Bioconjugate Chem. 1998, 9, 507−511
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A New Photoactivatable Reagent Capable of Transferring a Radiolabel to Target Proteins. Application to the Human Growth Hormone-Rat Liver Prolactin Receptor Interaction Ne´stor T. H. Masckaucha´n, Jose´ M. Delfino,* and Horacio N. Ferna´ndez† Instituto de Quı´mica y Fisicoquı´mica Biolo´gicas, Universidad de Buenos Aires, CONICET, Buenos Aires, Argentina. Received November 14, 1997; Revised Manuscript Received March 15, 1998
A new photoactivatable cross-linking reagent, 1-(2′-dithiopyridyl)-2-(5′-azidosalicylamido)ethane (ASDPE), was synthesized. This probe can be easily labeled with 125I in the azidosalicylamido ring and contains an activated disulfide bridge. After reaction of [125I]ASDPE with proteins, the radiolabeled moiety of the probe becomes attached to cysteine residues. Upon partial reduction of human growth hormone (hGH) with dithiothreitol, its C-terminal disulfide bond between residues 182 and 189 was cleaved and the nascent thiol groups were modified with [125I]ASDPE to yield [125I]ASET-hGH [1-(thiohGH)-2-(3′-[125I]iodo-5′-azidosalicylamido)ethane]. After binding of this hormone derivative to rat liver microsomes, followed by photolysis and subsequent reduction of disulfide bridges, the specific transfer of the radiolabeled moiety to prolactin receptor (PRL-R) was achieved. Partial purification of the radiolabeled receptor by size exclusion chromatography was performed. We anticipate that [125I]ASDPE will be generally useful in pursuing structural and functional studies of target proteins which interact specifically with protein ligands.
INTRODUCTION
After their introduction as photoaffinity reagents in 1969 (1), aryl azides became the most commonly used photoreactive compounds in biochemistry, mostly due to the simplicity of their synthesis (2). Cleavability of the reagent is an additional advantage, because it allows further analysis of the target of the cross-link by separating the photoreacted (and radiolabeled) head, which remains attached to the acceptor molecule, from the rest of the ligand. Certain photoactivatable probes of the azidosalicylamido kind, which may be cleaved by reduction of a disulfide bridge, have been employed to transfer a radioactive label between peptidic species (3, 4). This technique shows great promise for both identification and purification of ligand-binding proteins and further study of the binding site by fragmentation and peptide analysis. Herein, we synthesized and characterized ASDPE,1 a new photoreagent useful for these purposes. Rat liver PRL-R consists of a single polypeptide chain with an Mr of 42K. This species, by far the most abundant PRL-R form present in this tissue, has been the subject of previous cross-linking studies (5, 6). At present, a complete view of the transduction pathways following the initial step of binding of a lactogenic hormone to the PRL-R still has to be elucidated. In this work, we demonstrated the transfer of a radiolabel from hGH, a natural ligand of PRL-R, derivatized with [125I]ASDPE to this receptor. This opens the possibility of * To whom correspondence should be addressed: IQUIFIB (UBA-CONICET), Facultad de Farmacia y Bioquı´mica, Junı´n 956, 1113 Buenos Aires, Argentina. Fax: (54-1) 962-5457. E-mail:
[email protected]. † Died August 2, 1995. 1 Abbreviations: ASDPE, 1-(2′-dithiopyridyl)-2-(5′-azidosalicylamido)ethane; PRL-R, prolactin receptor; hGH, human growth hormone; [125I]ASET-hGH, 1-(thio-hGH)-2-(3′-[125I]iodo-5′-azidosalicylamido)ethane.
