Labeling of tyrosines in proteins with [15N]tetranitromethane, a new NMR reporter for nitrotyrosines

William J. Skawinski, Foluso Adebodun, Jung T. Cheng, Frank Jordan, Richard Mendelsohn

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

Abstract

Lysozyme and ribonuclease were used as model proteins to explore the feasibility of detecting protein-bound nitrotyrosines by 15N-NMR spectroscopy. The reporter group was introduced via synthesized [15N]tetranitromethane. Several experiments for detection of the 15N resonance in the model [3-15N]nitrotyrosine demonstrated a substantial pH-dependence of the chemical shift. When lysozyme was nitrated, either two or three 15N resonances were detected, depending on the extent of nitration. The pH-dependence of the detected resonances clearly described an apparent microscopic pK in accord with reported values, while addition of Gd(III) gave selective line broadening, indicating that the 15N reporter group could also monitor relative distances from paramagnetic sources. Nitration of ribonuclease showed five 15N resonances, of which three persisted in the purified monomer. The pH-dependence of these resonances also described apparent microscopic pK values. The [3-15N]nitrotyrosine model was reduced to the [3-15N]aminotyrosine and its 15N resonance was easily monitored by several methods, including selective population inversion. When the protein-bound nitrotyrosines were similarly reduced, much sample decomposition resulted, a possible result of photooxidation, and/or reduction of disulfide bond(s), thereby making interpretation difficult.

Original languageEnglish (US)
Pages (from-to)297-308
Number of pages12
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume1162
Issue number3
DOIs
StatePublished - Mar 26 1993

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Biophysics
  • Structural Biology
  • Biochemistry

Keywords

  • Aminotyrosine
  • NMR, N-
  • Nitrotyrosine
  • Nitrotyrosyl lysozyme
  • Nitrotyrosyl ribonuclease A
  • Tetranitromethane

Fingerprint

Dive into the research topics of 'Labeling of tyrosines in proteins with [<sup>15</sup>N]tetranitromethane, a new NMR reporter for nitrotyrosines'. Together they form a unique fingerprint.

Cite this