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[84] A homologous series of photoreactive peptidyl-tRNAs for probing the ribosomal peptidyltransferase center
[84]
PHOTOREACTIVE PEPTIDYL-tRNA DERIVATIVES
707
causes irreversible inactivation of peptidyltransferase activity by selectively alkylating cysteine-SH groups in proteins L2 and L27 as identified by amino acid analysis, and gel electrophoretic separation of the ribosomal proteins2 Iodoamphenicol prepared from iodoacetyl-N-hydroxysuccinimide ester and chloramphenicol base was reported to label protein L16. ~ In unpublished experiments it has been shown that irradiation with each of the two photoaffinity labeling reagents resulted in the irreversible inactivation of peptidyltransferase. The presence of erythromycin in the irradiation mixture with p-azidochloramphenicol protected against inactivation. It thus appears that these analogs react with the ribosome at functionally significant sites. The modified ribosomal components in these two cases have not been identified, however. Finally, a photoinduced reaction of unmodified chloramphenicol with the ribosome has been observed." The reaction is expressed in the labeling of several ribosomal proteins and in a pronounced shift in the electrophoretic mobility of protein L197 °
Acknowledgments We are indebted to Mr. D. Haik for ribosome preparalions. This work was supported by a grant from the United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel. O. Pongs, R. Bald, and V. A. Erdmann, Proc. Natl. Acad. Sci. U.S.A. 70, 2229 (1973). 9 N. Sonenberg, A. Zamir, and M. Wilchek, Biochem. Biophys. Res. Commun. 59, 693 (1974). 1oM. Israel, N. Sonenberg, M. Wilchek, and A. Zamir, in preparation.
[84] A Homologous Series of Photoreactive Peptidyl-tRNAs f o r P r o b i n g the Ribosomal Peptidyltransferase Center B y N. SONENBERG, M. WILCHEK, and A. ZAMIR
Aminoacyl- and peptidyl-tRNA are the natural substrates of ribosomal peptidyltransferase. Derivatives of these substrates modified chemically or with photoreactive groups can therefore be used as affinity labeling reagents for the location of ribosomal components at, or close to, the peptidyltransferase active center. Here we describe the synthesis of photoreactable derivatives of Phet R N A of the general structure: (AP) (Gly)~-Phe-tRNA (n = 0, 2, 4). 1 Abbreviations: (a) (AP) = p-azido-N-tBoc-Phe; (b) tBoc = tert-butyloxycarbonyl.
708
NUCLEIC ACIDS AND RIBOSOMAL SYSTEMS
[84]
These peptidyl-tRNA analogs bind reversibly, in the presence of poly (U), to 70 S ribosomes and attach covalently upon irradiation to a specific site(s) on the 23 S RNA of the 50 S subunit.
Experimental Procedures Synthesis of Reagents
p-Azido-N-tBoc-Phe- [3H]Phe-tRNA (AP)Phe-tRNA is synthesized according to Scheme 1 by coupling-" the N-hydroxysuccinimide ester of p-azido-N-tBoc-phenylalanine 3 with N-t8o¢ IOt
N-t8oc "3
c~-c.-coo.
.
c~-~.-
-
2)NON3 N H 2 - - ~ C H 2- CH-COOH
N-tBo¢ NH
H2
C=O I O-tRNA
N-tBo¢ NO2-~ \~P-CH2-/H-COOH~
(AP) Phe-fRNA SCHEME 1
(3H)Phe-tRNA (unfraetionated). In a standard preparation, 3.5 nmoles of (3H)Phe-tRNA are dissolved in 3.0 ml of 0.2 M triethanolamine-HC1 (pH 8.0) or 0.2 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (pH 8.0), and 600 mg of the N-hydroxysuccinimide ester of p-azido-N-tBoc-phenylalanine in 30 ml of freshly distilled dimethyl sulfoxide are added. The mixture is incubated for 2 hr at 30 °, chilled to 0 °, and 3.7 ml of 50% dichloroacetic acid are added. After 2 hr at 0 °, the mixture is centrifuged and the pellet is washed once with dimethylformamide and twice with ethanol. The dried pellet is finally dissolved 2y. Lapidot, D. Eilat, S. Rappoport, and N. deGroot, Biochem. Biophys. Res. Commun. 190, 558 (1970). 3A. Schwyzer and M. Caviezel, Helv. Chim. Acta 54, 1395 (1971).
