Bhxonjtgate Chem. 1994, 5, 205-212
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Pitfalls in Characterization of Protein Interactions Using Radioiodinated Crosslinking Reagents. Preparation and Testing of a Novel Photochemical 1251-Label Transfer Reagent' Troels Koch,? Elisabeth Suenson,' Birgitte Korsholm,' Ulla Henriksen,*'+and Ole Buchardtt Research Center for Medical Biotechnology, Department of Organic Chemistry, The H. C. 0rsted Institute, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark, and Department of Clinical Biochemistry, Section for Hemostasis and Thrombosis, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. Received December 7, 1993' ~
~~
Much attention has been focused on the study of protein interactions with radioiodinated photocrosslinking reagents, and pitfalls in using this methodology are discussed. A new photochemical and cleavable heterobifunctional crosslinking reagent, succinimidyl N-l4-(2-hydroxybenzoyl)-N-l1-(4azidobenzoyl)-9-oxo-8,11,14-triaza-4,5-dithiatetradecanoate (SHAD) was prepared, and its potential as alabel transfer reagent was tested in model systems. SHAD was radioiodinated, and the labeled reagent (1251-SHAD)was converted to an amide (1251-HADM,as a mimicry of conjugation to protein 1) and photolyzed. When compared to the widely used SASD reagent (sulfosuccinimidyl2-[[(4-azidosalicy1)aminolethyll-l,3-dithiopropionate,Pierce), SHAD has a number of decisive advantages. The amide of 1251-SASD(1251-ASDM)was generated and photolyzed, and it was found that at least 50% of the radioactivity is released from lZ5I-ASDMafter 3 min of irradiation, whereas only approximately 10% is liberated from 1251-HADMunder similar conditions. Furthermore, lZ5I-HADMwas photolyzed in the presence of excess amine (mimicry of crosslinking to protein 2), and the product was cleaved by reduction (mimicry of label transfer). The transformations in the course of photolysis were monitored by UV spectroscopy and TLC analysis, and a high degree of reagent cleavage upon reduction was demonstrated. 1251-SHADwas used to crosslink Lys78-plasminogen and fibrin. 1251-SHADwas conjugated to Lys78plasminogen in the dark. Fibrinogen and thrombin were added, and Lys78-plasminogenwas crosslinked to the fibrin clot by exposure to light. Irradiation for 5 min caused very little labeling of fibrin not crosslinked to Lys78-plasminogen;the total recovery of radioactivity was high, and the efficiency of the crosslinking was 30%. Under reducing conditions, it was found that all radioactivity was depleted from the Ly~78-plasminogen-~~~I-HAD conjugates in the dark, and label transfer showed that the labeling of the a-chain was significantly higher than that of the 0-and y-chains. Analogous experiments with 1251-SASDrevealed that this reagent is much less suitable for photocrosslinking and label transfer studies than 1251-SHAD. The reactions were followed by polyacrylamide gel electrophoresis.
INTRODUCTION Chemicalcrosslinking (irreversibleformation of covalent bonds) has become increasingly popular in identifying the components of protein-protein interactions. The use of photoactivatable probes allows determination of the temporal course of dynamic protein interactions, and crosslinking with heterobifunctional reagents and subsequent cleavage of the linker, resulting in label transfer, is a powerful tool in mapping macromolecular proteinprotein interactions (Schwartz et al., 1982;Ji, 1983;Denny and Blobel, 1984; Wollenweber and Morrison, 1985; Sarensen et al., 1986). Label transfer from the primary protein to the secondary protein domain@) includes at least four successive steps: (1)introduction of the label into the reagent, (2) conjugation of the labeled reagent to the primary protein or ligand, (3) crosslinkingto secondary, affinity protein@),and (4)cleavage of the linker to provide label transfer. In order to obtain meaningful results, it is of utmost importance that the affinity proteinb) or
* Corresponding author.
t Research Center for Medical Biotechnology, University of Copenhagen. * Department of Clinical Biochemistry, Rigshospitalet. a Abstract published in Advance ACS Abstracts, April 15,
1994.
This paper is dedicated to the memory of the late Dr. Elisabeth Suenson.
