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Bioconjuflte Chem. 1003, 4, 300-304
Labeling of Immunoglobulins with Bifunctional, Sulfhydryl-Selective, and Photoreactive Coumarins Janina Baranowska-Kortylewicz and Amin I. Kassis' Department of Radiology, Nuclear Medicine Division, Harvard Medical School, Shields Warren Radiation Laboratory, 50 Binney Street, Boston, Massachusetts 02115. Received October 27, 1992
4-(Bromomethyl)-6,7-dimethoxy-2-oxo-2H-benzopyran (la) and 4-(bromomethyl)-7-methoxy-2-oxo2H-benzopyran (lb) and their iodo analogs (2) reacted selectively with the sulfhydryl groups generated in a limited reduction of the hinge disulfide bonds of an immunoglobulin G (IgG) giving proteins labeled with a fluorescent and/or radioactive moiety. Quantitative alkylation of free thiol groups was obtained with 1 and 126'127I-2. Photodimerization of these adducts produced IgG with covalently joined heavy chains.
Photoreactive reagents for modification of proteins usually carry two distinct reactive moieties, one photosensitive, such as an azide, and the second conventional, such as an active ester, imidate, or active halogen. The covalent bond formation between side chains of the amino acid backbone of proteins and these modifying reagents relies predominantly on the nucleophilicity of the eamino group of lysine. The modification at more specific sites involves sulfhydryl groups of cysteine residues obtained by the reduction of either intra- or interchain disulfide bridges and, in glycoproteins,oxidized carbohydrate chains ( I ) . Only a few classes of photosensitive groups are currently available in protein-modifying compounds such as azides, diazo derivatives, and benzophenones. Nitrenes and carbenes generated from these compounds upon photolysis react indiscriminately with proteins and solvents. The reaction is nonselective and does not require any specific functional groups. This broad reactivity may not be desirable, however, when a precise reaction site is needed. This is particularly evident in the case of antibodies and enzymes where random covalent modification of protein always involvesthe risk of reacting amino acid residues in or near the binding site (2). Immunoglobulins in their monomeric form consist of four polypeptide chains, two identical heavy chains (HI1 and two identicallight chains (L),per macromolecule. Each heavy chain is covalently connected to one light chain (HL), and in turn these two HL halves are linked to each other by disulfide bonds. Cysteine residues of immunoglobulins,particularly those that link the two heavy chains, offer a potential site for modification in areas of an antibody remote from the antigen-binding site. The access to these cysteine sulfhydryls requires breaking of the intramolecular disulfide bridges, thus producing two halves of the protein now monofunctional and with an altered structure. The commonly applied sulfhydryl-reactive reagents are based on maleimide derivatives or employ active halides. None of these is capable of rejoining the two HL portions to produce intact IgG. Except for the elegant approach developed by Mitra and Lawton (31,
* Author to whom correspondence should be addressed.
1 Abbreviations: H, heavy chain of IgG; L, light chain of IgG; HL, half IgG obtained during limited reduction of disulfide bonds; IgG, immunoglobulin G; BSA, bovine serum albumin; ITLC, instantthin-layerchromatography;SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PBS, phosphatebuffered saline, pH 7.4; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; DMF,N,N-dimethylformamide.
1043-7802/93/2904-0300$04.oo/o
which utilizes equilibrium transfer alkylating cross-linking reagents, there are no compounds that can modify IgG at the hinge sulfhydryls and then connect two heavy chains together at the same site. In an attempt to develop site-selective bifunctional reagents for modification of sulfhydryl groups of cysteine at the hinge of immunoglobulins, we have considered 4-(bromomethy1)coumarins as thiol-alkylating and photoreactive reagents. This class of compounds offers a combination of an active halide, the 4-(bromomethylene) substituent, as a conventional group reactive toward thiols, and a photosensitive moiety, the 3,4-bond of the coumarin ring, known to produce photodimers upon irradiation with incident light of 340-400-nm wavelength. This is a region of the electromagnetic spectrum to which proteins are transparent, so the