Proc. Nad. Acad. Sci. USA Vol. 81, pp. 5286-5290, September 1984 Biochemistry "25I-labeled crosslinking reagent that is hydrophilic, photoactivatable, and cleavable through an azo linkage (heterobifunctional reagent/carrier-free specific activity/sulfosuccinimide / azide/transfer of radioactive label from derivatized protein A to serum IgG)

JOHN B. DENNY AND GUNTER BLOBEL Laboratory of Cell Biology, The Rockefeller University, New York, NY 10021 Contributed by Gunter Blobel, March 7, 1984

ABSTRACT A radioactive crosslinking reagent, N-[4-(P- protein-SH groups (13, 14). In addition, they cannot be used azido-m-[1251]iodophenylazo)benzoyl]-3-aminopropyl- in the presence of reducing agents. We therefore have N'-oxysulfosuccinimide ester, has been synthesized. The rea- prepared a photoactivatable crosslinker that is cleavable gent is photoactivatable, water-soluble, cleavable through an through an azo linkage, as in reagents described previously azo linkage, and labeled with 125I at the carrier-free specific (15-17), but with the added advantages of the high specific activity of 2000 Ci/mtnol. Any protein derivatized with the radioactivity of 125I and a sulfonate group to render the com- reagent is thus converted into an '251-labeled photoaffinity pound water-soluble. Photoactivatable crosslinking reagents probe. Crosslinks are formed following photolysis with 366- have been prepared that contain 35S (18) and 125I (19), but in nm light, and cleavage by sodium dithionite results in the do- the latter case the reagents were not cleavable and were not nation of radioactivity to the distal partner in crosslinked com- prepared at the carrier-free specific activity of 2000 Ci/mmol plexes. The newly labeled proteins are then analyzed by gel (1 Ci = 37 GBq). electrophoresis and autoradiography. The compound was pre- In this paper we have selected the protein A-IgG interac- pared by iodination of N-[4-(p-aminophenylazo)benzoylJ-3- tion (20) as a model system with which to test the crosslink- aminopropionic acid using carrier-free Na'25I and chlora- ing reagent. We show that radioactive label is transferred to mine-T, followed by azide formation and conversion to the wa- the heavy chain of IgG when derivatized protein A-Sepha- ter-soluble sulfosuccinimide ester. As a model system, protein rose is photolyzed in the presence of human serum and sub- A-Sepharose was derivatized with the reagent under subdued sequently treated with sodium dithionite. In addition, the ra- light. Each derivatized protein A molecule contained only one dioactive labeling that occurs following photolysis with 366- crosslinker. The derivatized protein A-Sepharose was then nm light is due to reactions mediated by the aryl and photolyzed in the presence of human serum and subsequently is not due to the incorporation of free 125I. This latter result treated with sodium dithionite. Analysis of the serum by gel is in agreement with those of others (19, 21-24). electrophoresis revealed that 1.1% of the radioactive label originally present on the protein A-Sepharose was transferred MATERIALS AND METHODS to the heavy chain of IgG, which was the most intensely labeled Aniline was purchased from Aldrich and was vacuum-dis- protein in the gel. The next most intensely labeled protein was tilled at 78°C prior to use. Tetrahydrofuran (gold label), diox- IgG light chain, which incorporated radioactivity that was ane (gold label), formaldehyde sodium bisulfite addition lowver by a factor of 3.6 than that of the heavy chain. These compound, p-aminobenzoic acid, sodium nitrite, and sodium results demonstrated the specificity of the derivatized protein azide were purchased from Aldrich and used without further A-Sepharose as a photoaffinity probe. Photolabeling of IgG purification. Dicyclohexylcarbodiimide and N-hydroxysuc- was the result ofnitrene-mediated reactions and was not due to cinimide were from Pierce, Na 251 was from Amersham, so- the incorporation of free 1251. dium dithionite and chloramine-T were from Fisher, TLC plates were from EM Laboratories (Elmsford, NY), and pro- Photoactivatable