Synthesis and characterization of N-(4-azidophenylthio)phthalimide: A cleavable, photoactivable crosslinking reagent that reacts with sulfhydryl groups
✍ Scribed by Robert B. Moreland; Paul K. Smith; Edward K. Fujimoto; Michael E. Dockter
- Publisher
- Elsevier Science
- Year
- 1982
- Tongue
- English
- Weight
- 499 KB
- Volume
- 121
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
The cleavable, photoaffinity label precursor, N-(4-azidophenylthio)phthalimide has been synthesized and purified. The recrystallized product was identified by infrared spectroscopy and nuclear magnetic resonance spectroscopy. The compound modifies free thiol groups to yield the corresponding S-4-azidophenylthio derivatives. In order to examine the biological applications of this compound, yeast iso-I-cytochrome c, containing a single free cysteine residue, was modified and characterized. The 102~S-(4-azidophenylthio)-iso-I-cytochrome c was found to contain 1 mol of label/m01 of cytochrome c. The photoaffinity labeling of purified, detergent-solubilized yeast cytochrome c oxidase was examined. Photolysis products of crosslinking could be analyzed on sodium dodecyl sulfate-polyacrylamide gels in the absence of reducing agents. The crosslinked products were readily cleaved by dithiothreitol. The use of this compound as a sulfhydryl-specific photolabile, bifunctional crosslinking reagent is discussed.