ligand(@are labeled by transfer from the reagent and not by unspecific labeling. The most widely used label is l%I, which is readily introduced in the reagent; the labeled ligands can conveniently be followed by autoradiography, and 1251 is easy to quantitate. The most widely used photochemical heterobifunctional, cleavable crosslinking reagents are l%I-labeled in an azidophenyl group. These reagents have recently been shown to have multiple drawbacks. The photocrosslinking efficiency of the aryl azide is low, and the radioiodo label of aromatic azido compounds is released during photolysis and incorporated into surrounding macromolecules in a diffusion-controlled, nonspecific manner. That is, macromolecules or macromolecular domains other than those involved in ligand-affinity interactions become labeled (Watt et al., 1989). Furthermore, nonspecific labeling of the ligands has also been observed prior to photolysis. This "dark-labeling" is mainly due to the widely adopted iodination procedure with Iodo-Beads and can be avoided by purification of the radiolabeled reagent prior to use. The photolability of the radioiodo label can be avoided (or significantly reduced) by separating the label site and the photoprobe (Henriksen and Buchardt, 1990). SHAD2 (Scheme 1)was prepared, and its properties were examined and compared to those of the commerciallyavailable SASD reagent (Scheme 1) which has been used extensively in biological label transfer studies (Schwartz et al., 1982; 0 1994 American Chemical Society
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Iodo-Beads (N-chlorobenzenesulfamideimmobilized on polystyrene) were purchased from Pierce Chemicals Co., Rockford, IL. Empore silica sheets were from 3M, St. Paul, MN. Silicagel, urea, SDS, and DTE were purchased from Merck, Darmstadt, Germany. Na1261in sodium hydroxide pH 7-11 (84 pM, 100 mCi/mL, carrier free) and 14C-methylated protein molecular weight markers were from Amersham, Buckinghamshire, England. Sephadex G 25F was from Pharmacia, Uppsala, Sweden. All standard chemicals were analytical grade and were used without further purification, except 3-aminopropanol which was distilled prior to use. TLC-solvents (v/v): solvent A, ethyl acetate/methanol 85/15; solvent B, chloroform/methanol/acetic acid 85/10/5. Phosphate buffer I: 6.93 mM, pH 8.00. Phosphate buffer 11: 52.8 mM, pH 8.25. Phosphate buffer 111: 0.5 M, pH 7.0. Proteins. Preparations of human fibrinogen, thrombin, and Lys-Pg were those described previously. The concentrations of Lys-Pg and fibrinogen were determined spectrophotometrically a t 280 nm (Suenson et al., 1984; Suensen and Thorsen, 1988). Aprotinin (Trasylol) was from Bayer, Leverkusen, Germany. All manipulations with label transfer reagents were carried out in the dark, or with Kodak safe light filter No. 6B. Photolysis was done with one UV tube (Amm 310 nm) "TL" 20W/12 (Phillips, Eindhoven, the Netherlands) at a distance of 10 cm with Pyrex filter. UV spectra were recorded on a Hewlett-Packard diode array spectrophotometer 8452 A. lH NMR spectra were recorded on a JEOL FX 9OQ spectrometer. Radiolabeled samples were counted in an LKB 1282Compugamma. Autoradiography of TLC plates and polyacrylamide gels was done with Agfa Cuprix RP-1 films in Agfa Cuprix MR 400 cassettes and developed in a Kodak RP X-Omat processor. Succinimidyl salicylate was prepared from salicylic acid (5.0 g, 36.2 mmol), NHS (4.58 g, 39.8 mmol), and DCC (8.95 g, 43.4 mmol) in T H F (80 mL) by a standard procedure (Koch et al., 1990). The product (7.81 g, 91 % ) was recrystallized from methanol: 'H NMR (CDC13) 6 9.49 (8,1H), 7.99 (d, lH), 7.59 (t, 1H), 7.07-6.88 (m, 2 H), 2.90 (s, 4 H). N-642-Hydroxybenzoyl)-N-3-(4-azidobenzoyl)-3,6diazahexanoic Acid. (Aminoethy1)glycine (Heimer et al., 1984) (6.58 g, 56 mmol) was dissolved in water, and EXPERIMENTAL PROCEDURES triethylamine (23.5 mL) was added. After the mixture was cooled in an ice bath, succinimidyl salicylate (13.1 g, Materials and Met hods. SASD [sulfosuccinimidyl 56 mmol) in THF (300 mL) was added during 1 h. 2- [ [ (4-azidosalicyl)amino]ethyl] -1,3-dithiopropionatel and 4-Azidobenzoyl chloride (10.2 g, 56 mmol) in THF (100 mL) was added dropwise, and the mixture was then Abbreviations used: DTE, dithioerythritol (erythro-1,4evaporated to half volume. The pH was adjusted to 10, dimercapto-2,3-butanediol);SASD, sulfosuccinimidyl 2-[ [ (4the mixture was filtered, and