crosslinking reagents have been success- tein A-Sepharose CL-4B and f-alanine were from Sigma. fully used to detect plasma membrane receptors for insulin NMR spectra were obtained on a Nicolet Fourier trans- (1), epidermal growth factor (2), human choriogonadotropin form spectrometer at 300 MHz. Chemical shifts were rela- (3), and the N-formyl chemotactic peptide (4) and to identify tive to an internal tetramethylsilane standard. IR spectra the nearest neighbors of many proteins, including fibronec- were obtained on a Perkin-Elmer model 237 B spectropho- tin (5), calmodulin (6), fibrinogen (7), glycopeptides (8), and tometer. ribosomal proteins (9) (for other previous studies, see refs. Preparation of N-[4-(p-Aminophenylazo)benzdyl]-3-gmino- 10-12). In some cases (1-4, 6, 8) relatively small proteins propionic Acid (VII). The reaction scheme for the prepara- and peptides were labeled with 125I, derivatized with a pho- tion of compound VII is shown in Fig. 1. toactivatable reagent, and crosslinked to their putative re- Sodium anilino N-methylenesulfohate (III) was prepared ceptors. However, crosslinks were not cleaved and high mo- by mixing 22 g (0.231 mol) of aniline (I) with 33.3 g (0.237 lecular weight complexes were assumed to contain the 125I- mol) of formaldehyde sodium bisulfite addition compound labeled peptide and its receptor. The above approach is more (II) in 100 ml of water at 70°C for 20 min with constant stir- difficult when used to detect receptors for larger pro- ring. The solution was then cooled on ice and the white crys- teins, due to the inability of crosslinked complexes to enter tals were collected by filtration, washed with , and vac- polyacrylamide gels. Cleavable, photoactivatable crosslink- uum-dried (yield, 66%). The N-methylenesulfonate group ing reagents were therefore developed (10-12), but most of protected the amino group from unwanted side reactions the reagents are cleavable through a bond and are during subsequent steps. therefore subject to mercaptan-disulfide interchange with A solution of diazotized p-aminobenzoic acid (IV) was prepared by dropwise addition of 3.6 g of sodium nitrite in 10 The publication costs of this article were defrayed in part by page charge ml of cold (4°C) water to an ice-cold suspension of 6.5 g payment. This article must therefore be hereby marked "advertisement" (0.047 mol) of p-aminobenzoic acid in 27.5 ml of 5.5 M HCl. in accordance with 18 U.S.C. §1734 solely to indicate this fact. After 15 min at 0°C, all of the above solution was added with 5286 Downloaded by guest on September 28, 2021 Biochemistry: Denny and Blobel Proc. Natl. Acad ScL USA 81 (1984) 5287 aminophenylazo)benzoyl]-3-aminopropionic acid (VII), was Ki6@> INH2 + HOCH2SO3 Na+ collected by centrifugation, washed with water, and vacu- (II) um-dried. The yield was 0.163 g (0.52 mmol) or 13% based (I) on the amount of compound VI used. The product at this stage was contaminated with 1-2% of @" NHcH2SO3 Na+ (III) compound VI. TLC on silica gel 60 in chloroform/methanol, 2:1 (vol/vol), was used to purify the desired compound VII (Rf = 0.45) from the contaminant VI (Rf = 0.66). NMR (com- HOOC A g2 i pound VII) (dimethyl-d6 ): 2.52 (t, 2H, CH2), 3.49 (IV) /A (q, 2H, CH2), 6.25 (s, 2H, aromatic NH2), 6.71 (d, 2H, aro- HOOC N-N j NHCH2SO3 Na+ (V) matic H), 7.71 (d, 2H, aromatic H), 7.81 (d, 2H, aromatic H), 7.99 (d, 2H, aromatic H), 8.65 (s, 1H, -NH) ppm (s = 1 1. NaOH 2. HC1 singlet, d = doublet, t = triplet, q = quartet). IR (compound VII) (potassium bromide): 3460 cm-' (N-H stretch of CONH), 3405 and 3375 cm-' (N-H HOOC ( >)N-N < NH2 (VI) stretch of NH2), 1760 cm-' (C==O stretch of COOH), 1605 cm'1 (N-H bend of NH2). | 1. DCC/POSu Preparation of N-[4-(p-Azido-m-[125I~iodophenylazo)ben- 2. P-alanine (pH 9.5) 3. HC1 zoyl]-3-aminopropyl-N'-oxysulfosuccinimide Ester (X). The reaction scheme for the preparation of the final crosslinker, compound X, is shown in Fig. 2. HOOCCH2CH2HN8 N-N NH2 (VII) To iodinate compound VII, 150 ,ul of a 5 mM solution of the compound in dioxane/water, 2:1 (vol/vol), was mixed FIG. 1. Preparation of