA pols and for understanding their biological functions in detail. Lithocolic acid (LCA), one of the bile acids, has been reported to promote tumorigenesis induced by N-methylN′-nitro-N-nitrosoguanidine in rats (6-8) and to possess inhibitory activities against eukaryotic DNA pol R and β (9-11). We previously found that both the hydrophilic * Corresponding author. Tel: +81-(0)4-7124-1501. Fax: +81(0)4-7123-9767. E-mail: [email protected]. † Genome and Drug Research Center. ‡ Department of Applied Biological Science. # Faculty of Pharmaceutical Sciences.

end (carboxyl group) and the hydrophobic region (steroidal skeleton) greatly influence the inhibitory activity of LCA, and that LCA binds to the 8-kDa DNA binding domain of DNA pol β (11). 1H-15N HMQC NMR analysis of the 8-kDa domain of pol β bound to LCA showed that LCA binds to the N-terminal 8-kDa domain of pol β as a 1:1 complex and interacts mainly with three amino acids (Lys 60, Leu 77, and Thr 79) in this domain (10, 11). In addition, we previously prepared biotinylated LCA derivatives and developed affinity chromatography for the separation of DNA pol R and β from a mixture of the two proteins (12). Recently, small molecules have been immobilized on solid surfaces to facilitate the purification, and to profile the cellular activities, of their binding proteins (13-18). Several matrixes for affinity purification have been reported, including a glycidyl methacrylate (GMA)covered GMA-styrene copolymer core (15), a poly(methacrylate) polymer (16, 17), and a poly(ethylene glycol) dimethylacrylamide (PEGA) copolymer (18), among others. Affinity purification of proteins is usually performed in aqueous solutions; however, the immobilization of bioactive compounds is usually performed in organic solvents. For this reason, we are interested in applying resins used for solid-phase organic synthesis to the development of methods for the affinity capture of proteins (16-18). Resins for solid-phase organic synthesis represent an ideal matrix for affinity purification because they are stable in both aqueous and organic media as well as under synthetic conditions (16, 17, 19). In addition to these attractive features, significant advances in solidphase synthesis, coupled with improvements in solid polymer supports and linkers, have made it possible to attach structurally complex and diverse compounds onto

10.1021/bc0497970 CCC: $30.25 © 2005 American Chemical Society Published on Web 12/31/2004

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the resins. Herein we report the application of resins used for organic synthesis to the detection and purification of DNA pol β. EXPERIMENTAL PROCEDURES

