An aliquot of 1 kg of
muscle was homogenized with 5 L of cold distilled water using a
mixer (Braun, Barcelona, Spain), and the pH was adjusted to 3 with
5 M HCl. The mixture was then left in an ice bath under constant
stirring for 60 min to ensure complete protein solubilization. The
mixture was centrifuged at 15,800 g/4 C/30 min (Sorvall Evolution
RC Centrifuge, Thermo Fisher Scientific Inc., Landsmeer