N-[4-(p-aminophenylazo)benzoyl]-3- with 10 ,ul (1 mCi, 0.5 nmol) of carrier-free Na1251, 89 ,ul of aminopropionic acid (VII). Aniline (I) reacts with formaldehyde so- water, 30 Al of 0.3 M H2SO4, and 31 ,ul of chloramine-T at 40 dium bisulfite addition compound (II) to give the adduct III. Reac- mg/ml in water (final pH = 1.9). The solution was centri- tion of III with diazotized p-aminobenzoic acid (IV) gives the azo fuged vigorously in a vortex for 5 sec and was then incubated compound (V), which is treated with NaOH to remove the N-methyl for 30 sec at room temperature, followed by the addition of sulfonate group. The resulting azo compound (VI) is converted to an 31 ,ul of NaHSO3 at 30 mg/ml in water. The solution was active ester by using N-hydroxysuccinimide (HOSu) and dicyclo- then immediately extracted three times with 0.5-ml portions hexylcarbodiimide (DCC) and then reacts with 18-alanine to give of ether. The ether extracts were combined and concentrat- compound VII. ed to 50 ,ul with a stream of N2, and the concentrate was then applied to a 0.2-mm-thick silica gel 60 plate with plastic stirring to an ice-cold solution of compound III (13.1 g, 0.063 backing (20 x 20 cm). The plate was developed in chloro- mol) in 175 ml of 0.86 M sodium acetate. The resulting yel- form/methanol, 2:1 (vol/vol), for 90 min, and the top half of low azo compound (V) precipitated after an overnight incu- the yellow spot of Rf = 0.45 along with the region just ahead bation at 4°C and was collected by filtration at that tempera- ture. To remove the N-methyl sulfonate group, the material col- H2N N-N 94NHCH2CH2COOH (VII) lected by filtration in the previous step (compound V) was dissolved in 200 ml of 1 M NaOH and was heated at 90°C for 1. chloramine-T, Na125 I, pH 1.9 80 min. The pH of the cooled solution was lowered to 2.7 by 2. NaHSO2 v. using 6 M HCl to convert the product to the free carboxylic 125I acid. The resulting red precipitate, 4'-aminoazobenzene-4- (VI), was collected by filtration, washed H2N N-N ( NCH2CH2COOH (VIII) with water, and vacuum-dried. The yield was 3.7 g of com- acid pound VI (33% based on the amount ofp-aminobenzoic 1. NaNO2 used). 2. It was desirable at this step to add a, 3-alanine spacer arm NaN3 to compound VI, which can be done by converting com- pound VI to an active ester and subsequently reacting the 125 active ester with ,B-alanine. One gram (4.15 mmol) of com- pound VI was dissolved in 150 ml of tetrahydrofuran/water, N3© N-N ? CH 2CH2COOK (IX) 2:1 (vol/vol). To this solution was added 0.48 g (4.15 mmol) of N-hydroxysuccinimide and 4.15 ml of 1 M dicyclohexyl- carbodiimide (4.15 mmol) in tetrahydrofuran. The solution DCC/Hcu (SO3) Na was stirred for 1 hr at room temperature, and the resulting 125i 1 dicyclohexylurea was removed by filtration. To the filtrate 0 so- Na+ was then added 30 ml of tetrahydrofuran (to maintain the active ester of VI in solution) and 10 ml of a 0.56 M solution N3 N-N ) LNN2CH2-0-N (X) of P-alanine in water. The pH was adjusted to 9.5 with trieth- ylamine and the solution was stirred for 1 hr at room tem- perature. The pH was then lowered to 1.0, which led to a precipitation of contaminating compound VI, while the de- FIG. 2. Preparation of N-[4-(p-azido-m-['25Iliodophenylazo)- When benzoyl]-3-aminopropyl-N'-oxysulfosuccinimide ester (X). Com- sired product, compound VII, remained in solution. pound VII (0.75 ,umol) reacts with 1 mCi of Na125I in the presence of the pH was increased to 2.0, an additional precipitate of chloramine-T at pH 1.9. The reaction is stopped by NaHSO3, and compound VI was obtained, which resulted in the further the iodinated compound (VIII) is purified from VII by TLC. Com- enrichment of compound VII in the supernatant. The pH pound VIII is then converted to the azide (IX) and subsequently to was adjusted to 7.0 and then slowly decreased until a precipi- the sulfosuccinimide ester (X) by using dicyclohexylcarbodiimide tate formed at pH 4.5. This light orange precipitate, N-[4-(p- (DCC) and N-hydroxysulfosuccinimide [HOSu(SO3)Na]. Downloaded by guest on September 28, 2021 5288 