Compounds 2 and 5 were prepared according to the reported procedures (12, 20-22). The determination of compound 2, 5, and LCA was performed by measuring 1H NMR, 13C NMR, IR, and MS data. 1H and 13C NMR spectra were recorded on a JEOL JNM-400 (Japan) or on a BRUKER DXR400 or DRX600 instrument (Germany). Chemical shifts were reported in δ, parts per million (ppm), relative to TMS as an internal standard or were calibrated by using residual undeuterated solvent as an internal reference. IR spectra were recorded on a JASCO FT/IR-410 spectrometer (Japan). Mass spectra were obtained on an ABI QSTAR Pulsar spectrometer equipped with an ESI ion source. UV irradiation was performed by an UM-453-A instrument (Ushio Inc., Japan). Column chromatography was carried out with Fuji Silisia PSQ100B silica gel (Japan). The immobilization of 2 and 5 on resins and cleavage of LCA from 3a were checked by analytical thin-layer chromatography (TLC). TLC was performed on precoated Merck silica gel 60 F254 plates (Germany), and compounds were visualized by UV illumination (254 nm) or by heating the plates to 150 °C after spraying them with phosphomolybdic acid in ethanol. THF was distilled from sodium/benzophenone; CH2Cl2 was distilled from P2O5; and DMSO was distilled from CaH2. All other solvent and reagents were obtained from commercial sources and used without further purification. Organic extracts were dried over MgSO4, filtered, and concentrated by using a rotary evaporator. Involatile oils and solids were vacuum-dried. Succinimidyl carbonate TentaGel resins (0.5 mmol/g) were purchased from Advanced ChemTech (USA); pNitrophenyl carbonate Merrifield resins (1.0 mmol/g) were obtained from Novabiochem (USA); and succimidyl carbonate Wang resins (1.0 mmol/g) were purchased from Aldrich (USA). The fluorescein amine isomer II-immobilized resins were scanned for fluorescence by Xioskop2 plus, ebg 100 ISOLATED-2 and AxioCam HRm instrumentation (Carl Zeiss Co., Germany). We used the following buffers: His-buffer A, containing 0.5 M NaCl, and 20 mM Tris-HCl (pH 7.9) buffer and 5 mM immidazole; His-buffer B, containing 0.5 M NaCl, and 20 mM Tris-HCl (pH 7.9) buffer and 60 mM immidazole; His-buffer C, containing 0.5 M NaCl, and 20 mM Tris-HCl (pH 7.9) buffer and 1 M immidazole; TEG buffer A, containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 10% (v/v) glycerol and 50 mM NaCl; TEG buffer B, containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 10% (v/v) glycerol and 1 M NaCl; TEG buffer C, containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 10% (v/v) glycerol and 100 mM NaCl; TEG buffer D, containing 50 mM TrisHCl (pH 7.5), 1 mM EDTA, 10% (v/v) glycerol and 500 mM NaCl; sucrose buffer A, containing 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA, 5 mM 2-mercaptoethanol, 0.01% Nonidet-40, protease inhibitors (1 µg/mL leupeptin, 1 µg/mL pepstatin A, and 1 mM phenylmethylsulfonyl fluoride) and 0.25 M sucrose; and sucrose buffer B, containing 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA, 5 mM 2-mercaptoethanol, 0.01% Nonidet-40, protease inhibitors, and 1 M sucrose. General Method for the Preparation of LCAimmobilized Resins. A suspension of each resin 1a-c (1.0 equiv), 3-(3′-aminopropionyl)lithocholic acid (2) (1.2

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equiv) and diidopropylethylamine (3 equiv) in DMSOacetone-CHCl3 (3:1:1, v/v) was stirred at 50 °C for 3 days. The processes of the reactions were checked by TLC. Each mixture was quenched by the addition of MeOH (1 mL) and then stirred at 50 °C for a further 1 h. Each mixture was filtered and washed with MeOH three times and dried in vacuo. General Method for the Preparation of Methyl Carbonate Resins. A suspension of each resin 1a-c (1 equiv) and K2CO3 (3 equiv) in MeOH was stirred at room temperature (rt) for 1 day. Each mixture was filtered and washed with MeOH three times. The resulting resins were dried in vacuo. General Determination of the Amount of LCA on the Resins. A suspension of each LCA-immobilized resin (50 mg) and 1 N NaOH aq (0.5 mL) in THF-MeOHH2O (3:1:1 v/v, 2.5 mL) was stirred at room temperature for 1 day. The processes of the reactions were checked by TLC. Each mixture was filtered and washed with EtOAc three times. The filtrate was acidified to pH 1 and extracted with EtOAc three times. The organic layer was dried (MgSO4) and evaporated. The residue was purified by silica gel chromatography to yield lithocholic acid (LCA). The determination of LCA was performed by measuring 1H NMR, 13C NMR, IR, and MS data. Preparation of the Photoaffinity Beads. To a solution of TentaGel-CO2Su (0.5 mmol/g, 56.0 mg) and 5 (20.0 mg, 0.056 mmol) in DMF (0.6 mL) was added i-Pr2NEt (0.02 mL, 0.11 mmol) at room temperature. The mixture was then stirred for 34 h at room temperature. The processes of the reactions were checked by TLC. After filtration, the beads obtained were washed with MeOH three times to yield photoaffinity beads. General Method for the Immobilization of Compounds on Photoaffinity Beads. A solution of photoaffinity beads (0.165 mmol/g, 30.0 mg) and substrate (5.0 µmol) in MeOH was stirred for 30 min. The solvent was removed under reduced pressure and the beads were dried in vacuo. After irradiation of the photoaffinity beads and substrate by a high-pressure mercury arc lamp for 1 h, the beads were washed with MeOH (3 times) and dried in vacuo. Expression and Purification of Six Histidine Tagged-DNA Polymerase β (pol β) (23). The cDNA encoding pol β from Rattus norvegicus was cloned into the expression vector pET28b(+), and the Escherichia coli BL21 (DE3) was transformed with the vector. A single colony was used to incubate 600 mL of LB medium containing 1% glucose and 50 µg/mL of kanamycin at 30 °C. The cells were grown overnight and used to incubate 6 L of the same medium at 30 °C. After 3 h of growth, 1 mM IPTG was added. After 4 h of growth at 30 °C, the cells were harvested by centrifugation at 4000 rpm for 10 min at 4 °C in a HITACHI 10A rotor. The cells were resuspended in His-buffer A, sonicated 20 times for 20 s, and centrifuged for 30 min at 15 000 rpm in a HITACHI 20-2 rotor (Japan) at 4 °C. The supernatant was loaded onto His-Bind Resin preequilibrated with Hisbuffer A. The column was washed with His-buffer B, and the bound proteins were eluted with His-buffer C. The fraction containing the six-histidine-tagged pol β protein was dialyzed against TEG buffer (containing 50% (v/v) glycerol and 100 mM NaCl) and stored at -20 °C until use. SDS-PAGE Analysis. Analyses by SDS-PAGE were generally done with 15% SDS-polyacrylamide gels and CBB staining. The eluted fractions of the resins from the nuclear extract were analyzed by SDS-PAGE (10-20% gel) and the gel was stained with silver.

