A Simple, rapid preparative method for isolating and purifying oxymyoglobin

A simple and rapid procedure was developed to isolate and purify preparative quantities (up to 200 mg) of reduced myoglobin (98% oxy) of high purity (>96%) from beef and pork muscles. This method involved fractional precipitation of a crude myoglobin extract with ammonium sulfate and purification with a single Chromatographic step on a Sephadex G-100 column. The metmyoglobin level of the myoglobin preparation was minimised by using muscle from freshly slaughtered animals (<48h post-mortem), trimming the muscle immediately before use to remove any oxidised myoglobin on the surface and carrying out all procedures at low temperature (0-5 °C) and alkaline pH (8.0-8.5). The purity of the myoglobin preparations was confirmed by ion exchange HPLC and SDS-polyacrylamide gel electrophoresis. The method was effective for skeletal muscle of both low myoglobin content (pork) and high myoglobin content (beef). The resulting purified myoglobin was very stable and there was little change in metmyoglobin level or autoxidation rate during 3 months' storage at 0 °C.