Biochemistry: Denny and Blobel Proc. NatL Acad ScL USA 81 (1984) of the spot were cut out, eluted with dioxane/water, 2:1 (vol- albumin or other serum proteins. A fresh 40-,/d aliquot of /vol), concentrated, and rechromatographed. This process human serum was added to each tube, followed by incuba- was repeated twice more, at which point the carrier-free 1251_ tion with occasional brief centrifugation in a vortex for an labeled compound (Rf = 0.48, compound VIII) was clearly additional 30 min at room temperature. In preparation for separated from the unlabeled compound (Rf = 0.45, com- photolysis, was bubbled into each tube for 5 min. pound VII). The yellow, radioactive spot of Rf = 0.48 (10% Tubes 1 and 2 were placed under a 1.5-cm-deep Pyrex Petri of the original 1 mCi of radioactivity was present in this spot) dish, and a UVL-21 lamp (Ultraviolet Products, San Gabriel, was cut out and eluted from the silica gel with 450 p.l of diox- CA) was then placed on top of the dish. The samples were ane/water, 2:1 (vol/vol). irradiated for 8 min at room temperature, with brief centrifu- The next step of the procedure was to diazotize compound gation in a vortex every 2 min to redistribute the Sepharose. VIII and convert the diazonium salt to the corresponding The Pyrex filter screened out light of wavelength <300 nm. aryl azide. The 450 pul of eluate obtained above was then To remove all proteins from the Sepharose beads other than mixed with 30 p.l of 0.3 M H2SO4 and was chilled in an ice those that had become covalently crosslinked, water (300 ,u1) bath for 15 min. Thirty microliters of ice-cold 0.1 M NaNO2 and 10 p.l of 25% NaDodSO4 were added to each tube. Tubes was added, and the mixture was incubated at 0C for an addi- 2 and 4 received 10 ,p1 of freshly prepared 1 M sodium dithio- tional 5 min. At this point, 30 ,ul of 0.1 M NaN3 was added, nite in water (the dithionite solution was passed through a and after 5 min at 0C the solution was extracted three times 0.2 pum-Nalgene filter before use to remove traces of insolu- with 0.5-ml portions of ether. The ether extracts were com- ble material), followed by incubation at room temperature bined and evaporated completely by using a stream of N2. for 25 min. Each tube then received 10 ,u1 of 1 M dithiothrei- The residue (compound IX) was immediately dissolved in 40 tol followed by adjustment of the pH to 8 and incubation for 1.d of dry dimethyl sulfoxide. 15 min. After vigorous centrifugation in a vortex to displace The final step in the synthesis was to convert compound bound material from the protein A-Sepharose, the beads IX to the sulfosuccinimide ester (X). To the solution of com- were removed by centrifugation and each supernatant re- pound IX in dimethyl sulfoxide was added 3 ,ul of 0.1 M N- ceived 125 ul of 10% Nonidet P-40 and 100 p1L of 100% hydroxysulfosuccinimide [prepared as described (25)] in dry Cl3CCOOH. Each Cl3CCOOH precipitate was washed with dimethyl sulfoxide and 5.2 ,ul of 0.1 M dicyclohexylcarbodii- acetone and then with 10% Cl3CCOOH. The precipitates mide in tetrahydrofuran, followed by incubation at room were suspended in 50 ,p1 of water, 50 p.1 of 25% NaDodSO4, temperature for 16 hr. The solvents were then completely 100 ,ul of 1 M Tris base, 30 ,ul of 1 M dithiothreitol, and 120 ,ul evaporated in a Savant Speed-Vac concentrator, and the re- of 50% glycerol. The samples were sonicated briefly to dis- sulting dried material was washed three times with 0.5-ml perse the large pellet of serum proteins and were placed in a portions of ether to remove any unreacted dicyclohexylcar- boiling water bath for 5 min. Twenty-five-microliter aliquots bodiimide. The residue contained the desired radioactive were analyzed by electrophoresis on a 10% polyacrylamide crosslinker (compound X), which was produced in 50% yield slab gel in NaDodSO4 as described (26), followed by staining based on the amount of compound IX used. The overall yield with Coomassie blue and autoradiography of the dried gel at of compound X, based on the original 1 mCi of Na'251, was -80°C for 94 hr using preflashed Fuji x-ray