Affinity Capture of Mammalian DNA Polymerase β

Western Blotting Analysis (24). For Western blotting analysis, purified proteins were first resolved on SDS-PAGE gel (10-20%) and then transferred to nitrocellulose membranes and probed with a primary antibody, DNA polymerase β Ab-3 rabbit polyclonal antibody (Lab vision corporation, USA). Anti-rabbit IgG conjugated with horseradish peroxidase (VECTOR Laboratories, Inc., USA) was used as a secondary antibody. The antigen-antibody complex was visualized using SuperSignal West Dura (PIERCE). The chemiluminescence of the bands corresponding to human DNA polymerase β was quantified using Luminescent image analyzer, according to the manufacturer’s instructions (LAS-1000 plus, Fuji Film, Japan). Affinity Purification Using the LCA-Immobilized TentaGel, Merrifield, and Wang Resins. All procedures were performed at 4 °C. Each resin (20 mg), preequilibrated with TEG buffer A, was added to a solution (1.0 mL) of proteins (pol β, 8 nmol; hemoglobin, 15 nmol; trypsin inhibitor, 15 nmol; lysozyme, 15 nmol) in TEG buffer A, and the batch adsorption of proteins to the proteins was carried out overnight. Hemoglobin, trypsin inhibitor, and lysozyme were purchased from Wako Pure Chemical Industry (Japan). The resin was collected by centrifugation, and 800-µL aliquots of each supernatant (flow through) were kept for analysis. The resins were then washed 4 times with 800 µL TEG buffer A, and 800-µL aliquots of each supernatant (wash) were kept for analysis. Elution was performed 3 times with 100 µL of TEG buffer A containing 1% Tween20 and 80µL aliquots of each supernatant (eluate) were kept for analysis. The resins were then boiled at 100 °C in 20 µL of SDS-PAGE loading buffer containing 1.25 mM DTT, 2.5% SDS, 100 mM Tris-HCl pH 6.8, 12.5% (v/v) glycerol and 0.01% BPB and 20-µL aliquots of each sample were kept for analysis. Determination of the Amount of pol β bound To the Photoaffinity Beads Immobilized pol β Inhibitors (25). Each resin (10 mg) was suspended in 800 µL of TEG buffer and then 200 µL of pol β predialyzed against TEG buffer C was added. The batch adsorptions of proteins were carried out overnight. The amount of the His- tagged pol β protein bound to the beads was determined by Bradford assay, with absorbance measured at 595 nm. Detection of pol β from Human T lymphoma Molt4 Cells by Using LCA-Immobilized TentaGel Resin. All procedures were performed at 4 °C. Frozen Molt4 cells (1 g) were crushed and suspended in 10 mL of sucrose buffer A. The Molt4 suspension was homogenized, and then centrifuged for 10 min at 9000 rpm in a HITACHI 20-2 rotor. The precipitate was then resuspended in 10 mL of sucrose buffer A. The suspension was layered in a thin band on top of 30 mL of sucrose buffer B, and then centrifuged for 30 min at 9000 rpm in a HITACHI 20-2 rotor. The precipitate was suspended in 5 mL of TEG buffer C containing 5 mM 2-mercaptoethanol, 0.01% Nonidet-40, and protease inhibitors, sonicated, and incubated on ice for 20 min, before being centrifuged for 20 min at 15 000 rpm in a HITACHI 20-2 rotor. The supernatant was further centrifuged for 30 min at 40 000 rpm in a HITACHI 70 Ti rotor (Japan). The LCA-immobilized TentaGel resin (10 mg) and nonimmobilized one (10 mg), which were preequilibrated with TEG buffer C, were added to the supernatant (5 mg; 1.2 mL) to adsorb proteins. After incubation of the sample for 3.5 h, aliquots of the supernatant were removed and kept for the analysis. These resins were first batch washed five times with 1 mL of TEG buffer C for 10 min

