New fluorogenic, photoactivable, heterobifunctional crosslinking thiol reagents

ANALYTICAL

BIOCHEMISTRY

140,63-68

(I 984)

New Fluorogenic, Photoactivable, Heterobifunctional Crosslinking Thiol Reagents’ TAKASHI

UENO,*** SETSUKO HIKITA,* DAISAKU Mmvo,* EISUKE SATO,~ YUICHI KANAOKA,~ AND TAKAMITSU SEKINE*,~

*DepaTtment of Biochemistry, School of Medicine, Junteno University, Bunkyo-ku, Tokyo 113, and tFaculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, Japan Received October 3, 1983 Properties of newly synthesized crosslinking reagents (ACM) and their applications to proteins are studied (ACM is the abbreviation for a series of photoactivable and heterobiinctional crosslinking thiol reagents, each of which has two reactive groups, maleimide and aside). These reagents bind specifically to the sulthydryl residues of proteins in the ftrst reaction step. Upon photoactivanon, the tide group of the coumarin ring reacts with side or main chains of the proteins, and thus intra- or intermolecular crosslinking can be elicited. In addition, the coumarin moiety of the reagents becomes highly tluorescent after photolysis. Therefore, the crosslinking products can be detected by tluorometry with high sensitivity in the pattern of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Reaction of ACM with rabbit muscle aldolase led to extensive crosslinking between subunits of the enzyme and maximally 25% of the total subunits were found to be crosslinked to the dimer.

Recently, Kanaoka et al. have designed and synthesized a series of heterobifunctional and photoactivable crosslinking reagents, ACM4 (4). ACM is an abbreviation for the azidocoumarin derivatives, each of which has a maleimide in the end opposite the azide (Fig. 1). We now have three ACM reagents with different spacer moieties (ACM-2, ACM-$ and ACM-SM). The maximally extended chain length between two reactive groups of ACMs was about 30 A. The reagents react readily and specifically with the sulthydryl residues of a protein under mild conditions. Upon photoactivation, the coumarin azide is converted to a highly reactive nitrene which

Chemical crosslinking is one of the most generalized tools in protein biochemistry. Its applicability for studying molecular architecture of proteins or topology of protein assemblies has been demonstrated in various systems (l-3). Among many crosslinkers, the heterobifunctional and photoactivable ones seem to be the most useful. These bind covalently to a specific amino acid residue in the first reaction step and, upon irradiation, react in a nonspecific manner with other amino acid residues. The results attained with these reagents could provide valuable information on the spatial relationship between a specific amino acid residue of a protein and amino acid residues of the same or another protein proximate to this residue.

4 Abbreviations used: ACM, axidocoumarin derivatives; ACM-2, N-[2-(7’-azidocoumarir&‘-carbonamide)ethoxycarboxymethyl]maleimide; ACM-Z, N-]2-(7’-azidocoumarin - 4’ - carbonamide)pentoxycarboxymethyl] maleimide; ACM-SM, N-[2-(7’-azidocoumarin-4’-acetomide)pentoxycarboxymethyl]maleimide; Me#O, dimethylsulfoxide; Pipes, 1,4-piperazinediethanesulfonic acid, SDS, sodium dodecyl sulfate; DTNB, 5,5’dithiobis(2nitrobenzoic acid).

’ This study was supported by Research Grants of Ministry of Education, Science, and Culture of Japan (Grants 457083 and 5657 108). ’ Present address: Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md. 20205. 3 To whom correspondence should be addressed. 63

0003-2697/84

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Copyri&t 0 1984 by Academic Press, Inc. All rights of reproduction in any form rwerved.

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UENO ET AL.