[84]
PHOTOREACTIVE PEPTIDYL-tRNA DERIVATIVES
709
in water and titrated to pH 6.0 with Tris chloride. The preparation contained, at most, 5% of unreacted Phe-tRNA as determined by paper electrophoresis following alkaline hydrolysis. 4
p-Azido-N-tBoc-Phe- (Gly), [3H]Phe-tRNA (n = 2, 4) 1. p-Nitro-N-tBoc-Phe-(Gly),COOH (n = 2, 4). To a solution of glycylglycine (1.60 g, 12 mmoles) in 50 ml of 0.45 M sodium bicarbonate is added the N-hydroxysuccinimide ester of p-nitro-N-tBoc-Phe (4.06 g, 10 mmoles) in 50 ml of dioxane. After 3 hr at room temperature, the solution is concentrated to remove dioxane and is acidified with 10% citric acid and extracted with ethyl acetate. The ethyl acetate solution is washed with water, dried with sodium sulfate, and concentrated to a small volume. On addition of petroleum ether, the product precipitates. Yield, 4 g; m.p. 95o-96 °. p-Nitro-N-tBoc-Phe-(Gly)4 COOH is synthesized similarly, using the same ratio of reagents as for the (Gly)_~ compound. The product precipitates upon addition of citric acid. Yield, 3 g; m.p., 168 °. 2. (AP) (Gly),-N-hydroxysuccinimide Ester (n = 2, 4). p-Nitro-NtBoc-Phe-(Gly)2 COOH (880 mg, 2 mmoles), dissolved in 100 ml of methanol, is hydrogenated in the presence of 100 mg of 10% paladium on charcoal for 2.5 hr. The product, p-amino-N-tBoc-Phe-(Gly)2COOH, after removal of the catalyst by filtration and evaporation of solvent, is dissolved in 2 ml of 2 N hydrochloric acid and 0.4 ml of water at 2 °, and sodium nitrite (145 mg, 2.1 mmoles) in 1 ml of water is added. After stirring for 1.5 hr at 2 °, the solution is filtered to remove insoluble materim and degassed under reduced pressure to expel excess nitrous acid. To this solution, sodium azide (130 mg, 2.0 mmoles) in 1 ml of water is added slowly. After stirring for 45 min at 0 °, the aromatic azide compound is extracted into ethyl acetate. The solution is evaporated to dryness to give (AP)(GIy)._,COOH. To prepare the N-hydroxysuccinimide ester, the product is dissolved in 5 ml of ethyl acetate, and N-hydroxysuccinimide (172 mg, 1.5 mmoles) is added followed by dicyclohexylcarbodiimide (310 mg, 1.5 mmoles) in 6 ml of ethyl acetate. The mixture is left overnight at 0 °. After removal of dicyclohexylurea, the solution is evaporated to dryness, yielding 350 mg of product. (AP) (Gly)~COOH is prepared similarly to the (Gly)._, compound. The (Gly)4 compound is, however, barely soluble in ethyl acetate. Therefore, at the end of the reaction, sodium hydroxide is added to adjust the pH to about 4.0 and the mixture is lyophilized. The residue is dissolved 4Z. Vogel, A. Zamir, and D. Elson, Proc. Natl. Acad. Sci. U.S.A. 61, 701 (1968).
