Partial Purification of Peroxiredoxin-2 From Porcine Skeletal Muscle

Abstract

Fresh meat quality is adversely affected by protein oxidation. However, a fundamental understanding of the diverse factors that influence protein oxidation in postmortem muscle remains elusive. Peroxiredoxin-2 (Prdx2), an antioxidant protein, is more abundant in tough meat based on instrumental tenderness; however, the role of Prdx2 in postmortem skeletal muscle is unknown. Therefore, the objective was to develop a method to purify Prdx2 from the diaphragm, psoas major, and longissimus lumborum. Proteins soluble at low ionic strength were extracted, dialyzed, clarified, and loaded onto a Q-Sepharose anion exchange column equilibrated with TEM (pH 7.4). In all preparations, Prdx2 eluted between about 75 and 115 mM NaCl. Immunoreactive fractions were dialyzed against TEM (pH 8.0), clarified, and loaded onto a DEAE-650S anion exchange column. In all preparations, Prdx2 eluted between approximately 55 and 75 mM NaCl. Immunoreactive fractions were concentrated and loaded onto a Superose-12 size exclusion column. Prdx2 was detected between 14 and 16 mL, and these fractions were concentrated and reduced with 0.5% 2-mercaptoethanol. A final pass over the Superose-12 column was conducted, and Prdx2 was detected in 2 peaks from 11–12 mL and 15–16 mL. Fractions 15–16 were pooled and retained for further experiments. The elution profile of Prdx2 in all 3 muscles was similar. The iden- tification of the primary protein was confirmed with liquid chromatography with tandem mass spectrometry. The purity of Prdx2 off the final Superose-12 column was approximately 33%, 52%, and 47% pure in the diaphragm, psoas major, and longissimus lumborum, respectively. This is the first report of a method to partially purify Prdx2 from skeletal muscle.

Keywords

• peroxiredoxin-2
• porcine
• anion exchange chromatography
• size exclusion chromatography