Bioconjugate Chem., Vol. 16, No. 1, 2005 99 Scheme 1. Preparation of LCA-immobilized Resinsa

a (a) TentaGel resins. (b) Merrifield resins. (c) Wang resins. R ) N-succinimidyl (1a and 1c) and R ) p-nitrophenyl (1b).

and 1-mL aliquots of each supernatant were kept. Next TEG buffer D (200 µL) was added to each resin, batch elution was carried out for 10 min, and 200-µL aliquots of each supernatant were kept. Then TEG buffer B (200 µL) was then added to each resin, batch elution was carried out for 10 min, and then 200-µL aliquots of each supernatant were kept for analysis. Finally, TEG buffer B (200 µL) containing 1% Tween20 was added to each resin, batch elution was carried out for 10 min, and 200µL aliquots of each supernatant were kept. RESULTS AND DISCUSSION

Initially, lithocholic acid (LCA) was immobilized on TentaGel resins (1a), Merrifield resins (1b), and Wang resins (1c). We previously found that the carboxyl group of LCA plays an important role in the inhibitory activity of LCA toward DNA pols (11, 12). Therefore, LCA was immobilized on the resins with the 3-hydroxyl group rather than the carboxyl group (12). Treatment of carbonate resins 1a-c and 3-(3′-aminopropionyl)lithocholic acid (2) with iPr2NEt in DMSO-acetone-CHCl3 gave LCA-immobilized resins 3a-c (Scheme 1). The amount of LCA immobilized was determined from the amount of LCA cleaved by hydrolysis from the resins. As a result, the amount of LCA immobilized on 3a-c was 0.14, 0.48, and 0.10 µmol/mg of resin, respectively. Methyl carbonate resins 4a-c were also prepared to examine the nonspecific binding of proteins. To test the ability of these affinity resins, the maximum amount of pol β (23) binding to each resin (10 mg) was determined by monitoring absorbance at 595 nm (Figure 1). After 3a-c (10 mg each) were equilibrated with a buffered solution of pol β (0.21 mg, 5.4 nmol), the amount of pol β that bound to the resins was determined by the colorimetric method reported by Bradford (25). From the amount of pol β that bound to 4b and 4c (0.014 and 0.012 mg, respectively), about 30% of the amount of pol β that bound to 3b and 3c was due to nonspecific binding. By contrast, only a small amount (0.003 mg) of pol β bound to 4a; thus, the nonspecific binding of pol β to resin 3a was far less (∼10%). We consider that the poly(ethylene glycol) (PEG) grafted on the TentaGel resin used in 3a and 4a prevented the nonspecific binding of pol β (26, 27).

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Figure 1. Pol β binding to resins 3a-c and 4a-c. Methyl carbonate resins (4a-c) were used as a negative control. The amount of pol β bound to each resin was determined by Bradford assay (25) by monitoring absorption at 595 nm.