the byproducts were extracted azidosalicyl)amino]ethyl]-l,3-dithiopropionate;lZI-SASD, iodinated sulfosuccinimidyl2-[[(4-azidosalicyl)aminolethyl]-1,3with EtOAc. After acidification, the aqueous layer was dithiopropionate; 126I-ASDM, iodinated 2-[[(4-azidosalicyl)extracted with three portions of EtOAc, and the combined amino]ethyl]-1,3-dithiopropanamide;SHAD,succinimidylN-14extracts were evaporated after drying. The product (15.2 (2-hydroxybenzoyl)-N-ll-(4-azidobenzoyl)-9-0~0-8,11,14-triaza-g, 71 5% ) was dissolved in EtOAc and precipitated with 4,5-dithiatetradecanoate; 126I-SHAD, iodinated succinimidyl petroleum ether: 1H NMR (DMSO-&) 6 12.2 (broad, 2 N -14-(2-hydroxybenzoyl)-N11-(4-azidobenzoyl)-9-oxo-8,11,14H), 8.81 (broad, 1H), 7.51 (t,1H), 7.42-6.80 (m, 8 H), 4.18 triaza-4,5-dithiatetradecanoate; '%I-HADM, iodinated N-14-(2hydroxybenzoyl)-N-ll-(4-azidobenzoyl)-9-oxo-8,11,14-triaza-4,5- (s, 2 H), 3.46 (m, 4 H); MS (EI) 383 (M+). Succinimidyl N-6-(2-hydroxybenzoyl)-N-3-(4-azidithiatetradecanamide;NHS, N-hydroxysuccinimide;DCC, N,"dicyclohexylcarbodiimide;THF, tetrahydrofuran; EtOAc, ethyl dobenzoyl)-3,6-diazahexanoatewas prepared as deacetate; DMSO-&, hexadeuteriodimethyl sulfoxide; MeOH, scribed for succinimidylsalicylate and used without further methanol; STB, Tris-HCl(O.05 M), NaCl(O.1 M), pH 7.7; SDS, purification. sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoreN-1442-Hydroxybenzoyl)-N-1 1- (I-azidobenzoyl)-gsis; NIHu, National Institutes of Health units; Lys-Pg, LysTe0~0-8,11,14-triaza-4,5-dithiatetradecanoic Acid. Sucplasminogen, plasmin-modified plasminogen, mainly with Ncinimidyl N-6-(2-hydroxybenzoyl)-N-3-(4-azidobenzoyl)terminal lysine (residues 78-790); Glul-Pg, native human 3,6-diazahexanoate (1 equiv) was dissolved in THF; plasminogen;t-PA, tissue-type plasminogen activator; K1+ 2 + 7-amino-4,5-dithiaheptanoic acid (2 equiv) (Schnaar et 3, K4, kringles 1-3 and kringle 4 of plasminogen;t-AMCA, trans(4-aminomethyl)cyclohexanecarboxylicacid. al., 1985)and triethylamine (4 equiv) were added dropwise. Wollenweber and Morrison, 1985; Srarensen et al., 1986). The design of SHAD and SASD is similar and the thermal probe and the cleavable linker are the same, but SASD is 1251-labeledin the photoprobe, whereas SHAD is 1251labeled exclusively in the hydroxyphenyl ligand. In order to obtain photoaffinity crosslinking with this type of reagent, several requirements must be met by the interacting proteins. The thermal probe is conjugated to proteins through lysine residues, and consequently, the primary protein must have an accessible lysine residue close to the binding domain but the residue must not be essential for the interaction studied. The photoprobe reacts with nucleophiles, and consequently, the secondary protein must have accessible nucleophilic side chains close to its binding domain. Furthermore, the time for the photocrosslinking reaction must be less than for dissociation of the protein complex; otherwise only so called "pseudo photoaffinity crosslinking" is observed. Another shortcoming is that the linker is cleaved under reductive conditions that also cleave disulfide bridges in the proteins. Furthermore, the distance between the thermal probe and the photoprobe is crucial in some cases. It has been shown that polyfluorination of the photoprobe (the phenylazide) changes the photochemical behavior of the azide (Soundararajan and Platz, 1990). Polyfluorinated aryl azides react, contrary to their nonfluorinated analogues by C-H bond insertion, and this type of photoprobe is more effective when the binding site of the target protein consists of hydrophobic amino acid residues (Crocker et al., 1990; Drake et al., 1992). The fibrinolytic system (Thorsen, 1992) has been extensively studied. It has been demonstrated that LysPg shows enhanced affinity for fibrin, compared to the native Glu-Pg, and has an important function in the t-PAmediated conversion of plasminogen into active plasmin (Thorsen et al., 1984;Suenson and Thorsen, 1988;Nesheim et al., 1990; Suenson et al., 1990; Fredenburgh and Nesheim, 1992). Fibrin, contrary to fibrinogen, binds both t-PA and plasminogen and stimulates plasminogen activation (Hoylaerts et al., 1982; Suenson et al., 1984; Bosma et al., 1988). The interaction between Lys-Pg and fibrin is consequently of great interest, and we used this system as a model for detailed testing of l25I-SHAD compared to 1251-SASD.
