Synthesis and Characterization of a Heterobifunctional Photoaffinity Reagent for Modification of Tryptophan Residues and Its Application to the Preparation of a Photoreactive Glucagon Derivative

Catherine D. Demoliou, Richard M. Epand

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Abstract

The synthesis of the heterobifunctional crosslinking reagent 2-nitro-4-azidophenylsulfenyl chloride (NAPSC1) is described. This reagent can be used to specifically attach a photoactivatable nitrophenyl azide to tryptophan- containing polypeptides and proteins lacking sulfhydryl groups. The sulfenyl chloride group of NAPSC1 reacts with the indole ring of tryptophan following second-order reaction kinetics in 50-100% acetic acid. The labeled product can be effectively photolyzed at wavelengths above 300 nm. The reaction of glucagon, a peptide hormone containing a single tryptophan residue at position 25 and no cysteine, with NAPSC1 gave one major product, the photosensitive derivative glucagon-NAPS. The structure and properties of the purified derivative were established by amino acid analysis, absorption spectroscopy, and photolysis. Only the tryptophan residue of this derivative was modified. The photosensitive glucagon was shown to activate the adenylate cyclase of hepatocyte plasma membranes to the same extent as the native hormone at equimolar concentrations. Glucagon-NAPS could be radiolabeled by the lactoperoxidase-catalyzed iodination of the peptide. A glucagon-specific antibody bound both radiolabeled glucagon and glucagon-NAPS peptides. The covalent labeling of protein molecules with radiolabeled glucagon-NAPS peptide upon photolysis was demonstrated. Glucagon-NAPS can be used as an effective photoaffinity probe for labeling the glucagon receptor site in plasma membranes of target cells.

Original languageEnglish
Pages (from-to)4539-4546
Number of pages8
JournalBiochemistry
Volume19
Issue number20
DOIs
Publication statusPublished - 1980

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Glucagon
Tryptophan
Derivatives
Peptides
Photolysis
Cell membranes
Labeling
Glucagon Receptors
Cell Membrane
Cross-Linking Reagents
Lactoperoxidase
Azides
Peptide Hormones
Halogenation
Absorption spectroscopy
Adenylyl Cyclases
Reaction kinetics
Acetic Acid
Cysteine
Chlorides

Cite this

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title = "Synthesis and Characterization of a Heterobifunctional Photoaffinity Reagent for Modification of Tryptophan Residues and Its Application to the Preparation of a Photoreactive Glucagon Derivative†",
abstract = "The synthesis of the heterobifunctional crosslinking reagent 2-nitro-4-azidophenylsulfenyl chloride (NAPSC1) is described. This reagent can be used to specifically attach a photoactivatable nitrophenyl azide to tryptophan- containing polypeptides and proteins lacking sulfhydryl groups. The sulfenyl chloride group of NAPSC1 reacts with the indole ring of tryptophan following second-order reaction kinetics in 50-100{\%} acetic acid. The labeled product can be effectively photolyzed at wavelengths above 300 nm. The reaction of glucagon, a peptide hormone containing a single tryptophan residue at position 25 and no cysteine, with NAPSC1 gave one major product, the photosensitive derivative glucagon-NAPS. The structure and properties of the purified derivative were established by amino acid analysis, absorption spectroscopy, and photolysis. Only the tryptophan residue of this derivative was modified. The photosensitive glucagon was shown to activate the adenylate cyclase of hepatocyte plasma membranes to the same extent as the native hormone at equimolar concentrations. Glucagon-NAPS could be radiolabeled by the lactoperoxidase-catalyzed iodination of the peptide. A glucagon-specific antibody bound both radiolabeled glucagon and glucagon-NAPS peptides. The covalent labeling of protein molecules with radiolabeled glucagon-NAPS peptide upon photolysis was demonstrated. Glucagon-NAPS can be used as an effective photoaffinity probe for labeling the glucagon receptor site in plasma membranes of target cells.",
author = "Demoliou, {Catherine D.} and Epand, {Richard M.}",
year = "1980",
doi = "10.1021/bi00561a001",
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T1 - Synthesis and Characterization of a Heterobifunctional Photoaffinity Reagent for Modification of Tryptophan Residues and Its Application to the Preparation of a Photoreactive Glucagon Derivative†

AU - Demoliou, Catherine D.

AU - Epand, Richard M.

PY - 1980

Y1 - 1980

N2 - The synthesis of the heterobifunctional crosslinking reagent 2-nitro-4-azidophenylsulfenyl chloride (NAPSC1) is described. This reagent can be used to specifically attach a photoactivatable nitrophenyl azide to tryptophan- containing polypeptides and proteins lacking sulfhydryl groups. The sulfenyl chloride group of NAPSC1 reacts with the indole ring of tryptophan following second-order reaction kinetics in 50-100% acetic acid. The labeled product can be effectively photolyzed at wavelengths above 300 nm. The reaction of glucagon, a peptide hormone containing a single tryptophan residue at position 25 and no cysteine, with NAPSC1 gave one major product, the photosensitive derivative glucagon-NAPS. The structure and properties of the purified derivative were established by amino acid analysis, absorption spectroscopy, and photolysis. Only the tryptophan residue of this derivative was modified. The photosensitive glucagon was shown to activate the adenylate cyclase of hepatocyte plasma membranes to the same extent as the native hormone at equimolar concentrations. Glucagon-NAPS could be radiolabeled by the lactoperoxidase-catalyzed iodination of the peptide. A glucagon-specific antibody bound both radiolabeled glucagon and glucagon-NAPS peptides. The covalent labeling of protein molecules with radiolabeled glucagon-NAPS peptide upon photolysis was demonstrated. Glucagon-NAPS can be used as an effective photoaffinity probe for labeling the glucagon receptor site in plasma membranes of target cells.

AB - The synthesis of the heterobifunctional crosslinking reagent 2-nitro-4-azidophenylsulfenyl chloride (NAPSC1) is described. This reagent can be used to specifically attach a photoactivatable nitrophenyl azide to tryptophan- containing polypeptides and proteins lacking sulfhydryl groups. The sulfenyl chloride group of NAPSC1 reacts with the indole ring of tryptophan following second-order reaction kinetics in 50-100% acetic acid. The labeled product can be effectively photolyzed at wavelengths above 300 nm. The reaction of glucagon, a peptide hormone containing a single tryptophan residue at position 25 and no cysteine, with NAPSC1 gave one major product, the photosensitive derivative glucagon-NAPS. The structure and properties of the purified derivative were established by amino acid analysis, absorption spectroscopy, and photolysis. Only the tryptophan residue of this derivative was modified. The photosensitive glucagon was shown to activate the adenylate cyclase of hepatocyte plasma membranes to the same extent as the native hormone at equimolar concentrations. Glucagon-NAPS could be radiolabeled by the lactoperoxidase-catalyzed iodination of the peptide. A glucagon-specific antibody bound both radiolabeled glucagon and glucagon-NAPS peptides. The covalent labeling of protein molecules with radiolabeled glucagon-NAPS peptide upon photolysis was demonstrated. Glucagon-NAPS can be used as an effective photoaffinity probe for labeling the glucagon receptor site in plasma membranes of target cells.

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