710
NUCLEIC A.CIDS AND RIBOSOMAL SYSTEMS
[84]
in ethanol and filtered to remove insoluble salts; product is obtained after evaporation of the ethanol. Thin-layer chromatography on silica gel with chloroform:methanol (3:1), or pure methanol, indicates the presence of a single product. In order to prepare the N-hydroxysuccinimide ester, 276 mg (0.5 mmole) of (AP) (Gly)4COOH are dissolved in 12 ml of dimethylformamide, and N-hydroxysuccinimide (56 rag, 0.5 mmole) is added, followed by dicyclohexylcarbodiimide (103 mg, 0.5 mmole) in 1.5 ml of dimethylformamide. The mixture is left overnight at 0 °. After removal of dicyclohexylurea, the dimethylformamide solution is used directly for coupling to Phe-tRNA. 3. (AP) (Gly)~-[3H]Phe-tRNA (n --- 2, 4). These compounds are prepared in a manner similar to that for (AP)[3H]Phe-tRNA as described above. The (Gly)2 derivative is prepared in dimethyl sulfoxide and the (Gly) 4 derivative in dimethylformamide. The preparation of (AP) (Gly)~[3H]Phe-tRNA contains between 15 and 25% of unreacted [3H]PhetRNA, whereas the preparation of (AP) (Gly)~-[3H]Phe-tRNA contains between 20 and 30% of unreacted [3H] Phe-tRNA. Reversible Binding of (AP)(Gly),-Phe-tRNA to 70 S Ribosomes (n = 0, 2, 4) The reaction mixture, in 0.15 M ammonium chloride, 30 mM magnesium acetate, and 50 mM Tris chloride (pH 7.4), contains the following per ml: 120-240 ~g of poly(U), 1.4 mg of 70 S ribosomes, and 640 pmoles of (AP) (Gly),-[~H]Phe-tRNA (n = 0, 2, 4). Reaction mixtures are incubated at 37 ° for 20 rain to allow formation of binding complexes. Covalent Binding of (AP)(GIy),-Phe-tRNA (n = 0, 2, 4) to 70 S Ribosomes Binding complexes are irradiated with a high-pressure mercury lamp (Hanovia, 450 watts) at an average distance of 8 cm for 5 rain in a water bath at room temperature. Light of wavelengths below 250 nm is eliminated by a Corex filter. Covalent binding is determined as described2 Comments The series of compounds described here is suitable for screening the ribosomal components involved in peptidyltransferase activity. All compounds described are peptidyl-tRNA analogs, and the shortest member ~N. Sonenberg, M. Wilchek, and A. Zamir, Proc. Natl. Acad. Sci. U.S.A. 72, 4332 (1975).
[85]
PUROMYCIN AND INITIATION FACTOR
711
in the series, (AP)Phe-tRNA, has been shown to bind preferentially to the ribosomal donor site2 The mode of reversible binding has not been determined for the longer peptidyl tRNAs. The compounds described are of potential value in characterizing the ribosomal environment at increasing distances from the site of interaction of the --CCA terminus of tRNA. With the same idea in mind, peptidyl-tRNAs of varying lengths with a bromoacetyl group blocking the terminal amino group have been previously synthesized and tested as affinity probes2 Information gathered so far with the present series indicates that the irradiation of poly (U)-directed ribosomal complexes of each of the compounds described results in a reaction with 23 S rRNA. Specifically, labeling takes place within the 18 S fragment that includes about 1700 nucleotides extending from the 3' end. The results point to the functional significance of rRNA in the peptidyltransferase center. Sequencing studies of the different modified rRNAs may clarify the mode of arrangement of the rRNA within the functional site. Labeling of 23 S rRNA by chemical and photoreactive analogs of peptidyl-tRNA has been reported from several other laboratories. 7-11 Acknowledgment We are indebted to Mr. I. Jaeobson for synthesizing some of the intermediate compounds. This research was supported by a grant from the United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel. oD. Eilat, M. Pellegrini, H. Oen, Y. Lapidot, and C. R. Cantor, J. Mol. Biol. 88, 831 (1974). 7L. Bispink and H. Matthaei, FEBS Lett. 37, 291 (1973). See also this volume [74]. 8A. S. Girshovich, E. S. Bochkareva, U. M. Kramarov, and Y. A. Ovchinnikov, FEBS Lett. 45, 213 (1974). See also this volume [77] and [78]. o M. Yukioka, T. Hatayama, and S. Morisawa, Biochim. Biophys. Acta 390, 192 (1975). 10A. Barta, E. Kuechler, C. Branlant, J. Sriwidada, A. Krol, and J. P. Ebel, FEBS Lett. 56, 170 (1974). See also this volume [81]. 1, j. B. Breitmeyer and H. F. Noller, J. Mol. Biol. 101, 297 (1976).