film. The distri- 5%. The residue also contained unreacted N-hydroxysulfo- bution ofradioactivity was quantified by cutting the dried gel succinimide, but this compound is innocuous in subsequent into 0.5-cm sections and assaying the sections in a Packard operations. Dry dimethyl sulfoxide (40 ,ul) was used to dis- Model 5110 spectrometer. solve the residue. As a control reaction, derivatized protein A-Sepharose Following the addition of NaN3, all steps were performed was treated exactly as above except that no serum was add- under subdued light (no overhead fluorescent light) until the ed. In a second control reaction, derivatized protein A-Seph- step at which dithiothreitol was added to prepare the sam- arose was incubated with purified rabbit IgG rather than hu- ples for gel electrophoresis. man serum and was then treated in the manner described Derivatization of Protein A-Sepharose. An aliquot (6.9 above. pmol, 2.7 x 107 cpm) of the carrier-free 1251I-labeled cross- linker in 20 ,ul of dimethyl sulfoxide was evaporated in the RESULTS Speed-Vac. To the residue was added 300 ,ul of a 50% slurry Fig. 1 shows the scheme used for the preparation of the in- of protein A-Sepharose CL-4B in 0.1 M NaHCO3 (pH 8), termediate compound N-[4-(p-aminophenylazo)benzoyl]-3- followed by incubation at room temperature for 30 min with aminopropionic acid (VII). The amino group of aniline (I) occasional agitation. The molar ratio of protein A to reagent was protected by reaction with formaldehyde sodium bisul- was 103. The compound reacts with protein amino groups fite addition compound (II), and the resulting adduct (III) during this step. The reaction was stopped by the addition of reacted with diazotized p-aminobenzoic acid to give the azo 100 ,ul of 1 M ,-alanine, and the protein A-Sepharose was compound (V). Removal of the protecting group at high pH then transferred to a column and washed once with 10 ml of yielded compound VI. A /-alanine spacer arm was then add- 0.1 M NaHCO3, twice with 10 ml of 50 mM NaHCO3 con- ed to compound VI to yield the desired intermediate, com- taining 2% (wt/vol) Nonidet P-40 and 0.5 M NaCl, and final- pound VII. This latter compound was iodinated at pH 1.9 by ly twice with 10 ml of 0.1 M NaHCO3. Thirty-four percent of using carrier-free Na1251 and chloramine-T and yielded com- the crosslinker had reacted covalently with the protein A- pound VIII. Since VII was present in excess over Na125I in Sepharose, and this incorporation was completely eliminat- the iodination reaction by a molar ratio of 1500:1, it was nec- ed if the reaction was performed in the presence of 0.1 M ,3- essary to separate the labeled and unlabeled compounds. We alanine. utilized TLC for this purpose but HPLC may be more conve- Photolysis of Derivatized Protein A-Sepharose in the Pres- nient. Compound VIII, purified by TLC, was subsequently ence of Human Serum. Derivatized protein A-Sepharose (75 diazotized and converted to the aryl azide IX, which was ,ul of a 50% slurry in 0.15 M NaCl/0.05 M NaHCO3; 1.8 x extracted into ether. Following evaporation of the ether, 106 cpm) was placed in each of four 1.5-ml Eppendorf tubes. compound IX was converted to the water-soluble sulfosuc- Each tube received 40 ,ul of human serum followed by incu- cinimide ester (X) by using dry dimethyl sulfoxide as sol- bation at room temperature for 15 min. The tubes were cen- vent. The water-soluble ester was synthesized by using N- trifuged briefly in a vortex every 3 min. The supernatants hydroxysulfosuccinimide, which was prepared from N-hy- were then removed to ensure the elimination of any traces of droxymaleimide as described by Staros (25). Compound X free crosslinker that had become bound noncovalently to can either be used immediately or stored for at least 1 wk in Downloaded by guest on September 28, 2021 Biochemistry: Denny and Blobel Proc. NatL. Acad. Sci. USA 81 (1984) 5289 dry dimethyl sulfoxide at room temperature. graph of the dried gel shown in Fig. 3A. Labeled heavy chain Thirty-four percent of the radioactive crosslinker was co- is seen in lane 2 of Fig. 3B, indicating