Figure 2. Inhibition of pol β adsorption to the resins by the addition of free LCA. The relative amount of pol β bound to 3a in the absence and presence of increasing concentrations of free LCA was determined. The amount of pol β bound to 3a in the absence of free LCA was arbitrarily taken to be 100%. Resin 4a was used as a negative control.

The total amount of LCA that was immobilized on 10 mg of 3a was 1.4 µmol, whereas the amount of pol β binding to 10 mg of 3a was only 0.81 nmol (0.032 mg). The low absorption capacity of 3a for pol β will be the low affinity between LCA and pol β. Our previous NMR experiments suggested that the 8-kDa domain of pol β binds to free LCA in a 1:1 ratio with a dissociation constant (KD) of 1.56 mM (10). Use of resins bearing the higher affinity inhibitor against pol β will improve the absorption capacity, and the greater the amount of 3a that is used, the greater the amount of pol β that will bind to 3a. Indeed, 1.8 nmol (0.070 mg) of pol β was absorbed onto 20 mg of 3a (data not shown). To confirm that the LCA immobilized on the surface of 3a specifically recognized pol β, we carried out competition assays of pol β adsorption to the resins in the presence of free LCA (Figure 2). The amount of pol β bound to 3a in the absence and presence of increasing amounts of LCA was determined. The results showed that the greater the amount of free LCA that was added, the greater the amount of pol β that was unbound. This competitive inhibition of binding between 3a and pol β by the addition of free LCA indicated that pol β bound to the LCA immobilized on 3a in a specific manner.

Kuramochi et al.

Figure 3. Relationship between the amount of LCA immobilized on 3a and the amount of pol β bound to the resin. The amount of LCA immobilized on TentaGel resins was as follows: lane 1, 0.16 µmol/mg of resin; lane 2, 0.14 µmol/mg of resin; lane 3, 0.09 µmol/mg of resin; lane 4, 0.07 µmol/mg of resin; lane 5, 0.04 µmol/mg of resin.

Figure 4. SDS-PAGE of input materials (pol β, hemoglobin, lysozyme, and trypsin inhibitor), flow-through, wash, elute, and resin during affinity purification with 3a. Lane 1, pol β; lane 2, hemoglobin; lane 3, trypsin inhibitor; lane 4, lysozyme; lane 5, mixture of these proteins; lane 6, flow-through; lane 7-10, wash; lane 11-13, elute; lane 14, resin; M, molecular weight marker.

We next examined the relationship between the amount of LCA immobilized and the amount of pol β bound to the resin (Figure 3). Resins, 3a with various concentrations of immobilized LCA (0.04 ∼ 0.16 µmol/mg resin), were prepared, and the amount of pol β bound to each resin was determined by Bradford assay. The amount of the LCA immobilized was determined by the amount of LCA cleaved by hydrolysis from the resins. The immobilization of 2 on 1a under various conditions was also examined; however, the maximum concentration of immobilized LCA was found to be only 0.16 µmol/mg of resin. This low reactivity is probably due to the insolubility of 2 in organic solvents. In Figure 3, the amount of pol β bound to the resin is plotted against the decreasing amounts of immobilized LCA. It can be seen that the more LCA that was immobilized on TentaGel resins, the more pol β that bound to the LCA-immobilized TentaGel resins; however, pol β binding seemed to reach a maximum when the LCA immobilized reached a concentration of 0.16 µmol/mg of resin. An example of the affinity purification of pol β from a mixture of proteins by 3a is shown in Figure 4. After 3a was equilibrated with a mixture of pol β, hemoglobin, trypsin inhibitor, and lysozyme, the resins were washed with TEG buffer A. Bound pol β was then eluted by the addition of 1% Tween20. Although the flow-through contained all of the charged proteins, which had various isoelectric points, pol β was certainly retained and purified by the LCA-immobilized resin. This result

Affinity Capture of Mammalian DNA Polymerase β

Bioconjugate Chem., Vol. 16, No. 1, 2005 101 Scheme 2. Preparation of Photoaffinity Beads (6)

Scheme 3. Photoaffinity Reaction of Photoaffinity Beads (7) and Fluorescein Amine Isomer II (8)