[85] Photoaffinity Labeling of Ribosomes with the Unmodified Ligands Puromycin and Initiation Factor 3 By BARRY S. COOPERMAN There are two general approaches to photoaffinity labeling. In the first, a radioactive photolabile ligand derivative is irradiated in the
PHOTOREACTIVE PEPTIDYL-tRNA DERIVATIVES
707
causes irreversible inactivation of peptidyltransferase activity by selectively alkylating cysteine-SH groups in proteins L2 and L27 as identified by amino acid analysis, and gel electrophoretic separation of the ribosomal proteins2 Iodoamphenicol prepared from iodoacetyl-N-hydroxysuccinimide ester and chloramphenicol base was reported to label protein L16. ~ In unpublished experiments it has been shown that irradiation with each of the two photoaffinity labeling reagents resulted in the irreversible inactivation of peptidyltransferase. The presence of erythromycin in the irradiation mixture with p-azidochloramphenicol protected against inactivation. It thus appears that these analogs react with the ribosome at functionally significant sites. The modified ribosomal components in these two cases have not been identified, however. Finally, a photoinduced reaction of unmodified chloramphenicol with the ribosome has been observed." The reaction is expressed in the labeling of several ribosomal proteins and in a pronounced shift in the electrophoretic mobility of protein L197 °
Acknowledgments We are indebted to Mr. D. Haik for ribosome preparalions. This work was supported by a grant from the United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel. O. Pongs, R. Bald, and V. A. Erdmann, Proc. Natl. Acad. Sci. U.S.A. 70, 2229 (1973). 9 N. Sonenberg, A. Zamir, and M. Wilchek, Biochem. Biophys. Res. Commun. 59, 693 (1974). 1oM. Israel, N. Sonenberg, M. Wilchek, and A. Zamir, in preparation.
[84] A Homologous Series of Photoreactive Peptidyl-tRNAs f o r P r o b i n g the Ribosomal Peptidyltransferase Center B y N. SONENBERG, M. WILCHEK, and A. ZAMIR
Aminoacyl- and peptidyl-tRNA are the natural substrates of ribosomal peptidyltransferase. Derivatives of these substrates modified chemically or with photoreactive groups can therefore be used as affinity labeling reagents for the location of ribosomal components at, or close to, the peptidyltransferase active center. Here we describe the synthesis of photoreactable derivatives of Phet R N A of the general structure: (AP) (Gly)~-Phe-tRNA (n = 0, 2, 4). 1 Abbreviations: (a) (AP) = p-azido-N-tBoc-Phe; (b) tBoc = tert-butyloxycarbonyl.
708
NUCLEIC ACIDS AND RIBOSOMAL SYSTEMS
[84]
These peptidyl-tRNA analogs bind reversibly, in the presence of poly (U), to 70 S ribosomes and attach covalently upon irradiation to a specific site(s) on the 23 S RNA of the 50 S subunit.
Experimental Procedures Synthesis of Reagents
p-Azido-N-tBoc-Phe- [3H]Phe-tRNA (AP)Phe-tRNA is synthesized according to Scheme 1 by coupling-" the N-hydroxysuccinimide ester of p-azido-N-tBoc-phenylalanine 3 with N-t8o¢ IOt
N-t8oc "3
c~-c.-coo.
.
c~-~.-
-
2)NON3 N H 2 - - ~ C H 2- CH-COOH
N-tBo¢ NH
H2
C=O I O-tRNA
N-tBo¢ NO2-~ \~P-CH2-/H-COOH~
(AP) Phe-fRNA SCHEME 1
(3H)Phe-tRNA (unfraetionated). In a standard preparation, 3.5 nmoles of (3H)Phe-tRNA are dissolved in 3.0 ml of 0.2 M triethanolamine-HC1 (pH 8.0) or 0.2 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (pH 8.0), and 600 mg of the N-hydroxysuccinimide ester of p-azido-N-tBoc-phenylalanine in 30 ml of freshly distilled dimethyl sulfoxide are added. The mixture is incubated for 2 hr at 30 °, chilled to 0 °, and 3.7 ml of 50% dichloroacetic acid are added. After 2 hr at 0 °, the mixture is centrifuged and the pellet is washed once with dimethylformamide and twice with ethanol. The dried pellet is finally dissolved 2y. Lapidot, D. Eilat, S. Rappoport, and N. deGroot, Biochem. Biophys. Res. Commun. 190, 558 (1970). 3A. Schwyzer and M. Caviezel, Helv. Chim. Acta 54, 1395 (1971).