studying modifications of the PRL-R occurring along its intracellular transit, including degradation and/or phosphorylation. In addition, a receptor radiolabeled in this way could serve as a tracer in pursuing its purification and in studying the association with other membrane or intracellular molecules. MATERIALS AND METHODS
General Procedures. The 1H NMR spectra were recorded on a Bruker WM 300 NMR spectrometer, FTIR spectra on a Bruker IFS 25 infrared spectrometer, mass spectra on a Shimadzu GCMS QP-1000 mass spectrometer, and UV/visible spectra on a JASCO 7850 spectrophotometer. Thin layer chromatography (TLC) was routinely performed on 5.0 cm × 1.6 cm plates of silica gel 60 F254 (E. Merck, Darmstadt, Germany). Different solvent systems were employed: solvent 1, benzene/acetic acid (9:1, v/v); solvent 2, tetrahydrofuran/ethanol/acetic acid (4.5:4.5:1); and solvent 3, dichloromethane/ethanol/ acetic acid (6:3:1). All reagents employed were of analytical grade and were purchased from Aldrich, Sigma, or Fluka. Na125I (15-17 Ci/mg) was purchased from New England Nuclear (Boston, MA). Samples were irradiated at 254 nm in quartz cuvettes or in a Petri dish with a Desaga UVIS setup 3 cm from the lamp for the times indicated. Synthesis and Characterization of Compounds. 5-Azidosalicylic Acid (3). 5-Aminosalicylic acid (1, 200 mg, 1.3 mmol) was dissolved in 7.5 mL of 0.94 M H2SO4 and the mixture cooled to 4 °C, and NaNO2 (110 mg, 1.6 mmol) dissolved in 1 mL of distilled water was then added. The reaction mixture was gently shaken at 0 °C for 20-30 min. The resulting diazonium salt (2) was not isolated. NaN3 (420 mg, 6.5 mmol) dissolved in 10 mL distilled water was added next. From this step on, care was taken to avoid direct sunlight; thus, the following synthetic procedures were performed under dim yellow light. The reaction was allowed to proceed for 48 h at 4
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508 Bioconjugate Chem., Vol. 9, No. 4, 1998
°C with gentle stirring. After the test for diazonium salt turned negative, compound 3 was filtered and purified by recrystallization from ethanol. After 24 h at 4 °C, the crystalline material was filtered and dried. The yield of product was 67%. TLC (silica, solvent 1): Rf ) 0.34. MS (EI, 70 eV): m/e (relative intensity) 180 (4.9, M + 1), 179 (4.6, M), 152 (3.2, M + 1 - N2), 151 (3.3, M - N2). UV (1 mM HCl in methanol): λ (nm) 371 (min), 330 (max), 290 (min), 257 (max). 1-(2′-Dithiopyridyl)-2-aminoethane (6). 2,2′-Dithiodipyridine (200 mg, 0.91 mmol) was dissolved in 0.5 mL of 10% (v/v) acetic acid in ethanol, and 2-aminoethanethiol hydrochloride (5, 50 mg, 0.44 mmol) dissolved in 1 mL of ethanol was then added. The reaction mixture was incubated at 25 °C for 16-24 h under a nitrogen atmosphere. At the end, the mixture was centrifuged and the clear supernatant was dried under vacuum and dissolved in 0.25 mL of methanol. The product 6 was precipitated by the addition of 0.5-0.75 mL of diethyl ether and left overnight at -20 °C. After filtration and drying under vacuum, large prismatic crystals of compound 6 were obtained. Further purification was achieved by an additional recrystallization step from the same solvent mixture. The yield of pure product was 55%. TLC (silica, solvent 2): Rf ) 0.57. MS (EI, 70 eV): m/e (relative intensity) 157 (5.5), 143 (16.4), 112 (6.3), 111 (34.2). 1-(2′-Dithiopyridyl)-2-(5′-azidosalicylamido)ethane (ASDPE, 7). A fresh batch of ASDPE can be synthesized starting from compounds 3 and 6, which can be stored for months in the dark at 4 °C. Compound 3 (40 mg, 0.22 mmol) was dissolved in 0.35 mL of tetrahydrofuran while the mixture was gently shaken. To this solution were added N-hydroxysuccinimide (27 mg, 0.24 mmol) and dicyclohexylcarbodiimide (50 mg, 0.24 mmol), and the reaction mixture was stirred for 24 h at room temperature. At the end, the precipitate was separated by filtration or centrifugation, and to the clear supernatant containing the N-hydroxysuccinimidyl ester of 5-azidosalicylic acid (4) were added pyridine (20 µL, 0.25 mmol) and a solution of compound 6 (49 mg, 0.22 mmol). The reaction was allowed to proceed under a nitrogen atmosphere while the mixture was gently shaken for 1 week at 25 °C. The progress of the reaction was followed by TLC using solvent 3. The workup procedure was as follows. The mixture was centrifuged, and the supernatant was separated. The remaining residue was washed with 0.4 mL of tetrahydrofuran, and this solution was pooled with the supernatant. Finally, the solvent was evaporated under reduced pressure. Purification was achieved by column silica gel chromatography employing straight chloroform as the solvent. Fractions (2-3 mL) were collected and analyzed by TLC; those containing compound 7 were pooled together, and the solvent was evaporated under reduced pressure. The yield of pure product was 91%. TLC (silica, solvent 3): Rf ) 0.95. 1H NMR (CDCl3, 300 MHz): δ 3.22 (m, 2H), 3.98 (m, 2H), 7.23-7.68 (complex, 5H), 7.85 (m, 1H), 8.74 (m, 1H), 9.28 (s broad, 1H), 12.53 (s broad, 1H). 1H NMR (CDCl3 and D2O): δ 3.22 (t, J ) 5.4 Hz, 2H), 3.97 (t, J ) 5.4 Hz, 2H), 7.23-7.68 (complex, 5H), 7.85 (m, 1H), 8.73 (m, 1H). FTIR (film): wavenumber (cm-1) 3337 (br, m), 2115 (s), 1643 (m), 1597 (m), 1577 (m), 1558 (m), 1492 (m), 1418 (s), 1291 (m). After photolysis in a chloroform solution, the bands at 2115 and 1291 cm-1 diminished significantly. MS (EI, 70 eV): m/e (relative intensity) 319 (4.1, M - N2), 176 (3.2), 135 (3.8), 134 (2.9), 112 (4.8), 111 (26.5), 79 (5.3), 78 (8.2). UV (ethanol): λ (nm) 338 (max), 312 (min), 275 (sh), 233 (max). Changes that occurred
Masckaucha´n et al.