radiation damage to the target can be minimized. A special feature of these reagents is that the cross-linking can take place only when two coumarins are in the immediate vicinity of each other, Le., during the reestablishment of the conformational folding of a protein. EXPERIMENTAL PROCEDURES All chemicals were reagent grade. Sodium [1251]iodide (specific activity 2200 Ci/mmol) in M NaOH was purchased from NEN (Du Pont, Billerica, MA). HPLC analyses were conducted on either a Cla reverse-phase column using CH3CN/H20 ( l / l , v/v) as solvent at a flow rate of 1 mL/min or a Maxisil5 silica column (250 X 10 mm; Phenomenex, Rancho Palos Verdes, CA) with CH2Cld hexane (80/20, v/v) as solvent a t a flow rate of 1mL/min with detection at 280 nm. The radioactive species were detected with a NaI(T1) 3-in. crystal detector. TLC was done on silica gel plates 6OF2u in CHCl3/CH30H (100/1, v/v) unless otherwise indicated. ITLC of labeled proteins was performed on silica gel impregnated fiber sheets in 80% aqueous methanol (Gelman Sciences Inc., Ann Arbor, MI). lHNMR spectra were recorded on a Varian T60 spectrometer and UV spectra on a Cary 17 spectrophotometer. SDS-PAGE gels (5-12 % gradient) were run according to the standard Laemmli procedure ( 4 ) . Gels of proteins labeled with a fluorescent moiety were photographed using illumination with a hand-held Model UVGL-25 Mineralight lamp at 366 nm (UVP, Inc., San Gabriel, CA). Melting points were taken on a FisherJohns melting point apparatus and are uncorrected. Elemental analyses were performed by Galbraith Laboratories (Knoxville, TN). 0 1993 American Chemical Society
Technlcal Notes
Iodination of Coumarin Derivatives (Scheme I). The iodination of both coumarin derivatives has been accomplished using a published procedure without modifications (5). To a shielded-from-light, stirred mixture of 3.34 mmol of coumarin la or lb and silver trifluoroacetate (3.34 mmol) in 75 mL of anhydrous CHCls was added, over a 30-min period, a solution of an equimolar amount of iodine (6.68 mmol) in 25 mL of CHCl3. This was followed by additional 1 2 solution (0.1 g/mL) until no decolorization was observed. The mixture was stirred for 4 h at room temperature or sonicated for 20-60 min. After the substitution was complete, as indicated by TLC, the precipitated AgI was removed by filtration. The filtrate was washed with 0.01 % aqueous NaHS03 and water and was dried over anhydrous MgSO4. The residue obtained after evaporation of CHCL was chromatographed on a flash silica gel column in CHCl3/CH30H (200/1, v/v) for 2a or CHCl3 for 2b,c. Radioiodination of Coumarin Derivatives. In a tightly stoppered vial a mixture of 1mg of Chloramine-T and 0.1-1.0 mCi of NalZ5I(specific activity 2200 Ci/mmol for no-carrier-added and 1 Ci/mmol for carrier-added preparations in M NaOH neutralized with equimolar amounts of M CH3COOH) in 0.1 mL of water was stirred for 30 min. Chloroform (0.5mL) was added to this mixture and the stirring continued for up to 30 min until most of the radioactivity was recovered in the chloroform. The organic layer was separated, dried over anhydrous MgS04, and transferred to a vial containing a stirred mixture of 3.34 pmol of 1 and 4.0 pmol of CF3COOAg in 0.1 mL of CHCl3. The mixture was allowed to react at room temperature with or without sonication and filtered to remove AgCl and the unreacted CF&OOAg, and the solvent was evaporated to dryness under nitrogen. The residue was dissolved in either 0.1 mL CHsCN/H20 ( l / l , v/v) (2a) or CHCl3 (2b,c) and filtered through a 0.22-pm Millipore filter. Compound 2a was purified on an HPLC Cla reverse-phase column, and compounds 2b and 2c on an HPLC silica column. The identity of these compounds was verified by comparing their HPLC and TLC properties with those of 1271-2, Limited Reduction of Proteins (Scheme 11). Immunoglobulin G (IgG, mouse or rabbit) at a concentration of 5 mg/mL (3.33 X 10-5 M) was dissolved in 0.01 M PBS (2.7 mmol KC1, 120 mmol NaCl), pH 7.6. The solution also contained 0, 10,20,50, or 100molar equiv of DTT and 0.002 M EDTA to chelate any metals that might catalyze the oxidation of free sulfhydryl groups (6).The reaction was allowed to proceed at room temperature for 1-2 h, depending on the required degree of reduction. Initially the extent of the reduction in the partially reduced IgG was quantitated with 2,2'-dithiodipyridine (7,8). Once the selectivity of 1 and 2 was established, these were