Detection of Mobile Proteins by Proton Nuclear Magnetic Resonance Spectroscopy in the Guinea Pig Brain Ex Vivo and Their Partial Purification

Abstract

Proton nuclear magnetic resonance (1H NMR) spectroscopy was used to study metabolites of the brain cortex ex vivo. The superfused brain cortex preparation was judged to be metabolically viable on the basis of the 31P NMR spectrum (intracellular pH of 7.23 +/- 0.03 and phosphocreatine/ATP ratio of 1.21 +/- 0.09). Using 1H NMR a group of previously unidentified signals was detectable at 0.94, 1.22, and 1.40 ppm with a water-suppressed spin-echo sequence. These signals had shorter spin-spin relaxation times (51-54 ms) than N-acetylaspartate and lactate (84-93 ms) and also smaller saturation factors, an indication of shorter spin-lattice relaxation times than the latter two low-molecular-weight metabolites. The unidentified signals also displayed homonuclear coupling to other spins in the methine region of the spectrum. Acid extraction of the brain slices or cortex from animals that were killed yielded a mixture of proteins that exhibited NMR properties matching the 1H NMR signals in the brain cortex. The molecular mass of these thermoresistant, "mobile" proteins, which contained proline plus hydroxyproline (9-16% of all amino acids), ranged between 8 and 40 kDa. These "new" assignments of 1H NMR-detectable compounds may influence interpretation of NMR data of some metabolites, as their signals are in the vicinity of the -CH3 1H NMR peaks of lactate and alanine.