that, upon photolysis, valently incorporated into protein when 46 nM reagent react- crosslinking occurred between derivatized protein A and ed at pH 8 with 50%o (vol/vol) protein A-Sepharose CL-4B in bound IgG and that following cleavage with sodium dithion- a total volume of 300 ,ul for 30 min at room temperature. The ite radioactive label remained with the heavy chain as it was protein A was in large excess over the reagent, so that each released from the Sepharose beads. A total of 1.1% of the derivatized protein A molecule contained only one cross- radioactivity originally present on the protein A-Sepharose linker. As a model system, the derivatized protein A-Sepha- was transferred to the IgG heavy chain. The second most rose was added to human serum to determine if radioactive intensely labeled protein was IgG light chain, which incorpo- label could be specifically transferred from protein A to the rated radioactivity that was lower by a factor of 3.6 than that heavy chain of IgG following photocrosslinking and cleavage of the heavy chain. Photolysis alone (Fig. 3B, lane 1) did not of the crosslinks with sodium dithionite. Protein A is known result in the appearance of labeled heavy chain on the gel to bind to the heavy chain of IgG near the CH2-CH3 interface because the heavy chain remained covalently crosslinked to (20). the protein A-Sepharose and was removed by centrifugation. The results of the experiment are presented in Fig. 3. Fig. Omission of photolysis and dithionite treatment (Fig. 3B, 3A shows the Coomassie blue-stained material in superna- lane 3) and treatment only with sodium dithionite (Fig. 3B, tants following removal of derivatized protein A-Sepharose lane 4) did not result in labeled heavy chain. Fig. 3B shows beads when the beads were first photolyzed (lane 1), photo- that radioactive label was transferred specifically from deri- lyzed and dithionite treated (lane 2), untreated (lane 3), or vatized protein A to IgG, since very little nonspecific trans- treated only with dithionite (lane 4). The pattern is of human fer of label to other serum proteins took place. serum proteins, and a closed arrow marks the position of the As a control reaction, derivatized protein A-Sepharose heavy chain of IgG, whereas an open arrow shows the posi- was treated exactly as above, except that no human serum tion of IgG light chain. Fig. 3B, lanes 1-4, is the autoradio- was added. No labeled protein appeared on the autoradio- graph following photolysis and treatment with sodium dith- A B ionite, demonstrating that the protein seen in Fig. 3B (lane 2) 1 2 3 4 is not labeled protein A that was released from the Sepha- 1 2 3 4 rose beads. When derivatized protein A-Sepharose was in- cubated without human serum but in the presence ofpurified rabbit IgG, an autoradiograph identical to that in Fig. 3B (lane 2) was obtained following photolysis and treatment with sodium dithionite (data not shown), which confirms that radioactive label was transferred from protein A to IgG in the experiment shown in Fig. 3. As a third control reac- tion, protein A-Sepharose was derivatized exactly as de- scribed in Materials and Methods with a reagent identical to compound X (Fig. 2), except that the aryl was not converted to an aryl azide. When this derivatized protein A- Sepharose was incubated with either human serum or puri- fied rabbit IgG and subsequently photolyzed and treated with sodium dithionite, no labeled proteins appeared on the autoradiograph (data not shown). DISCUSSION The crosslinker described in this paper, N-[4-(p-azido-m- FIG. 3. Analysis of human serum samples incubated with deriva- [1251]iodophenylazo)benzoyl]-3-aminopropyl-N'-oxy- tized protein A-Sepharose. Protein A-Sepharose was derivatized sulfosuccinimide ester, has been synthesized at a specific with the radioactive crosslinker (X) and was incubated with human activity of 2000 which is the of serum. The samples, each containing 1.8 x 106 cpm of protein A- Ci/mmol, specific activity Sepharose, were then treated as shown below, followed by removal carrier-free Na'25J. Such high specific