used to measure the number of free sulfhydryl groups. Partially reduced, not alkylated HL was separated on a Sephadex G-100 column, with 0.1 M propionic acid containing 0.002 M DTT as eluent. Labeling of Proteins with Radioiodinated Coumarins. To the solution of partially reduced IgG (HL) isolated from a Sephadex G-100 column was added an equal volume of 0.1 M carbonate buffer, pH 8, saturated with argon, and the mixture was concentrated by sizeexclusion filtration on Centricon microconcentrators (Amicon Corporation, Danvers, MA). Buffer exchange was repeated three times. After the final filtration the protein content was adjusted to 1 mg/mL, and variable amounts of 1261-2 (0.1-1 mCi) in 10pL of DMF were added to 0.1 mg of protein depending on the degree of substitution
Bioconlugete Chem., Vol. 4, No. 4, 1993 501
desired. The reaction was continued for 30 min at room temperature. Radiolabeled proteins were chromatographed on a Sephadex G-25 column with PBS as the eluent. The protein fraction collected in the void volume was concentrated on a Centricon filter and the incorporation of radioiodine was determined by trichloroacetic acid precipitation or ITLC in CH30H/H20 (80/20, v/v). Over 98% of lZ5Iwas associated with protein. The average conjugation yield was 12 % Labeling of Proteins with Fluorescent Coumarins. To the crude reaction mixture obtained after the limited reduction of IgG was added 1 either absorbed on Celite (see below) or dissolved in DMF (1mg/mL) (the reagent was used in sufficient molar excess to remove DTT still present in the reaction mixture and to allow at least an additional 2 molar equiv to react with the protein). The mixture was stirred for 1h and transferred to a Sephadex G-25 column, and the labeled protein was elutedwith PBS. Alternatively, when purified HL fragments were used, the reaction mixture was filtered through 0.2/0.8-pm Acrodisc PF filters (Gelman Sciences,Ann Arbor, MI). Satisfactory labeling was achieved in all cases 10min after the initiation of the reaction (the visibility of fluorescence of a l-pg protein band on polyacrylamide gels was used as the criterion). Reaction times of 60 min or longer resulted in complete alkylation of free sulfhydryl groups. Adsorption of 1 on Celite. To 100 mg of 1 dissolved in 100 mL of chloroform was added 400 mg of Celite which had been dried at 300 "C for 10 min. The chloroform was evaporated to dryness, and the residual powder was dried in vacuo overnight and stored shielded from light in a desiccator at room temperature (9). The reagent is stable and reactive toward thiols even after 6 months of storage. Photodimerization (Scheme 111). A solution of coumarin-S-alkylated HL fragments (3) in PBS (1 mg/ mL) was placed in quartz cuvettes and irradiated from a distance of 20 cm with a General Electric long-wave UV plant light at 0-4 "C for 0-180min. Aliquots of the reaction mixture were analyzed on areducing 5-12 5% gradient SDSPAGE. Typically after 120 min, H fragments were not detectable on gels either with Coumassie Blue staining or by fluorescence. HL labeled with more than six molecules of 1 or 1aa/1271-2 produced the covalently cross-linked HH as a major product, but the presence of protein fragments of molecular mass greater than 100 000 Da on SDS-PAGE run under reducing conditions and the quenching of fluorescence associated with L indicated, in addition, production of covalently cross-linked HL and LHHL molecules.
.
RESULTS The iodination of 4-(bromomethyl)-6,7-dimethoxy-2oxo-2Wbenzopyran (la)and 4-(bromomethyl)-7-methoxy2-oxo-W-benzopyran (lb) was accomplished in the modified Pr6vost reaction as described previously (5,10-12). Trifluoroacetyl hypoiodite generated in situ from I2 or IC1, the latter obtained by oxidation of NaI with N-chlorosuccinimide or Chloramine-T, substituted the 3-position of the lactone portion of the coumarin la,producing 3-iodo4(bromomethyl)-6,7-dimethoxy-2-oxo-W-benzopyran (2a), and the 3- or 6-position of lb, giving a mixture of 3-iodo and 6-iodo regioisomers (Scheme I). When the reaction mixture was sonicated, the time required to achieve quantitative substitution was substantially reduced from about 4 h to 20 min. The radioiodinations were conducted under identical conditions. Attempts to iodinate 1 in the absence of silver salt using 12, IC1, or NaI with a variety of oxidants traditionally used in radioiodinations resulted in the recovery of the unchanged starting material.