Bioconjupte Chem., Vol. 5, No. 3, 1994 207
Label Transfer: Plasmlnogen/Fibrln Interactions
Scheme 1. Reaction Sequence for the Synthesis of 129-HADM and the Structure of SASD
OH
*)p-Azidobenzoylchloride
n
U
'
0
.' 0
H
~
N
~
O
H
12510dinationof SHAD. To SHAD (3.75 pmol) in The mixture was stirred for 15 min, evaporated, and acetonitrile (600 pL) was added sodium phosphate buffer suspended in water. The pH was adjusted to 10 with I11 (3.75 pL), NaI (100 mM) in acetonitrile (75 pL), and triethylamine, and the byproducta were extracted with Na1261(84 pmol, 30 pL, 100 mCi/mL) in NaOH (pH 8-11). EtOAc. After acidification, the aqueous layer was exChloramine-T (15pmol) in acetonitrile (300pL) was added, tracted with three portions of EtOAc, and the combined and the mixture was placed on a rocking table for 10 min extracts were evaporated after drying and used without at room temperature. The iodinated reagent was purified further purification. by TLC on Empore Sheets (10 X 10cm). A 330-pL portion Succinimidyl N-14-(2-hydroxybenzoyl)-N-l I-( 4azidobenzoyl)-9-oxo-8,11,14-triaza-4,5-dithiatetra- of the reaction mixture was applied on each sheet in elongated bands, and the sheets were eluted with solvent decanoate (SHAD) was prepared from N-1442-h~droxybenzoyl)-N-ll-(4-azidobenzoyl)-9-oxo-8,11,14-triaza- A. The fastest moving band was cut out and extracted twice with EtOAc (10 mL). The extracts were combined, 4,5-dithiatetradecanoic acid (1.05 g, 1.92 mmol), NHS and the solvent was evaporated with a stream of nitrogen; (0.243 g, 2.11 mmol), and DCC (0.475 mg, 2.30 mmol) in 63 76 was obtained as purified iodinated label transfer THF (20 mL) by the standard procedure. The product reagent, and 31% of the total iodide used was incorporated. (0.674 g, 55 % ) was passed through a silica gel column After evaporation the residue was dissolved in acetonitrile (elution with EtOAc:MeOH, 98:2). The product should (1.5 mL). be stored a t -20 OC: lH NMR (CDCld 6 7.55-6.88 (m, 8 12510dinationof SASD. To SASD (0.1pmol) dissolved H), 4.08 (8, 2H), 3.71 (m, 6 H), 3.05 (s,4 H), 2.84 (s,4 H); in acetonitrile (50 pL) was added phosphate buffer I11 (10 MS (FAB+) 644 (M + 1).
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pL), sodium iodide (5 pL, 84 pM containing 30 pCi of Na1251),and 1Iodo-Bead. The mixture was incubated for 2 min at room temperature. Preparation of 1251-HADMand Characterization of Its Transitions during Photolysis. To 1200 pL of the solution of 1251-SHADwas added 3-aminopropanol (200 pmol), and after 2 min the mixture was purified by Empore Sheets as described above (as eluent was used solvent B). The fastest moving band was excised, and 3.9 pmol of purified material was isolated. 1251-HADM(0.15 pmol) in acetonitrile (12 pL) was added to phosphate buffer I (1500 pL) and photolyzed. UV spectra were recorded, and aliquots (50 pL) were taken at time intervals of 0, 10, 20,40,60,120,180,240,600,1200,1800 s and applied on analytic TLC plates (eluent solvent B). The spots were excised and counted, and the total recovered radioactivity was calculated. Simulation of Label Transfer. 1251-SHADwas dissolved in acetonitrile (500 pL, 0.1 mM), DTE (to 100 mM) was added, and the mixture was refluxed for 2 min. To 1251-SHADin acetonitrile (20 pL, 7.63 mM) was added acetonitrile (80 pL), sodium phosphate buffer I(1.4 mL), and 3-aminopropanol (2 pL). After 2 min, the mixture was irradiated for 4 min, DTE (to 100 mM) was added, and the mixture was refluxed for 2 min. The reaction was followed by TLC (solvent B). Conjugation of 1251-SHADand 1251-SASDto LysPg. To 1251-SHAD(76.3 nmol) in acetonitrile (10pL) was added Lys-Pg (3.68 nmol) in sodium phosphate buffer I1 (150 pL). The mixture was incubated for 30 min on a rocking table, purified by gel filtration on Sephadex G 25 F (5 mL), and eluted with STB a t 10 mL/h; only 0.85% of the radioactivity was found in nonconjugated material. When 1251-SASDwas conjugated in the same way, 5.6% of the radioactivity was found in the nonconjugated material. The specific radioactivity was 274 MBqlpmol for the L ~ s - P ~ - ~ ~ ~ I conjugate - H A D and 418/pmol for the L ~ s - p g - l ~ ~ I - Aconjugate. sD CrosslinkingAssay. Aprotinin (5 pL 380 pM, plasmin inhibitor), L ~ s - P ~ - ~ ~ ~ I - H orAL D ~ s-- p g - l ~ ~ I - AconjusD gate (30 p L 1.5 pM), fibrinogen (60 pL 7.5 pM), and thrombin (25 pL 6 NIHu/mL) were mixed in STB (180 pL). The mixture was incubated for 10 min in the dark at room temperature and irradiated for 5 min, and urea (to 6 M) and SDS (to 35 mM) were added to quench the reactions and dissolve the clot. The conjugation and crosslinking were analyzedby SDS (3-5%) PAGE (Weber and Osborn, 1975). The gels were autoradiographed(Weiselet al., 1994),and the bands were subsequently excised and counted. Label Transfer. Conjugation and crosslinking were performed as described above. Aliquots (100 pL) were taken, DTE (to 100 mM) was added, and the mixtures were refluxed for 2 min. The reactions were analyzed by SDS (7.5 % ) PAGE. The gels were autoradiographed, and the bands were subsequently excised and counted. RESULTS AND DISCUSSION
Iodination of SHAD. The iodination was preferentially performed in organic solvents instead of water in order to minimize hydrolysis of the succinimidyl probe (Shephard et al., 1988). In pilot experiments, the degree of reagent iodination was judged from autoradiographs of TLC analyses of the reaction mixtures. Acetonitrile and acetone were compared as solvents, and Iodo-Beads and nonimmobilized chloramine-?' were compared as oxidizing agents. Optimal results were obtained using chloramine-?' in acetonitrile. Nonimmobilized chloramine-?' gave no-
A
B
0.57
0.20
1
Figure 1. Autoradiographyof the TLC analysisinEtOAc/MeOH (85/15) of (A) I25I-SHAD (reaction mixture) after 10 min incubation a t room temperature and (B) purified 1251-SHAD.