Figure 1. Time course of photolysis of ASDPE. UV spectra of compound 7 (t ) 0 s) and the photolysis reaction mixture at the times (in seconds) indicated with numbers. The sample was irradiated at 254 nm in ethanol. The inset shows the decay of the absorbance at 233 nm as a function of the time of photolysis. The data were adjusted with the equation A ) A1 exp(-kt) + A2, where A1 is the difference between the absorbance values at 233 nm for t ) 0 and t ) ∞, A2 is the absorbance at t ) ∞, and k is the apparent rate constant for the decay in absorbance where A1 ) 0.496 ( 0.015, A2 ) 1.483 ( 0.010, and k ) 0.060 ( 0.004 s-1.
upon photolysis of this compound (Figure 1) are discussed in the Results and Discussion. 1-(2′-Dithiopyridyl)-2-(3′-[125I]iodo-5′-azidosalicylamido)ethane ([125I]ASDPE, 8). ASDPE (2 µL of a 2.5 mM solution in CHCl3) was dried in the bottom of a 3 mm × 40 mm tube under a nitrogen stream, and 10 µL of a 0.3 M sodium phosphate buffer (pH 7.4), 0.5 mCi of Na125I (1.5 µL), 2 µL of distilled water, and 2 µL of 1.2 mg/mL chloramine T were successively added. The mixture was stirred, and the reaction was allowed to proceed for 15 min. Then, 2 µL of dimethylformamide and 30 µL of benzene were added. After vigorous shaking and centrifugation, the organic phase containing the product (8) was separated carefully and dried under a nitrogen stream. The solid residue was dissolved in 100 µL of anhydrous ethanol. There was no need for purification, since an autoradiography of a TLC analysis of the reaction mixture showed a single spot (8) with a mobility similar to that of unlabeled ASDPE. The radioiodinated probe (35 Ci/mmol) was used immediately for the derivatization of hGH. Modification of hGH with [125I]ASDPE. hGH (200 µg) was dissolved in 200 µL of 10 mM sodium phosphate buffer (pH 8.4), and 2 µL of 25 mM dithiothreitol was added under an inert atmosphere. After 40 min at room temperature, the reaction mixture was sampled on a Sephadex G-100 column (Vt ) 0.9 mL) using 10 mM sodium phosphate buffer (pH 7.0) and 0.01 mM EDTA as the eluent. Fractions of 200 µL were collected under nitrogen. A sample of the fraction (50 µL) containing reduced hGH was tested for the content of thiol groups by estimating the absorbance at 343 nm after (i) the addition of 5 µL of 5 mM 2,2′-dithiodipyridine, (ii) incubation for 15-20 min, and (iii) dilution with water to a 600 µL final volume. The remaining partially reduced hGH (150 µL) was reacted with iodoacetamide (5.5 µL of a 1 mM solution, equivalent to a 0.5-fold molar
Photoreagent Capable of Transferring a Radiolabel
content of thiol groups) over the course of 30 min at room temperature. The resulting partially reduced and blocked hGH was modified with [125I]ASDPE as follows. The probe (100 µL of the ethanolic solution) was dried under a nitrogen stream; the residue was dissolved in anhydrous ethanol (1 µL) and propylene glycol (1 µL), and the hGH solution (10 µL) was then added. After being carefully shaken, the mixture was incubated at room temperature for 1 h under nitrogen. Next, 60 µL of iodoacetamide-treated BSA [1 mg/mL BSA in 10 mM sodium phosphate buffer (pH 7.0) and 0.01 mM EDTA, reacted with a 0.05 mM iodoacetamide solution] was added. Finally, [125I]ASET-hGH was