Figure 5. Partial purification of pol β from Molt4 nuclear extracts by the resin 3a. (A) SDS-PAGE analysis of the eluted samples from the resin 3a and 4a. The gel was stained with silver: lane 1, nuclear extract; lane 2, elute from 4a with TEG buffer B containing 1% Tween20; lane 3, elute from 3a with TEG buffer B containing 1% Tween20; M, molecular weight marker. (B) Western blotting analysis for pol β. Proteins were transferred to a nitrocellulose membrane, and pol β was detected with anti-pol β polyclonal antibody. Lanes were the same as those for panel A.

indicates that only pol β specifically bound to the LCAimmobilized resins. To test whether the LCA-immobilized resins can retain pol β from many kinds of proteins, the capture of pol β from a crude nuclear extract of human T lymphoma Molt4 cells by using 3a was examined (Figure 5). Resins 3a and 4a were respectively incubated with the nuclear extract (5 mg). After the resins were washed with TEG buffer C, TEG buffer D, and TEG buffer B, the bound fraction was eluted with 1% Tween20. Figure 5A shows SDS-PAGE analysis of the nuclear extract (lane 1) and the fractions eluted from 4a and 3a (lanes 2 and 3, respectively). Although the total proteins in the fraction eluted from 3a was obviously less than those in the nuclear extract (lane 1 versus lane 3), it was difficult to identify the band corresponding to pol β. However, pol β was successfully detected by Western blotting using an anti-pol β polyclonal antibody (Figure 5B) (24). Thus, pol β was certainly retained from the crude nuclear extract by 3a and eluted with 1% Tween20 (lane 3, Figure 5B). On the other hand, pol β was not detected by Western blotting in the eluted fraction from 4a (lane 2, Figure 5B). These observations suggest that pol β specifically recognized LCA on the surface of 3a. Furthermore, the signal intensity of the band in lane 1 was approximately equal to that in lane 3 in Figure 5B (lane 1/lane 3 ) 100: 99). Unfortunately, it was unable to identify how pure the eluted fraction from the resin 3a was because the amount of the proteins in the eluted fractions was too little to determine the protein concentration by Bradford assay. Indeed, several nonspecific binding proteins were observed in the eluted fraction, but it is possible to concentrate pol β from the crude nuclear extract by 3a. These results suggest that a combination of the inhibitor-

immobilized resins and classical column chromatography may facilitate the purification of pol β from a crude extract. Nonspecific binding of proteins to 3a would be due to hydrophobic interactions between proteins and the matrix of 3a (16). As shown in Figure 1, LCA-immobilized Merrifield resins (3b) and Wang resins (3c) exhibited much more nonspecific protein binding. The matrixes of 3b and 3c are more hydrophobic than that of TentaGel (3a). Introduction of hydrophilic spacers on 3a could reduce the nonspecific binding proteins (15, 17). To facilitate the immobilization of compounds on the resins, we prepared photoaffinity beads (6), that is, TentaGel resin beads with a photoreactive group at the free termini (20-22, 28). Small molecules can be immobilized to such groups on a glass slide by a photolabeling reaction, as described in the small-molecules microarrays reported by Kanoh et al. (28) We therefore considered that, by using this methodology, small molecules, proteins, oligosaccharides, and DNA could be immobilized on the resin by UV irradiation. Photoaffinity beads (6) were prepared according to Scheme 2, in which a highly reactive carbene would be generated following the UV irradiation of 6 and would react with compounds in a nonspecific manner. To check immobilization on the photoaffinity beads (6) following a photoreaction, the fluorescein amine isomer II (7) was immobilized on TentaGel resin by UV irradiation (Scheme 3). A solution of 6 and 7 in MeOH was stirred for 30 min at room temperature (rt). The solvent was removed under reduced pressure, and the beads were dried in vacuo. After irradiation of the solution of 6 and 7 by a high-pressure mercury arc lamp for 1 h, the beads were washed with MeOH (3 times) and dried in vacuo.