[84]
PHOTOREACTIVE PEPTIDYL-tRNA DERIVATIVES
709
in water and titrated to pH 6.0 with Tris chloride. The preparation contained, at most, 5% of unreacted Phe-tRNA as determined by paper electrophoresis following alkaline hydrolysis. 4
p-Azido-N-tBoc-Phe- (Gly), [3H]Phe-tRNA (n = 2, 4) 1. p-Nitro-N-tBoc-Phe-(Gly),COOH (n = 2, 4). To a solution of glycylglycine (1.60 g, 12 mmoles) in 50 ml of 0.45 M sodium bicarbonate is added the N-hydroxysuccinimide ester of p-nitro-N-tBoc-Phe (4.06 g, 10 mmoles) in 50 ml of dioxane. After 3 hr at room temperature, the solution is concentrated to remove dioxane and is acidified with 10% citric acid and extracted with ethyl acetate. The ethyl acetate solution is washed with water, dried with sodium sulfate, and concentrated to a small volume. On addition of petroleum ether, the product precipitates. Yield, 4 g; m.p. 95o-96 °. p-Nitro-N-tBoc-Phe-(Gly)4 COOH is synthesized similarly, using the same ratio of reagents as for the (Gly)_~ compound. The product precipitates upon addition of citric acid. Yield, 3 g; m.p., 168 °. 2. (AP) (Gly),-N-hydroxysuccinimide Ester (n = 2, 4). p-Nitro-NtBoc-Phe-(Gly)2 COOH (880 mg, 2 mmoles), dissolved in 100 ml of methanol, is hydrogenated in the presence of 100 mg of 10% paladium on charcoal for 2.5 hr. The product, p-amino-N-tBoc-Phe-(Gly)2COOH, after removal of the catalyst by filtration and evaporation of solvent, is dissolved in 2 ml of 2 N hydrochloric acid and 0.4 ml of water at 2 °, and sodium nitrite (145 mg, 2.1 mmoles) in 1 ml of water is added. After stirring for 1.5 hr at 2 °, the solution is filtered to remove insoluble materim and degassed under reduced pressure to expel excess nitrous acid. To this solution, sodium azide (130 mg, 2.0 mmoles) in 1 ml of water is added slowly. After stirring for 45 min at 0 °, the aromatic azide compound is extracted into ethyl acetate. The solution is evaporated to dryness to give (AP)(GIy)._,COOH. To prepare the N-hydroxysuccinimide ester, the product is dissolved in 5 ml of ethyl acetate, and N-hydroxysuccinimide (172 mg, 1.5 mmoles) is added followed by dicyclohexylcarbodiimide (310 mg, 1.5 mmoles) in 6 ml of ethyl acetate. The mixture is left overnight at 0 °. After removal of dicyclohexylurea, the solution is evaporated to dryness, yielding 350 mg of product. (AP) (Gly)~COOH is prepared similarly to the (Gly)._, compound. The (Gly)4 compound is, however, barely soluble in ethyl acetate. Therefore, at the end of the reaction, sodium hydroxide is added to adjust the pH to about 4.0 and the mixture is lyophilized. The residue is dissolved 4Z. Vogel, A. Zamir, and D. Elson, Proc. Natl. Acad. Sci. U.S.A. 61, 701 (1968).