FIG. 1. Structures of the fluorogenic, thiol-specific, heterobifunctional crosslinking reagents, ACM. (A) ACM-2, (B) ACMS, (C) ACM-SM.

can react with many groups of proteins to form intra- and intermolecular crosslinking. Furthermore, coumarin moieties of ACM become highly fluorescent after photolysis, and thus crosslinked products can be clearly and easily detected in a variety of analytical techniques. We have utilized these crosslinkers to study the structural relationship between heavy chain and light chain of skeletal myosin (5) or between subunits of hemoglobin (6). In this paper, we describe some fundamental properties of ACM, especially in relation to their reactivity, and fluorescence properties, and report an application to aldolase. MATERIALS

AND METHODS

Chemicals. Three ACM compounds, N-[2(7’ - azidocoumarin - 4’ - carbonamide)ethoxy carboxymethyl]maleimide (ACM-2, Cr8Hr3NsO,), N-12-(7’~azidocoumarin4’-carbonamide)pentoxycarboxymethyl]maleimide (ACM5, C2,Hi9NS07), and N-[Z(7’~azidocoumarin - 4’ - acetoamide)pentoxycarboxymethyl] maleimide (ACM-SM, CZ2H2,NS07), were synthesized as described elsewhere (4). Solid ACMs are relatively stable and can be stored for several months, even at room temperature,

if they are kept protected from the light. These reagents were dissolved in dimethylsulfoxide (Me$O) and stored at -20 or -50°C in the dark. Stock solutions were used within 4 weeks of their preparation. Rabbit muscle aldolase (Type IV) was obtained from Sigma Chemical Company. 1,4-Piperazinediethanesulfonic acid (Pipes), N-acetylcysteine, quinine sulfate, and Me*SO (spectroscopic grade) were purchased from Wako Pure Chemical Industries, Ltd. Quinine sulfate was recrystallized twice from ethanol-water mixtures.Silica gel plates for thin-layer chromatography (silica gel 60) were purchased from E. Merck, A. G. All other reagents used were of analytical grade. Reaction of ACM with N-acetylcysteine. Each of the ACMs was reacted with 50-fold molar excess of N-acetylcysteine in the dark. The reaction medium (0.5 ml) contained 10 mM Pipes-Na (pH 6.8) 20 pM ACM, and 1 mM N-acetylcysteine. The medium was kept protected from the light and at O’C for 16 h. Reaction of ACM with aldolase. A crystalline suspension of rabbit muscle aldolase in 2.5 M (NH&SO4 was dialyzed against 1 liter of 10 XnM Pipes-Na (pH 6.8) for 8 h with two changes of the buffer. The concentration of the enzyme was determined by absorbance at 280 nm (7). Unless otherwise stated, the aldolase ( 1.5 mg) was added to the medium in a total volume of 0.5 ml containing 10 mM Pipes-Na (pH 6.8) and ACM in various concentrations. For a control, aldolase was added to the same medium containing MeZSO but not ACM. In all experiments, the final concentration of Me$O was retained below 10%. The reaction mixture was incubated at 0°C for 16 h in the dark. After the incubation, 50fold molar excess of N-acetylcysteine was added to the medium in order to stop the reaction. Then the medium was subjected to photoactivation.

Photolysis and crosslinking experiments. Photolysis of ACM adducts with N-acetylcysteine and ACM-modified aldolase was carried out with continuous irradiation from a mercury lamp (Ushio De&i Co., 100 W). Usually, a 0.5-ml sample cooled on an ice bath was