activity allows the of the protein A-Sepharose beads under denaturing conditions and incorporation of a large amount of radioactivity even though Cl3CCOOH precipitation of the supernatants. The precipitates were a given protein may be modified at only a single site. redissolved in 370 A.l of buffer and 25-1.l aliquots were analyzed by In the present paper the interaction between protein A and gel electrophoresis and autoradiography. (A) Coomassie blue- the heavy chain of IgG (20) was used as a model system to stained gel. Lane 1, photolysis only; lane 2, photolysis followed by test the ability of compound X to photocrosslink and transfer treatment with sodium dithionite; lane 3, no treatment; lane 4, treat- radioactive label. Derivatized protein A-Sepharose was pho- ment only with sodium dithionite. (B) Autoradiograph of the gel in tolyzed in the presence of human serum, and the proteins A. Conditions for lanes 1-4 correspond to those given in A. The were subsequently treated with sodium dithionite and ana- closed and open arrows show the positions of the heavy and light Radioac- chains of IgG, respectively. The gel was cut into sections and the lyzed by gel electrophoresis and autoradiography. radioactivity in each section was determined. The radioactivity in tive label was transferred to IgG and predominantly to the lane 4 was considered background and was subtracted from the re- heavy chain. True photoaffinity labeling occurred, since sults obtained for lane 2. The gel section containing the heavy chain very little nonspecific transfer of radioactive label to other in lane 2 contained 1552 cpm, whereas the corresponding section in serum proteins took place. lane 4 contained 271 cpm. The light chain gel section in lane 2 con- It is important to note that dithiothreitol cannot be used tained 492 cpm, whereas the corresponding section in lane 4 con- during crosslinking procedures because it rapidly reduces tained 139 cpm. Subtraction of the background levels of radioactiv- the aryl azide moiety of compound X to the corresponding ity in lane 4 from those in lane 2 yielded 1281 cpm for the heavy as shown for other azides However, 2-mer- and a ratio of 3.6. The amine, aryl (27). chain, 353 cpm for the light chain, heavy/light because re- slight darkening of the autoradiograph at the position of the heavy captoethanol and glutathione are suitable they chain in B (lane 4) is an artifact due to a gel crack and was not ob- duce aryl azides much more slowly (27). served when aliquots of the samples were analyzed on subsequent Photolysis of iodinated organic compounds with 254-nm gels. light has been shown to cleave carbon-iodine bonds yielding Downloaded by guest on September 28, 2021 5290 Biochemistry: Denny and Blobel Proc. NatL Acad Sci. USA 81 (1984)

carbon and iodine radicals (28), but this clearly did not occur 5. Perkins, M. E., Ji, T. H. & Hynes, R. 0. (1979) Cell 16, 941- under our conditions using 366-nm light, which is in agree- 952. ment with previous studies (19, 21-24). No radioactive pro- 6. Andreasen, T. J., Keller, C. H., LaPorte, D. C., Edelman, teins were present in the Sepharose eluate after photolysis A. M. & Storm, D. R. (1981) Proc. Natl. Acad. Sci. USA 78, alone (Fig. 3B, lane 2782-2785. 1), indicating that random labeling of 7. Bennet, J. S., Vilaire, G. & Cines, D. B. (1982) J. Biol. Chem. protein by released iodine radicals did not occur. In addition, 257, 8049-8054. protein A-Sepharose was derivatized with a reagent identical 8. Baenziger, J. U. & Fiete, D. (1982) J. Biol. Chem. 257, 4421- to compound X, except that the aryl amine was not coverted 4425. to the aryl azide, and photolysis of this material in the pres- 9. Maassen, J. A. (1979) Biochemistry 18, 1288-1292. ence of human serum yielded no labeled IgG heavy chain 10. Das, M. & Fox, C. F. (1979) Annu. Rev. Biophys. Bioeng. 8, (data not shown). Thus, incorporation of radioactive label 165-193. following photolysis with 366-nm light is due to nitrene-me- 11. Ji, T. H. (1979) Biochim. Biophys. Acta 559, 39-69. diated reactions and not to labeling via iodine radicals. 