purified by gel filtration on a Sephadex G-100 column (Vt ) 100 mL) equilibrated with 10 mM sodium phosphate buffer (pH 6.0) and 0.05 mM EDTA. To minimize radiolysis and/or reduction, this photoderivative was prepared immediately before use. For the purpose of characterization, ASET-hGH was synthesized from unlabeled ASDPE and hGH in the same fashion. UV (25 mM NaHCO3, pH 8.0): λ (nm) 351 (max), 311 (min), 270 (max), 257 (min). Binding of [125I]ASET-hGH to Rat Liver Microsomes. Microsomes (1.2 mg of protein), prepared as described by Bonifacino et al. (7), were incubated in flatbottom plastic tubes in the presence of 3.5 ng of [125I]ASET-hGH (approximately 9 × 104 cpm) in a total volume of 160 µL with or without a 1000-fold molar excess of unlabeled hGH. Samples were run in triplicate. The incubation medium was 25 mM Tris-HCl, 10 mM CaCl2, 10 mM MgCl2, and 0.25 mg/mL BSA (pH 7.5). Samples were incubated in the dark for 3 h at 25 °C. Binding was stopped by addition of 0.5 mL of cold buffer, and the suspension was then irradiated at 254 nm for 6 min while it was gently shaken and finally centrifuged for 5 min at 2500g. The resulting pellet was suspended in 0.5-1 mL of buffer and kept at -20 °C until it was used. The protein content was evaluated (8) after boiling a sample for 20 min in 1 M NaOH. Partial Purification of PRL-R after Radiolabel Transfer with [125I]ASET-hGH. Microsomes (0.5 mL, 0.9 mg of protein) bound to [125I]ASET-hGH were centrifuged, and the pellet obtained was dissolved in 0.5 mL of 42 mM Tris-HCl buffer (pH 7.0), 1.8% (w/v) SDS, 3 mM EDTA, and 0.8% (v/v) 2-mercaptoethanol. The mixture was incubated overnight at room temperature and then applied to a Sephadex G-100 column (Vt ) 14 mL), equilibrated with 3.5 mM Tris-HCl buffer (pH 7.0), 0.15% (w/v) SDS, and 0.25 mM EDTA. Fractions (220 µL) were collected, freeze-dried, and analyzed by SDSPAGE. Gel Electrophoresis and Autoradiography. Samples (0.10-0.15 mg of protein) were boiled at 100 °C for 3 min in the presence of sample buffer [1.5% (w/v) SDS, 5% (v/v) glycerol, 25 mM EDTA, and 35 mM TrisHCl buffer (pH 7.3)] and 5% (v/v) 2-mercaptoethanol. Proteins were separated by SDS-PAGE on 6.5 to 12% T [1.5% C and 0.2% (w/v) SDS] gradient gels (9). Gels were then stained with Amido Black, destained, and dried. Radioactive bands were visualized by exposure of gels on Kodak X-Omat XAR-5 films at -70 °C for 5-20 days with the aid of intensifying screens. Western Blot Analysis. After SDS-PAGE [10% T, 1.5% C, and 0.1% (w/v) SDS], rat liver microsomes were blotted onto a nitrocellulose membrane for 2 h at 100 V in 25 mM Tris and 193 mM glycine buffer with 20% (v/ v) methanol. After being blocked for 2-3 h with 4% (w/ v) dry fat-free milk in buffer [PBS and 0.1% (v/v) Tween 20 (pH 7.3)], the nitrocellulose membrane was rinsed four times for 10 min with buffer and incubated overnight at
Bioconjugate Chem., Vol. 9, No. 4, 1998 509 Scheme 1. Synthesis of [125I]ASDPEa
a (a) NaNO , aqueous H SO , 0 °C; (b) NaN , aqueous H SO , 2 2 4 3 2 4 4 °C; (c) N-hydroxysuccinimide, dicyclohexylcarbodiimide, tetrahydrofuran, 25 °C; (d) 2,2′-dithiodipyridine, acetic acid/ ethanol, 25 °C; (e) pyridine/tetrahydrofuran, 25 °C; (f) Na125I, chloramine T, sodium phosphate buffer, pH 7.4.