1
2
3
4
5
6
7
8
Figure 2. Autoradiography of the TLC analysis in CHCld MeOH/AcOH (85/10/5) of the time course of photolysis of l%IHADM. Lane 1: 1251-HADM(0.15 pmol) in phosphate buffer I (1500 pL). Lanes 2-8 represent photolysis for 10,20,40,60,120, 180, and 240 s, respectively.
tably higher yields and fewer byproducts than Iodo-Beads. When iodide and chloramine-?' were present in equimolar concentrations, iodide was oxidized to iodine, and no reagent iodination was detected. Increasing the chloramine-?' concentration to a 2 M excess accomplished aromatic electrophilic substitution (iodination) by giving rise to a more highly reactive iodine species (iodine chloride). Iodination of SHAD on a preparative scale with nonradiolabeled NaI and chloramine-?' showed that no iodine is incorporated into the azidophenyl ligand and that the hydroxyphenyl ligand is iodinated almost exclusively in the 5-position. 1251-SHADwas purified after the iodination procedure to remove hydrolyzed reagent (the compound with Rf = 0.2 in Figure 1A) and excess of iodination reagent that otherwise will be directly incorporated into tyrosine residues in a subsequent protein modification step. Autoradiography of the TLC analysis of 1251-SHAD is shown in Figure 1. Upon solvent evaporation and storage a t -20 "C, the radiolabeled reagent is stable for at least 3 weeks. Reagent Photolysis and Resultant Deiodination. The photostability of the radioiodine substituent of 1251SHAD was investigated and compared to that of lEI-SASD. In order to simulate label transfer conditions, 1251-SHAD was reacted with excess 3-aminopropanol prior to photolysis in the aqueous system. The time course of
Label Transfer: Plasminogen/Fibrin Interactions
Bioconjugate Chem., Vol. 5, No. 3, 1994 209
A
A' b
,7?*"
3
4
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E
a
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0
~
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0
I
.
I
200
.
,
300
. 400
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.
500
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.
600
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4
5
6
7 0
7 0
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Figure 3. Recovered radioactivity: (0) total radioactivity recovered for 1261-HADMand its photolysis products. The spots displayed in Figure 2 were excised and counted. (A) Total radioactivityrecovered for '=I-ASDM and its photolysisproducts from a similar experiment (the autoradiography is not shown). The results are representative of three trials.
?
1
2
3
4
5
Figure 4. Autoradiography of the TLC analysis of simulated label transfer with '261-SHAD. Lane 1: nonphotolyzed1251-SHAD in acetonitrile (0.1 mM, 500pL). Lane 2: nonphotolyzedreaction mixture containing 1251-SHAD(7.63 mM, 20 pL), acetonitrile (80 pL), phosphate buffer I (1.4 mL), and 3-aminopropanol(2 pL), incubated for 2 min; this mixture is used in the following experiments. Lane 3: photolysis of the reaction mixture for 4 min. Lane 4 nonphotolyzed reaction mixture after reduction with DTE (100 mM). Lane 5: reduction of the photolyzed reaction mixture with DTE (100 mM).