12. Peters, K. & Richards, F. M. (1977) Annu. Rev. Biochem. 46, The use of aryl azides as photolabels 523-551. has been reviewed 13. Lomant, A. J. & Fairbanks, G. (1976) J. Mol. Biol. 104, 243- (29-33) as well as the nature of the products obtained from 261. aryl (34, 35). It is clear that the nature of the photo- 14. Gilbert, H. F. (1982) J. Biol. Chem. 257, 12086-12091. products depends on whether the aryl nitrene is in a singlet 15. Jaffe, C. L., Lis, H. & Sharon, N. (1980) Biochemistry 19, or triplet state (34, 35). Singlet aryl nitrenes rearrange and 4423-4429. react with groups such as -OH and -NH in proteins (34, 35). 16. Jaffe, C. L., Lis, H. & Sharon, N. (1979) Biochem. Biophys. It was concluded that most of the aryl azide reagents cur- Res. Commun. 91, 402-409. rently in use react primarily via a singlet nitrene (35). Al- 17. Fasold, H., Klappenberger, J., Meyer, C. & Remold, H. (1971) though the presence of an iodine atom in compound X great- Angew. Chem. Int. Ed. Engl. 10, 795-801. ly enhances the rate of 18. Schwartz, M. A., Das, 0. P. & Hynes, R. 0. (1982) J. Biol. intersystem crossing from the singlet Chem. 257, 2343-2349. to the triplet state (36), it is presently not clear whether a 19. Ji, T. H. & Ji, I. (1982) Anal. Biochem. 121, 286-289. singlet or triplet nitrene is responsible for the covalent incor- 20. Lancet, D., Isenman, D., Sjodahl, J., Sjoquist, J. & Pecht, I. poration of label seen in Fig. 3B (lane 2). However, com- (1978) Biochem. Biophys. Res. Commun. 85, 608-614. pound X does recombine to give azo linkage in high yield 21. Rashidbaigi, A. & Ruoho, A. E. (1981) Proc. Natl. Acad. Sci. when photolyzed in free solution (unpublished results), and USA 78, 1609-1613. such azo compound formation is characteristic of triplet ni- 22. Bayley, H. & Knowles, J. R. (1980) Ann. N. Y. Acad. Sci. 346, trenes (34). 55. 23. Bercovici, T. & Gitler, C. (1978) Biochemistry 17, 1484-1489. Note Added in Proof. We have found that quantitative cleavage of 24. Spiess, M., Brunner, J. & Semenza, G. (1982) J. Biol. Chem. such low concentrations of azo linkages requires two to three suc- 257, 2370-2377. cessive additions of sodium dithionite at 15-min intervals, rather 25. Staros, J. V. (1982) Biochemistry 21, 3950-3955. than a single addition. Each addition should yield a final dithionite 26. Blobel, G. & Dobberstein, B. (1975) J. Cell Biol. 67, 835-851. concentration of 0.2 M. 27. Staros, J. V., Bayley, H., Standring, D. N. & Knowles, J. R. (1978) Biochem. Biophys. Res. Commun. 80, 568-572. We thank Drs. Charles Jaffe and Reid Gilmore for many helpful 28. Wolf, W. & Kharasch, N. (1965) J. Org. Chem. 30, 2493-2498. discussions, Dr. Gregory Conner for providing the human serum, 29. Staros, J. V. (1980) Trends Biochem. Sci. 5, 320-322. and Ms. Gisele Nimic for typing the manuscript. This work was sup- 30. Chowdhry, V. & Westheimer, F. H. (1979) Annu. Rev. Bio- ported by National Institutes of Health Grant GM27155. J.B.D. is chem. 48, 293-325. supported by U.S. Public Health Service Postdoctoral Fellowship 31. Bayley, H. & Knowles, J. R. (1977) Methods Enzymol. 46, 69- GM09329. 114. 32. Reiser, A. & Wagner, H. M. (1971) in The Chemistry of the 1. Yip, C. C., Yeung, C. W. T. & Moule, M. L. (1978) J. Biol. Azido Group, ed. Patai, S. (Wiley, New York), pp. 441-501. Chem. 253, 1743-1745. 33. Knowles, J. R. (1972) Acc. Chem. Res. 5, 155-160. 2. Das, M., Miyakawa, T., Fox, C. F., Pruss, R. M., Aharonov, 34. Iddon, B., Meth-Cohn, O., Scriven, E. F. V., Suschitzky, H. A. & Herschman, H. R. (1977) Proc. Nati. Acad. Sci. USA 74, & Gallagher, P. T. (1979) Angew. Chem. lnt. Ed. Engl. 18, 2790-2794. 900-917. 3. Ji, I. & Ji, T. H. (1981) Proc. Natl. Acad. Sci. USA 78, 5465- 35. Nielsen, P. E. & Buchardt, 0. (1982) Photochem. Photobiol. 5469. 35, 317-323. 4. Schmitt, M., Painter, R. G., Jesaitis, A. J., Preissner, K., 36. McGlynn, S. P., Azumi, T. & Kinoshita, M. (1969) Molecular Sklar, L. A. & Cochrane, C. G. (1983) J. Biol. Chem. 258, Spectroscopy of the Triplet State (Prentice-Hall, Englewood 649-654. Cliffs, NJ), pp. 261-283. Downloaded by guest on September 28, 2021