302 Bloconjugate Chem., Vol. 4, No. 4, 1993
Baranowska-Kortylewicz and Kassis
Scheme I
1
A
fer +
2
3
4
5
6
7
8
-205 -118 97.4
No * I
1 . Chloramine-1
2. Cf,CoOk
- 66 - 45 - 29
R~ CH,O
0
a
R-OCH,;
0 R=I
R=H; R'=l R=l; R = H
The reductive cleavage of the exposed disulfide bonds in IgG was carried out with DTT in PBS (6). The reducing agent was used in 10-100 molar excess compared to the moles of protein. The disulfide bridge in rabbit IgG was reduced quantitatively with 10-50 molar equiv of DTT and reaction times not extendingbeyond 2 h. The reaction with interchain disulfide bonds in the vicinity of the hinge region went to completion with minimal reduction of H-SS-L bridges, as indicated by gel electrophoresis under nonreducing conditions and by free sulfhydryl determination. For mouse IgG, 100molar equiv of DTT and longer reaction times were required to generate HL halves of the IgG molecule since this protein contains several hinge disulfide bonds. The number of free sulfhydryl groups in the reduced proteins was estimated with 2,2'-dithiodipyridine using the extinction coefficient of 7600 cm-l M-l at 343 nm in PBS (7,8). When the selectivity of 1for thiol groups in proteins was confirmed, the degree of protein alkylation was measured at 350 nm ( E 10 120 cm-l M-l for la and E 9600 cm-l M-l for lb). Under these conditions, 2-6 thiol groups on average were generated per molecule of rabbit IgG and 8-10 per molecule of mouse IgG. Extinction coefficients (E) used to measure the degree of thiol substitution were determined for model compounds ben4- [S-(2'-aminoethyl) thio] -6,7-dimethoxy-2-oxo-2Hzopyran and its 7-methoxy analog. The conjugation of coumarins 1 to reduced IgG was carried out either in a heterogeneous mixture using 1 adsorbed on Celite or in a homogeneous solution by adding the reagent dissolved in DMF to the solution of protein in PBS (Scheme 11). The reactions proceeded at approximately the same rate. The use of solid 1 allowed for a simple purification using only filtration through a 0.2-pm filter. For homogeneous reactions it was necessary to use gel filtration chromatographyon a Sephadex G-25column (1 X 15 cm) to remove the unreacted reagent (some precipitation of coumarin occurred at higher coumarin to protein ratios). The removal of excess DTT proved unnecessary in either case since, with the amounts of 1 equal to the molar excess of DTT plus 2 molar equiv for each free SH, quantitative alkylation was achieved (the alkylation product of DTT with 1 is insoluble in buffers and most organic solvents). The labeling of protein with Scheme I1
B
-205 -116 97.4
- 66 - 45 -29 Figure 1. Reducing 5-12 % SDS-polyacrylamide electrophoresis of partially reduced rabbit IgG (HL)conjugated to la, irradiated with UV light at 0-4 "C in PBS, and treated at 95 "C with mercaptoethanol-containing buffer. (A) Gel stained with Coomassie Brilliant Blue following irradiation of 3 for 0 (lane l),5 (lane 2), 10 (lane 5), 20 (lane 3), 40 (lane 4), 60 (lane 6), and 80 (lane 7) min. In lane 8: intact IgG exposed to a 500 molar excess of la,passed through Sephadex column, irradiated for 120 min, and heated at 95 "C with reducing buffer. In the unmarked lane are SDS-PAGE molecular mass markers: myosin, 205 kDa; @-galactosidase,116kDa; phosphorylase b, 97 kDa; BSA, 66 kDa; ovalbumin, 45 kDa; and carbonic anhydrase, 29 kDa. (B) Same gel photographed under illumination with a hand-held UV light.
high specific activity 12sI-2required the separation of HL chains on a Sephadex column to avoid wasting the radioiodinated reagent in the alkylation of unreacted DTT. The incorporation of radioiodocoumarin into the protein was not as efficient for no-carrier-added preparations as it was for either 1 or carrier-labeled 2. This is probably attributable to the dilution effect. Trichloroacetic acid precipitation and ITLC indicated that typically about 1276 of the total added radioiodinated 2 was covalently bound to HL. Photodimerization of the adduct 3 or 12s/1271-3 gave IgG with covalently joined heavy chains (Figure 1A,B;Scheme 111). The reaction proceeded without sensitization at 0-4 "Cand approached completionafter 120min. For proteins alkylated with six or more coumarin residues, the fluorescence of covalently linked chains persisted even after 120 min. SDS-PAGE of aliquots of the reaction mixture treated with mercaptoethanol at 95 "C was used to determine the progress of photodimerization (Figure lA,B). Under these conditions all disulfide bridges are reduced; therefore the appearance of new bands with molecular masses other than those of the light and heavy chains is a good measure of the extent of the formation of new covalent bonds not reduced with mercaptoethanol,
Bloconjugate Chem.. Vol. 4, No. 4, 1993 903
Technical Notes
Scheme I11 n
Y 3 -