photolysis was examined by UV spectroscopy(not shown) as well as by TLC analysis (Figure 2). UV spectroscopy showed typical azide photodegradation,yielding isosbestic points. These were maintained after 3-4 min of photolysis and thereafter slowly faded out. Photolysis was almost complete after 4 min, judging from both UV spectroscopy and TLC analysis. On the basis of the autoradiograms, the 1251content in the organic species was determined (Figure 3). The radioactivity of the products and the unchanged amide during photolysis was determined and related to the activity of unphotolyzed amide. Eighty-five to 90% of the total reagent radioactivity is recovered in the products after 3-5 min of irradiation. Figure 3 shows that the deiodination is more pronounced in the initial minute of photolysis and decreases with product formation. The products may act as internal filters or quench the excited state that leads to deiodination, thus interfering with the
B
B'
M W 1 2 3 4 5 6 7 8 7 8 Figure 5. PanelA autoradiographyofSDS ( 3 4 % )PAGE under nonreducing conditions of Lys-Pg conjugated to 12SI-SASD(lanes with uneven numbers) or to 1251-SHAD(laneswith even numbers) and crosslinking of the Lys-Pg conjugates with fibrin. M,: reduced mixture of 14C-labeledprotein molecular weight standards (M, = 200,92.5,69, and 46 kDa). Lane 1: Lys-Pg-129-ASD conjugates, formed from 1251-SASD(73.9 nmol) and Lys-Pg (3.68 nmol) in phosphate buffer I1 (150 pL) after 30 min incubation in the dark and purified by gel filtration. Lane 2: L ~ s - p g - l ~ ~ I HAD conjugates, formed as described under lane 1. Lane 3: L ~ s - P ~ - ' ~ ~ I - Aconjugates SD irradiated for 5 min. Lane 4: LysPg-1251-HAD conjugates irradiated for 5 min. Lane 5: Lys-Pg1251-ASDconjugates (0.15 pM), aprotinin (6.3 pM), fibrinogen (1.5 pM), and thrombin (0.5 MIHu/mL) in STB (300 pL) incubated for 10 min in the dark. Lane 6: L ~ s - P ~ - ' ~ ~ I - H A D conjugates, aprotinin, fibrinogen, and thrombin in STB as described under lane 5. Lane 7: the mixture described under lane 5 irradiated for 5 min. Lane 8: the mixture described under lane 6 irradiated for 5 min. nF-L Lys-Pg-1251-reagent-n fibrin complexes (n> 3). 3F-L Lys-'261-reagent-3 fibrin complexes. 2F-L: Lys-Pg-1251-reagent-2 fibrin complexes. F-L: Lys-Pg1251-reagent-fibrincomplexes. nL/F: n Lys-Pg complexes or/ and fibrin (n > 3). 3L: Lys-Pg-'261-reagent trimer. 2L: LysPg-1251-reagentdimer. L: L~s-Pg-l~~I-reagent conjugates. Panel A': as panel A but stained with Coomassie Brilliant Blue R250 (only lanes 7 and 8 are shown). Panel B: autoradiography of SDS (7.5%) PAGE under reducing conditions. M,: reduced mixture of 1%-labeled protein molecular weight standards (M, = 200,92.5,69,46,30,21.5, and 14.3 kDa). The lanes correspond to those in panel A, except that all mixtures have been reduced with DTE prior to SDS-PAGE. L Lys-Pg. a-,8-, and y-: the three polypeptide chains from reduced fibrin. Panel B': as panel B but stained with Coomassie Brilliant Blue R250 (only lanes 7 and 8 are shown).
photodeiodination. Furthermore, a small amount of diiodinated reagent is present, and this compound loses iodine much faster than the monoiodinated reagent (Davidson et al., 1984). We performed a similar experiment with 1251-SASDand found that after 2 min only 50 % was retained in the organic species. Further irradiation unexpectedly increased the recovered radioactivity by about 20%. This may be explained by massive initial 1251-release(Watt et al., 1989) and subsequent photo-
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Table 1. Recovered Radioactivity from SDS-PAGE of the Products from Crosslinking Lys-Pgand Fibrin with 'SI-SASD and 129-SATD. nonreducing conditions reducing conditions laneb nF-LC 2F-L nL F-L nL/F 3L 2L L totald frct 1-4e L ai3y-chain frct 12-13f totald 1 2 3 4 5 6 7 8
5 1.5
3
2
1
1
2 1 0.5
4 4 2 0.5 2 4
1 4 0.5 3 3 6 2 3 7 4 1 4.5 0.5 3.5 0.5 1 1.5 3
49 56 69 75 9 5 9 0 6 2 64 74 89 97 16 56 40 94
2 2 1
6.5 0.5 11 7 7 0.5 8.5 8
0.5
1.5 2.5 2 2.5 1.5 3.5 2 4
10
3 17 13
12 5
4 1 1 5 23 2.5 10 6 1.5 1.5 2 18 5 23 a Percentage of the applied radioactivity corrected for gel background; the first part of the table is from SDS (3-5%) PAGE under nonreducing conditions, the second part from SDS (7.5%) PAGE under reducing conditions. The values given are from individual experiments, but the same trend was observed in a series of experiments. * The lanes correspond to those in Figure 5A and B. Corresponds to the complexesgiven in the legend to Figure 5. d Total recovered radioactivity form the lanes. e High molecular weight unidentified proteins. f Low molecular weight unidentified compounds. 15 12
7 10