NEW FLUOROGENIC

HETEROBIFUNCTIONAL

CROSSLINKERS

65

placed 10 cm directly under the light source RESULTS and exposed to the light filtered through a Fluorogenicity ofACM andfluorescencesof Iilter (9300 nm) for 10 min. Crosslinking the ACM- adducts with N-acetylcysteine. products of aldolase were analyzed by SDSFluorogenicity is the most characteristic proppolyacrylamide gel electrophoresis (8). The irerty of ACM. Addition products of ACM with radiated and control samples were solubilized various sulfhydryl compounds or the reagents in a solution containing 0.1 M Na-phosphate alone fluoresce intensely upon photoactiva(pH 7.0) and 2% SDS. These samples were tion. This means that photolysis of the azide then incubated on a boiling water bath for 1 group of ACM is the key reaction not only to min and electrophoresed in 0.1% SDS and nitrene formation but also to fluorescence de7.5% polyacrylamide slab gels. velopment. Titration of suljhydryl groups. For deterWhen ACM or the adducts of ACM with mining the number of sulfhydryl groups of N-acetylcysteine were irradiated with a merACM-modified aldolase, the samples (0.5 mg cury lamp as described under Materials and protein) were incubated in medium (3 ml) Methods, they fluoresced instantly. The flucontaining 4 M guanidine-HCl, 1 mM EDTA, orescence development was very rapid and 67 PM DTNB, and 33 mM phosphate (pH reached nearly the maximal level within 5 min. 8.0) and the release of 3-carboxylato-4~nitroFigure 2 and Table 1 summarize the corrected thiophenolate anion (4i2 om = 13,600 M-’ tluorescence spectra, excitation and emission cm-‘) was measured at 412 nm (9). ACMmaxima, and some other optical parameters treated aldolase solutions which contained of photoactivated adducts. The excitation added, excess iV-acetylcysteine were extenspectra of the adducts of the three ACMs are sively dialyzed against 10 mM Pipes-Na (pH very similar to each other with excitation 6.8) containing 1 mM EDTA before assay. maxima around 360 nm. The emission spectra Optical measurements.Absorption spectra of the adducts of ACM-2 and ACM-5 are alof ACM adducts were measured with a Hitachi most identical but distinguishable in their Type 200-20 spectrophotometer. Fluorescence emission maxima (ACM-2, 550 nm; ACM-5, was measured with a Hitachi Type 850 fluorescence spectrophotometer. The fluores- 546 nm). On the other hand, the adduct of has its emission maximum farcence quantum yield was determined based ACM-5M shifted to the blue as compared with those of on the comparative method described by ParACM-2 and ACM-5 (Fig. 2). In addition, the ker and Rees (lo), with quinine sulfate as a reference, using a value of 0.55 as an absolute quantum yield (11). The concentration of standard quinine was determined by absorbance at 345 nm (12). Thin-layer chromatography. Thin-layer chromatography of ACM was carried out in the dark at room temperature. Stock ACM solutions were diluted 50 fold with ethanol, and aliquots of 1 to 2 ~1were charged on silica gel plates. The samples were developed by either of the following two solvent systems: WAVELENGTH (nm) CHC13-ethanol-water (65:25:4) or ethyl acFIG. 2. Correct4 fluorescence spectra of the adducts etate-benzene (3: 1). The spots corresponding of ACM with N-acetylcysteine. The corrected excitation to ACM could be easily detected as intensely and emission spectra of each adduct in 10 mM pipes-Na fluorescent spots when exposed to ultraviolet (pH 6.8) were measured and normalized for comparison. . * . , ACM-2; - *-, ACM-5; -, light. ACM-SM.

66

UENO ET AL. TABLE OPTICAL

PARAMETERS

OF ACM

1

ADDUCTS

WITH N-ACETYLCYSTEINE

Crosslinking reagent Parameter

ACM-2

ACM-5

ACM-5M

c at 365 nm (M-’ cm-‘) Excitation maximum (nm) Emission maximum (nm) Quantum yield

10,410 366 550 0.026

10,750 357 546 0.016

13,390 350 (348) 460 (450) 0.52

Note. Absorbance and fluorescence of the photolyzed ACM adducts dissolved in 10 mM Pipes-Na (pH 6.8) were measured at 25°C. Corrected emission spectra of adducts (l-2 PM) and of quinine sulfate in 1 N H2S04 (1 PM) excited at 365 nm were measured for determining tluorescence quantum yield. Values in parentheses were obtained with free.ACM-SM.

adduct of ACM-5M is characterized by its exceedingly high quantum yield. The value of 0.52 was almost comparable to that of quinine in HzS04 (11) and much larger than those of other ACM adducts (Table 1). Reaction of aldolase with ACM. When protein-bound ACM molecules are photolyzed intra- and/or intermolecular crosslinking can be elicited. Crosslinking was studied precisely with rabbit-muscle aldolase, a tetrameric enzyme comprising four 40,000-Da subunits. Figure 3 shows a typical result of experiments in which aldolase was modified with ACM-SM in ratios between 0 and 4 mol/mol