4 e.g. photodimers. The individual heavy chains were not detectable after approximately 2 h of irradiation. The major product was two covalently linked heavy chains (molecular mass of about about 100000 Da), but the quenching of the fluorescence associated with the light chain and the appearance of faint bands with molecular masses over 100 000 Da on reducing SDS gels indicated production of covalently cross-linked HHL (about 125 000 Da) and LHHL (about 150 000 Da) fragments. There were no isolated HL fragments (molecular mass about 75 000 Da) present, indicating that the heavy chains reacted preferentially. The control sample of protein was not reduced with DTT but was directly exposed to 500 molar equiv of 1, then passed through the Sephadex column and irradiated for 120 min. There was neither fluorescence associated with thus treated protein nor any detectable cross-links formed (lane 8 in Figure lA,B). The photodimerization of 125/1271-3 resulted in considerable loss of iodine (about 20 % -40 7% ) but produced products identical to those observed for 3. DISCUSSION
Various maleimide and iodoacetamide derivatives of coumarin have been applied as fluorescent probes in histochemistry (13, 141, to measure thiol levels in viable cells in flow cytometry (15,161,as energy transfer donors or acceptors (17,18), and to prepare fluorescent enzyme substrates (19).Of the two derivatives of coumarin used in this study, 4-(bromomethyl)-7-methoxycoumarinhas been reported to S-label thiouridine of tRNA in aqueous solution (20, 21) and to react with pyrimidines found in serum (22). The 6,7-dimethoxy analog has been used as a sensitive fluorescent label for chromatographic detection of carboxylic acids in organic solvents (23). Nevertheless, the bromine in 4-(bromomethy1ene)-substitutedcoumarins is relatively inert. Evidently, it is not replaced by phenols,
alcohols, or amines, but it easily reacts with thiols (21). Owing to their reactivity toward sulfhydryls, 1 and 2 provide an alternative to the maleimides typically used to label cysteine residues in proteins. Both compounds can be radioiodinated (5). The mixture of the original compound and its radiolabeled analog can be used to label proteins, permitting a sensitive, dual detection. The efficiency of sulfhydryl labeling is quantitative when a slight excess of coumarin is used, and we have successfully used this method to determine the number of free thiols on several proteins. This has been tested with DTTtreated human and bovine serum albumins, avidin, and several immunoglobulins. Detection with either UV light at 350 nm or fluorescence is possible. With a quantum yield of over 0.6 in phosphate buffer for 4-substituted, noniodinated 1 (23),these compounds may be useful in rapid assays of the immunointegrity of partially reduced antibodies tagged at a site remote from the antigen-binding domain. A special feature of these reagents is their ability to form photodimers. The chemistry resembles that of psoralen-DNA interactions (24). Comparison of the behavior of BSA and half-IgG, both labeled with either la or lb, suggests that noteworthy photodimerization can be achieved only when two reacting coumarins are in close vicinity to each other. Labeled albumin, when irradiated with the UV light, reacted slowly and reducing gel electrophoresis on polyacrylamide of samples irradiated for 8 h showed only a small amount of high molecular mass aggregates (data not shown). On the other hand, the photoreaction of 3 gave after 10 min detectable crosslinking of two heavy chains (Figure 1). The effect of distance on the efficiencyof photodimerization is presently being tested in our laboratory on model compounds and denatured HL fragments. The contrasting behavior of free coumarin and 3 gives a good indication of the importance of the ideal arrangement of two photoreacting benzopyran rings. When an aqueous solution of 2-oxo-2H-benzopyran was irradiated for 4 weeks, only about 1%of the photodimer was produced (25). A similar outcome was observed when the reaction was carried out in ethanol (26). These two reports (25,26) also give some information on the stereochemistry of 4. The only observed product in protic solvents was the cishead-to-head dimer. The reactions run in benzene gave modest yield of trans-head-to-head along with traces of trans-head-to-tail dimers. Our reactions are conducted in aqueous solution without sensitizers. Therefore the cis-head-to-head-dimer seems to be the most probable stereoisomer. It is unlikely that the protein environment is sufficiently hydrophobic [the hinge region of immunoglobulins is proline- and cysteine-rich (27)l and that coincidentally a tryptophan residue is located close enough to allow for the energy transfers required to produce the trans isomer. Although the precise knowledge of the isomer formed has no bearing on the fact of the formation of photodimer 4, this issue is also being studied with model compounds. ACKNOWLEDGMENT
This research was supported by DOE Grant DE-FGO286ER60460 and NIH Grant R 0 1 CA 15523. The authors wish to thank Dr. Zbigniew P. Kortylewicz for helpful discussions and Mr. Robert M. Berman for excellent technical assistance.
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