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Figure 6. Microscopy (left) and the fluorescence (right) images of resins 6 and 8. Scheme 4. Photoaffinity Reaction of Photoaffinity Beads (6) and pol β Inhibitors 9a-c

Figure 7. Amount of pol β bound to resins 10a-c. Resin 6 was used as a negative control.

The resulting beads (8) were scanned for fluorescence by using a fluorescence microscope (Figure 6). The strong yellowish green fluorescence signals produced by 8 indicated that the fluorescein amine II (7) had been immobilized on the surface of resin 6 by the photoaffinity reaction. Because LCA (9a), SQDG (9b) (29-31), and epolactaene (9c) (32-34) inhibit the activities of pol β in a dosedependent manner, each of these molecules was immobilized on the photoaffinity beads (6) in the same way to obtain the inhibitor-immobilized TentaGel resins

10a-c (Scheme 4). The maximum amount of pol β binding to 10a-c (10 mg) was determined by monitoring absorbance at 595 nm (Figure 7). Whereas only 0.0075 mg of pol β bound to 6, pol β was absorbed onto all of the inhibitor-immobilized resins. The amount of pol β bound to 10a-c (10 mg) was 0.024, 0.037, and 0.019 mg, respectively. The immobilization of 9a-c on 6 proceeded in a nonspecific manner; however, some of the immobilized 9a-c did not lose the ability to bind pol β. As mentioned above, the carboxylic group plays an important role in the interaction of LCA andpol β. Therefore, LCA was immobilized on 3a through its 3-hydroxyl group. By contrast, LCA was immobilized in a nonspecific manner on 10a-c. However, there was not much difference between the amount of pol β binding to 3a and that binding to 10a (0.032 mg for 3a; 0.024 mg for 10a). These findings show that the immobilization of protein inhibitors by a photoaffinity reaction will be effective not only for detecting the interactions between small molecules and proteins, but also for purifying proteins via these interactions. Finally, the affinity purification of pol β from a mixture of proteins containing pol β, hemoglobin, trypsin inhibi-

Affinity Capture of Mammalian DNA Polymerase β

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applicable to the affinity capture of other proteins based not only on small molecule-protein interactions but also protein-protein interactions, DNA-protein interactions, and so on. Studies of the use of resins in further proteomics applications are currently under way. ACKNOWLEDGMENT

This work was supported in part by a Grant-in-aid of the JSPS. LITERATURE CITED

Figure 8. (A) SDS-PAGE of starting materials (pol β, hemoglobin, lysozyme, and trypsin inhibitor), flow-through, wash, elute, and resin during affinity purification with resin 10a. (B) SDS-PAGE of starting materials (pol β, hemoglobin, lysozyme, and trypsin inhibitor), flow-through, wash, elute, and resin during affinity purification with resin 10b. Lane 1, flow-through; lane 2-5, wash; lane 6-8, elute; lane 9, resin; M, molecular weight marker.

tor, and lysozyme was also successfully achieved with photoaffinity beads 10a and 10b (Figure 8). CONCLUSION

We report here the application of resins normally used in solid-phase organic synthesis to the affinity capture of DNA pol β. LCA, an inhibitor of a mammalian DNA polymerase β, was immobilized on Merrifield resins, Wang resins, and TentaGel resins, and the batch affinity purification of pol β from a mixture of proteins using these LCA-immobilized resins was examined. Among the resins, TentaGel resins were found to be the most effective for both purifying pol β and reducing nonspecific protein absorption. Furthermore, with the LCAimmobilized TentaGel resin, it was possible to concentrate pol β from a crude cell extract. To facilitate the immobilization of compounds on TentaGel resins, we also designed and prepared photoaffinity beads containing a photoreactive group at the free termini of the TentaGel resin. The immobilization of compounds on the resins was easily achieved by UV irradiation. LCA, C18-β-SQDG, and epolactaene, which all inhibit pol β, were immobilized on the photoaffinity beads by photoreaction. Pol β was successfully bound by all three of the immobilized pol β inhibitors. The batch affinity purification of pol β from a protein mixture was also achieved by the use of these resins. We believe that these inhibitor-immobilized resins will facilitate the purification of pol β from a crude extract. As this methodology for the affinity capture of proteins is based on ligand-receptor interactions, it will be

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