ACM -5M

ADDED

(mol/ml

subunit)

PIG. 3. Reaction of aldolase with ACM-SM. Rabbit muscle aldolase was reacted with various concentrations of ACM-SM, as described in the text. After reaction, photolyxed samples were dialyzed extensively against 1 liter of 10 mM Pipes-Na (pH 6.8) containing 1 mM EDTA for 24 h with three changes of the dialyzing medium. ACM-5M bound to the dialyzed enzyme was determined by absorbance at 365 nm. 0, Sulthydryl residues; 0, bound ACM-SM.

subunit. The amount of bound ACM-SM increased with an increase in the amount of added ACM-SM, while the remaining sulfhydry1 residues decreased in mirror image. As can be seen in Fig. 3, maximally two sullhydryl residues of 7 mol/mol subunit (13,14) were modified by ACM-5M under these conditions. Crosslinked products of ACM-modified aldolase were investigated with SDS-polyacrylamide gel electrophoresis (Fig. 4). The main band corresponding to dimer as well as minor bands presumably corresponding to trimer and tetramer could be clearly observed in the pattern of protein staining and in the fluorograph. No crosslinked products were found with the control samples labeled by ACM but not photoactivated. There seemed to be no significant difference among the three types of ACM, though the oligomer bands appeared to be more evident in the samples labeled with ACM-2 or ACM-5 than in the one labeled with ACM-SM. The yield of crosslinked dimer increased with increase in the amount of ACM-5M added and reached as much as 25% of the total subunits (Fig. 5). Stability and storage of ACM. Information on stability and conditions for storage of stock solutions of ACM is necessary for applying the reagents to proteins. As described in the previous section, photolysis of the azide group of ACM results in fluorescence development irrespective of the reaction of the maleimide. Therefore, the basic fluorescence of ACM solutions kept in the dark could be an indication

NEW FLUOROGENIC

-

a

+-+-+-+

b

C

HETEROBIFUNCTIONAL

d

FIG. 4. Crosslinking of aldolase with ACM. Aldolase was modified with a fourfold molar excessof each ACM. The samples with (+) or without (-) photoactivation were dissolved in 0.1 M Na-phosphate (pH 7.0) containing 2% SDS and were electrophoresed in a 0.1% SDS-7.5% polyacrylamide slab gel. After electrophoresis, the fluorograph of the gel was taken under uv illumination and then the gel was stained with 0.25% Coomassie brilliant blue in 25% ethanol-7% acetic acid and destained with 25% ethanol-7% acetic acid. (A) Fluorograph, (B) Coomassie blue staining pattern. Aldolase modified with ACM-2 (a), ACM-5 (b), ACM-5M (c), and control (d).

of spontaneous degradation of the azide groups of ACM. Aliquots of stock solutions were diluted with ethanol and the fluorescence intensities excited at 365 nm were compared with or without photoactivation. The fluorescence intensity of a fresh solution of ACM5M before photoactivation was found to be less than 1.5% of the maximal intensity after photoactivation. Even after storage for 2 months at -20°C the basic fluorescence was only 3% of the maximal value after photolysis.

CROSSLINKERS

67

Similarly, fluorescence intensities of the fresh solutions of ACM-2 and ACM-5 were less than 2% of those fully activated, but they became as much as 15% after storage for more than 2 weeks. Thus, concerning the stability of azide groups of the reagents, ACM-5M seems more stable than ACM-2 or ACM-5. Thin-layer chromatography of ACM provided additional information on the stability of ACM. The Rf values of ACM-2, ACM-5, and ACM-5M were found to be 0.86, 0.89, and 0.84, respectively, in CHCls-methanolwater (65:25:4), and 0.71, 0.80, and 0.56 in ethyl acetate-benzene (3: 1). A few minor spots were often found after prolonged storage (more than 1 month at -20°C). Using a standard substance we confirmed that none of these spots is attributable to the cleavage products at ester bonds. However, it is assumed that at least one or two of them are degraded products of ACM, since these components increase with the time of storage. Also, the temperature affected production of these spots. Storage is more effective at -50°C than at -20°C in preventing the production of these components. Therefore, it is recommended that the stock solutions of ACM should be stored at -50°C in the dark and be used within 4 weeks of their preparation. DISCUSSION