Scheme 2. Pathways for the Plasminogen and Fibrin Crosslinking Experiments
Lys-Pg-'"I-HAD-Fibrin
DTEi F
iJ Fibrin
Labeling of Fibrin
chemical iodination of organic compounds in the reaction mixture, including photolyzed ASDM species and impurities. Preparative photolysis of iodinated N-(4midobenzoyl)tyrosine gave products with iodine retained (Henriksen and Buchardt, 1990), but a similar experiment with I-HADM revealed that iodine is released under these conditions. Furthermore, iodinated N-salicylyl-N'-(4azidobenzoyl)-1,6-hexanediaminewas photolyzed preparatively, and no iodine was observed in the products, indicating that it is the carbonyl group of the salicylyl ligand that is responsible for the deiodination on prolonged irradiation. Cleavage of 1251-HADMand thePhotolysis Products by Reduction. In order to simulate label transfer the reagent must be cleaved. This is in most cases done by adding mercapto compounds to the label transfer mixture. DTE was added to the native 1251-HADMand to the photolysis products (Figure 4). As seen from the autoradiograms, the disulfides are readily cleaved, yielding compounds with a slightly higher Rf value. Apparently only one product is formed on photolysis in the presence of 3-aminopropanol (Figure 4, lane 3), which corresponds
Lys-Pg-'"I-HAD-Lys-Pg
I
D DTE
cLys-Pg
Labeling of Lys-Pg
to reaction of the initially generated dehydroazepine with the amino group of 3-aminopropanol (Nielsen and Buchardt, 1982;Shields et d.,1988;Henriksen and Buchardt, 1990). Formation of Lys-Pg Conjugates with 1251-SHAD and 1251-SASD:Irradiation and Reduction of the Conjugates. Autoradiography of SDS-PAGE analyses of the reaction profiles is shown in Figure 5, lanes 1-4,and the recovered radioactivity is summarized in Table 1.1251SHAD and 1251-SASDwere conjugated to Lys-Pg (reaction A in Scheme 2) and 72% of the applied radioactivity was found in the bands representing the Lys-Pg-l251-HAD conjugates, versus 54 % for the Lys-Pg-l25I-ASD conjugates. Three to 4 % of the radioactivity was found in bands presumably representing two crosslinked Lys-Pg. This is unexpected since the conjugates were not exposed to light. Disulfide exchange may be responsible for this dimer formation, since it is known that disulfide interchange occurs spontaneously in neutral and alkaline solution and is promoted by thiols and thiourea (Eager and Savige, 1963). After reduction of the L ~ S - P ~ J ~ ~ I -conjugates, ASD 6.5% of the radioactivity was still found in Lys-Pg indicating incomplete reduction or nonspecific labeling.
Label Transfer: Plasminogen/Fibrin Interactions
Bioconjugate Chem., Vol. 5, No. 3, 1994 211
as well as removal of oxidant and free radioiodide from As expected, only 0.5 % was found in Lys-Pg after reduction the reagent after iodination potentially inhibit nonof the Lys-Pg-1251-HAD conjugates (reaction B in Scheme crosslinker related direct incorporation of radioiodine into 2), indicating that the reduction of the disulfide is complete protein tyrosine residues. In theory, both methodological and that no nonspecific labeling of Lys-Pg occurs with this reagent. After irradiation of the L ~ S - P ~ - ~ ~ ~ I - H A D sensitivity and specificity are thus markedly increased for and the L ~ s - P ~ - ' ~ ~ I - Aconjugates, SD 40 and 29%, reSHAD compared to SASD. These potential advantages spectively, of the radioactivity were still found in Lys-Pg, hold in parallel protein crosslinking experiments, indicatmeaning that most of the photoprobes have reacted with ing that the described TLC analyses of novel crosslinking the solvent, but complexes containing two (pathway C in reagents is highly advantageous in predicting their use in Scheme 21, three, or more Lys-Pg were also observed, protein systems and in interpreting the results. indicating a certain affinity between Lys-Pg molecules. Fibrin has binding sites for both plasminogen and t-PA. After reduction, most of the recovered radioactivity was, The latter is bound to fibrin at the distal end of the coiledas expected, found in Lys-Pg (reaction D in Scheme 2). coil regions, connecting the D-domains with the central Photocrosslinking of Fibrin and the L y ~ - P g - l ~ ~ 1 - E-domain (Schielen et al., 19911, and it seems reasonable HAD and Lys-Pg-lzaI-ASDConjugates and Reduction to assume that plasminogen binds to fibrin in the vicinity of the Complexes (Label Transfer). Autoradiography of the activator. Although the yield of label transfer is of SDS-PAGE analyses of the reaction profiles is shown low, we conclude that the labeling of the a-chains of fibrin in Figure 5, lanes 5-8, and the recovered radioactivity is is significantly higher than for the 8- and y-chains, summarized