room temperature with T6 mouse IgG anti-PRL-R mAb (4 g/mL, purchased from Affinity Bioreagents, Golden, CO) (10) in buffer with 1% (w/v) BSA. Next, the membrane was rinsed four times for 15 min with buffer and incubated for 1 h with a donkey anti-immunoglobulin G antibody [1/5000 dilution in buffer and 2% (w/v) BSA]. Finally, the membrane was rinsed in PBS and revealed by an enzymic chemiluminescent assay (ECL, Amersham International). RESULTS AND DISCUSSION
Among the wide variety of photoactivatable reagents available, only a relatively limited set has the added capability of carrying a radioactive label and of transferring it between molecules. Herein, we describe the synthesis and use of the novel photoactivatable probe ASDPE (7, Scheme 1). ASDPE includes the photoreactive azidosalicylamido headgroup at one end and the thiol-reactive function dithiopyridine at the other end. The linkage between these moieties is accomplished through a short ethylene bridge. ASDPE can be radiolabeled with 125I to yield [125I]ASDPE (8, Scheme). In turn, this compound is reacted with natural cysteine residues present in proteins or those introduced by sitedirected mutagenesis, to yield a stable disulfide bridge. Next, the derivatized protein interacts with its target molecule, and upon photolysis, the azido group generates a highly reactive nitrene species which inserts readily into different functional groups present in the environment around the headgroup. At variance with other reagents of this class (3, 4, 11), it is noteworthy that the phenol group para with respect to the azido group orients the substitution of the bulky 125I in meta, thus minimizing steric hindrance in the addition reactions of the nitrene. The expectation is that this feature will result in improved yields of photoadduct. A convergent strategy for the synthesis of ASDPE is described in Scheme 1. The starting materials were 5-aminosalicylic acid (1) and mercaptoethylamine (5). We attempted to optimize yields at each step to avoid unnecessary purifications whenever possible. For analytical purposes, a small sample of each compound was routinely purified before the final characterization was carried out. The azido group was built in place of the amino substituent in 1 through a diazonium salt intermediate 2. On the other hand, 5 was reacted with 2,2′-
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Figure 2. Reductive cleavage of ASDPE. (A) UV spectrum of a 0.05 mM solution of ASDPE in 5 mM sodium phosphate buffer (pH 7.4) (curve 1). To this solution was added 2-mercaptoethanol (1 mM final concentration), and the scanning of the spectrum was repeated after 1 min (curve 2) and 16 min (curve 3). (B) Autoradiography of a thin layer chromatogram (silica, chloroform) of a 0.2 mM solution of [125I]ASDPE in ethanol (Rf ) 0.35, lane a) and its reaction mixtures with 0.2 mM 2-mercaptoacetic acid (Rf ) 0.0, lane b) or 0.2 or 2 mM 2-mercaptoethanol (Rf < 0.1, lanes c and d, respectively).
dithiodipyridine and the resulting amine 6 was coupled to 5-azidosalicylic acid (3), conveniently activated as the N-hydroxysuccinimidyl ester 4, to yield ASDPE (7). The reagent, as a dry powder, is stable in the dark for months. Finally, an efficient protocol, involving a two-phase reaction, for radiolabeling ASDPE was developed to yield 8. Thus, our reagent is able to withstand the oxidative conditions of this reaction medium with little or no appearance of byproducts (Figure 2B, lane a). The UV spectrum of ASDPE (Figure 1) showed characteristic absorption bands with maxima at 233 and 338 nm and a shoulder at 275 nm, all these transitions related to the azido group (2). Upon irradiation, the first band decayed exponentially with a half-life of 11.6 ( 0.8 s in our photolysis setup (Figure 1, inset), while there were lesser changes in the region of 250-290 nm. Although there was a concomitant decrease of the band at 338 nm, accurate measurement in this spectral region was hampered by the appearance of UV-absorbing byproducts of the photolysis at longer irradiation times (>20 s). This is consistent with the lack of a clear isosbestic point at around 280 nm. The FTIR bands at 2115 and 1291 cm-1, characteristic of the azido group (12), disappeared after photolysis (not shown). The reactivity of ASDPE with thiol groups was tested against selected mercaptans. The reaction of ASDPE with 2-mercaptoethanol produced an almost immediate (