The results obtained from the experiments described in this paper showed that ACMs are

3 mi

FIG. 5. Dependence of crosslinking between aldolase subunits on the concentration of ACM-SM. The aldolase samples crosslinked with ACM-5M were electrophoresed, as described in the legend to Fig. 4. 0, Monomer; 0, dimer.

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UENO ET AL.

highly applicable as thioldirected heterobifunctional crosslinkers to the investigation of protein-protein interactions. Rabbit muscle aldolase, used in this study as a model protein, is a tetrametric enzyme. Its high content of reactive sulthydryl residues (3 mol/mol subunit) enables us to apply ACM to crosslinking study. Furthermore, since much information on crosslinking between aldolase subunits has been reported ( 15- 17), comparison of our data with other studies is possible. Photolysis of ACM-modified aldolase results in extensive crosslinking between the subunits. That this crosslinking might originate from any of the reactive sullhydryl residues of the enzyme was supported by the data for the titration of sulthydryl groups with DTNR (Fig. 3). The main product was dimer and the amount of crosslinked dimer reached as much as 25% of the total of the subunits. In addition, slight but significant amounts of trimer or tetramer were found. These results are quite similar to those obtained after crosslinking with a bifunctional iodoacetamide analog (16) and are also consistent with those of other studies in which various imidoesters were applied ( 15,17). We have utilized ACM for investigating the interaction between light chain and heavy chain of skeletal myosin (5) and between the subunits of hemoglobin (6). Moreover, significant crosslinking was found between the subunits of yeast hexokinase and between Ca’+-ATPase molecules in the sarcoplasmic reticulum.5 One of the most unique and useful prop erties of ACMs is their fluorogenicity. In the mechanism the strong fluorescence of the coumarin ring itself is greatly reduced by attachment of the azide group and regained as the result of the opening of the azide ring by photoactivation. ACM-5M was originally designed from the fact that 7-aminocoumarin4-acetic acid fluoresces much more strongly than its carboxylic derivatives based on the 5 T. Ueno and T. Sekine, unpublished observation.

interaction between the aromatic ring and the carboxyl group. Therefore, ACM-5M is the most interesting because of its exceedingly high efficiency of fluorescence. Its high quantum yield of fluorescence, together with its large molecular extinction coefficient, indicates that the detection limit of ACM-5M is much lower than that of ACM-2 or ACM-5, and that ACM-5M would thus be the most sensitive probe as a tracer for detecting intra- or intermolecular crosslinkage of a protein. Detection of small amounts of crosslinked products in the SDS-polyacrylamide gel electrophoretogram by fluorometry is now under investigation. REFERENCES 1. Weld, F. (1972) in Methods in Enzymology (Gimbexg V., ed.), Vol. 28, pp. 623-651, Academic Press, New York. 2. Peters, K., and Richards, F. M. (1977) Annu. Rev. Biochem. 46, 523-55 1. 3. Ji, T. H. (1979) Biochim. Biophys. Acta 559, 39-69. 4. Kanaoka, Y., Kobayashi, A., !&to, E., Nakayama, H., Muno, D., and Sekine, T. (1983) Chem. Pharm. Bull., in press. 5. Sekine, T., Muno, D., Hikita, S., and Kanaoka, Y. (1982) Int. Congr. Biochem. Abstr., 339. 6. Muno, D., Chiba, Y., Tanaka, T., Sate, E., Kanaoka, Y., and Sekine, T. (1982) Japan. B&hem. Sot. Abstr., 809. [in Japanese] 7. Donovan, J. W. (1964) Biochemistry 3,67-74. 8. Weber, K., and Osbom, M. ( 1969) J. Biol. Chem.

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