A novel reversible thiol-specific spin label: Papain active site labeling and inhibition

Lawrence J. Berliner, Jacob Grunwald, H. Olga Hankovszky, Kalman Hideg

Research output: Article

221 Citations (Scopus)

Abstract

A new, highly reactive, thiol-specific spin label, (1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)methanethiosulfonate was synthesized. Its unique specificity was demonstrated with the active thiol protease, papain, which was stoichiometrically inhibited within 5 min, resulting in a conformationally sensitive spectrum, which was identical over the pH range 4.5-7.5. The spin-label modification yielded a mixed disulfide between Cys 25 of papain and the 3-methylpyrroline nitroxide which was rapidly and completely reversed by exposing the labeled papain to mild concentrations of dithiothreitol. The concentration of released nitroxide corresponded exactly to the number of reactive thiol groups in the original enzyme. Full enzymatic activity was restored after the spin label was removed. This spin label is useful as a sensitive thiol titrating agent as well as a specific conformational probe of thiol site structure by virtue of its minimal rotational freedom and distance from the covalent disulfide linkage to the macromolecule under study.

Original languageEnglish
Pages (from-to)450-455
Number of pages6
JournalAnalytical Biochemistry
Volume119
Issue number2
DOIs
Publication statusPublished - jan. 15 1982

    Fingerprint

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this