in Table 1. The assignment of the bands was indicating that the plasminogen binding domain is located performed as described by Weisel et al. (1994). The Lyson the a-chain, close to the coiled-coil regions. Pg conjugates were mixed with fibrinogen in the dark, and clot formation was initiated with thrombin. A total ACKNOWLEDGMENT of 93 % of the applied radioactivity was found in the LysPg-125I-HAD conjugates, and no nonspecific labeling of fibrin was observed. After reduction, no significant This work was supported by the Danish Natural Science P-, radioactivity was observed in either Lys-Pg or the CY-, Research Counsel (Grant No. 11-8133to T.K.). We thank and y-chains of fibrin. On the contrary, significant Dr. Michael Egholm for suggestingthe use of (aminoethy1)radioactivity was observed both in Lys-Pg and in the a-, glycine and for donating a sample. P-, and y-chains when the Lys-Pg-1251-ASD conjugates were reduced in the presence of fibrin. LITERATURE CITED Photocrosslinking was accomplished by irradiation of the clot, but approximately 45% of the radioactivity was Bosma, P. J., Rijken, D. C., and Nieuwenhuizen, W. (1988) still found in the L ~ s - P ~ - ' ~ ~ I - Hconjugates AD (40% in Binding of tissue-type plasminogen activator to fibrinogen noncrosslinked Lys-Pg), and only approximately 40% in fragments. Eur. J. Biochem. 172,399-404. the L~s-Pg-l~~I-HAD-fibrin complexes (pathway E in Crocker, P. J., Nobuyuki, I., Rajagopalan, K., Boggess, M. A,, Scheme 2). The high percentage of radioactivity found in Kwiatkowski, S., Dwyer, L. D., Vanaman, T. C., and Watt, D. noncrosslinked Lys-Pg could be expected, since only S. (1990)Heterobifunctional crosslinkingAgents Incorporating Perfluorinated Aryl Azides. Bioconjugate Chem. I,419-424. photoprobes close to the fibrin binding site@)of Lys-Pg Davidson, R. S.,Gooden, J. W., and Kemp, G. (1984)The are effective in the crosslinking process. The K1 + 2 + Photochemistry of Aryl Halides and Related Compounds, in 3, K4, and Vala3-plasminogen domains are all modified Advances in Physical Organic Chemistry 20 (V. Gold, and D. with 125I-SASD (Weisel et al., 1994). Complex formation Bethell, Eds.) pp 191-233,Academic Press, London. between modified Lys-Pg and fibrin is inhibited by Denny, J. B., and Blobel, G. (1984) 1261-labeledcrosslinking t-AMCA, showing that lysine binding sites are involved reagent that is hydrophilic, photoactivable, and cleavable in a photoaffinity crosslinking process (Weiselet al., 1994). through anazo-linkage. Proc. Natl. Acad. Sci. U.S.A. 81,5286Label transfer was then performed by reduction of the 5290. Drake, R. R., Slama, J. T., Wall, K. A., Abramova, M., D'Souza, complexes (reaction F in Scheme 2), and 6 5% of the applied C., Elbein, A. D., Crocker, P. J., and Watt, D. S. (1992) radioactivity was found in the a-chain, 1.6 5% in the @-chain, Application of an N-(4-Azido-2,3,5,6-tetrafluorobenzoyl)tyand 1.7% in the y-chain when 125I-SHAD was used as rosine-Substituted Peptide as a Heterobifunctional Crossphotocrosslinker. A total of 8% of the radioactivity was Linking Agent in a Study of Protein 0-Glycosylation in Yeast. found in Lys-Pg (from cleavage of crosslinked Lys-Pg Bioconjugate Chem. 3,69-73. molecules). Similar results were obtained when 1251-SASD Eager, J. E., and Savige, W. E. (1963)Photolysis and Photowas used as crosslinker. The yield of label transfer to the oxidation of Amino Acids and Peptides-VI. A study of the a-, P-, and y-chains of fibrin is expected to be low since Initiation of Disulfide Interchange by Light Irradiation. most of the radioactivity is found in Lys-Pg complexes Photochem. Photobiol. 2, 25-37. before reduction. However, the observation that the Fredenburgh, J. C., and Nesheim, E. (1992)Lys-plasminogen Is a Significant Intermediate in the Activation of Glu-plasminlabeling of the a-chain of fibrin is four times the labeling ogen during Fibrinolysis in Vitro. J.Biol. Chem. 267,26150of the P- and y-chains is conclusive, since it was demon26156. strated that no nonspecific labeling of fibrin takes place Heimer, E. P., Gallo-Torres, H. E., Felix, A. M., Ahmad, M., when 1251-SHADwas used as photocrosslinker. Lambros, T. J.,Schiedl, F., andMeienhofer, J. (1984)Synthesis CONCLUSION
Substitution of the radioiodine into a separate hydroxybenzoyl group, or better a hydroxyphenyl group, instead of the photoreactive phenyl azide of SASD radically decreases photoinduced liberation of radioiodine. The yield of reduction of the disulfide linker is almost quantitative, a feature that is essential for high methodological sensitivity. Markedly reduced photodeiodination
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