710
NUCLEIC A.CIDS AND RIBOSOMAL SYSTEMS
[84]
in ethanol and filtered to remove insoluble salts; product is obtained after evaporation of the ethanol. Thin-layer chromatography on silica gel with chloroform:methanol (3:1), or pure methanol, indicates the presence of a single product. In order to prepare the N-hydroxysuccinimide ester, 276 mg (0.5 mmole) of (AP) (Gly)4COOH are dissolved in 12 ml of dimethylformamide, and N-hydroxysuccinimide (56 rag, 0.5 mmole) is added, followed by dicyclohexylcarbodiimide (103 mg, 0.5 mmole) in 1.5 ml of dimethylformamide. The mixture is left overnight at 0 °. After removal of dicyclohexylurea, the dimethylformamide solution is used directly for coupling to Phe-tRNA. 3. (AP) (Gly)~-[3H]Phe-tRNA (n --- 2, 4). These compounds are prepared in a manner similar to that for (AP)[3H]Phe-tRNA as described above. The (Gly)2 derivative is prepared in dimethyl sulfoxide and the (Gly) 4 derivative in dimethylformamide. The preparation of (AP) (Gly)~[3H]Phe-tRNA contains between 15 and 25% of unreacted [3H]PhetRNA, whereas the preparation of (AP) (Gly)~-[3H]Phe-tRNA contains between 20 and 30% of unreacted [3H] Phe-tRNA. Reversible Binding of (AP)(Gly),-Phe-tRNA to 70 S Ribosomes (n = 0, 2, 4) The reaction mixture, in 0.15 M ammonium chloride, 30 mM magnesium acetate, and 50 mM Tris chloride (pH 7.4), contains the following per ml: 120-240 ~g of poly(U), 1.4 mg of 70 S ribosomes, and 640 pmoles of (AP) (Gly),-[~H]Phe-tRNA (n = 0, 2, 4). Reaction mixtures are incubated at 37 ° for 20 rain to allow formation of binding complexes. Covalent Binding of (AP)(GIy),-Phe-tRNA (n = 0, 2, 4) to 70 S Ribosomes Binding complexes are irradiated with a high-pressure mercury lamp (Hanovia, 450 watts) at an average distance of 8 cm for 5 rain in a water bath at room temperature. Light of wavelengths below 250 nm is eliminated by a Corex filter. Covalent binding is determined as described2 Comments The series of compounds described here is suitable for screening the ribosomal components involved in peptidyltransferase activity. All compounds described are peptidyl-tRNA analogs, and the shortest member ~N. Sonenberg, M. Wilchek, and A. Zamir, Proc. Natl. Acad. Sci. U.S.A. 72, 4332 (1975).
[85]
PUROMYCIN AND INITIATION FACTOR
711
in the series, (AP)Phe-tRNA, has been shown to bind preferentially to the ribosomal donor site2 The mode of reversible binding has not been determined for the longer peptidyl tRNAs. The compounds described are of potential value in characterizing the ribosomal environment at increasing distances from the site of interaction of the --CCA terminus of tRNA. With the same idea in mind, peptidyl-tRNAs of varying lengths with a bromoacetyl group blocking the terminal amino group have been previously synthesized and tested as affinity probes2 Information gathered so far with the present series indicates that the irradiation of poly (U)-directed ribosomal complexes of each of the compounds described results in a reaction with 23 S rRNA. Specifically, labeling takes place within the 18 S fragment that includes about 1700 nucleotides extending from the 3' end. The results point to the functional significance of rRNA in the peptidyltransferase center. Sequencing studies of the different modified rRNAs may clarify the mode of arrangement of the rRNA within the functional site. Labeling of 23 S rRNA by chemical and photoreactive analogs of peptidyl-tRNA has been reported from several other laboratories. 7-11 Acknowledgment We are indebted to Mr. I. Jaeobson for synthesizing some of the intermediate compounds. This research was supported by a grant from the United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel. oD. Eilat, M. Pellegrini, H. Oen, Y. Lapidot, and C. R. Cantor, J. Mol. Biol. 88, 831 (1974). 7L. Bispink and H. Matthaei, FEBS Lett. 37, 291 (1973). See also this volume [74]. 8A. S. Girshovich, E. S. Bochkareva, U. M. Kramarov, and Y. A. Ovchinnikov, FEBS Lett. 45, 213 (1974). See also this volume [77] and [78]. o M. Yukioka, T. Hatayama, and S. Morisawa, Biochim. Biophys. Acta 390, 192 (1975). 10A. Barta, E. Kuechler, C. Branlant, J. Sriwidada, A. Krol, and J. P. Ebel, FEBS Lett. 56, 170 (1974). See also this volume [81]. 1, j. B. Breitmeyer and H. F. Noller, J. Mol. Biol. 101, 297 (1976).
[85] Photoaffinity Labeling of Ribosomes with the Unmodified Ligands Puromycin and Initiation Factor 3 By BARRY S. COOPERMAN There are two general approaches to photoaffinity labeling. In the first, a radioactive photolabile ligand derivative is irradiated in the