Separation And Purification Of Human Myoglobin

Myoglobin which exists mainly in the cytoplasm of the skeletal muscles and myocardium, is a peculiar protein. Its molecular is constituted of a polypeptide chain and a prosthetic group, 153 amino acid residue composed the polypeptide chain and the prosthetic group was haema-chrome. And its molecular weight is 16.7KD. The myoglobin tertiary structure is constituted of 8 α -spiral segments. The main physiological function of the myoglobin reserves the oxygen in the muscles tissue and provides the energy for the muscle activity. When the myocardium cell is necrosis at acute myocardial infarction (AMI), the myoglobin quantity in blood will exceed the normal upper limit, and get up to the high peer after 9 to 12 hour, and return to the normal value after 24 to 36 hours. So the negative myoglobin value is helpful to exclusion AMI. We mash 200g the refrigerant skeletal muscles of the human thigh which have been removed connective and adipose tissue. After centrifuged we get the supernatant, by passing the Sephacryl S-100 HR gel column we primarily separate the protein. The collection solution is identified the tube No. 10 to 14 which contain major 17KD protein after SDS-PAGE. After DEAE-Sephacel chromatography, dialyzing in 4℃ distilled water removing the ion and concentrating with PEG20000,we identify the concentrated solution which only have 17KD protein. The protein isidentified as human myoglobin after Western blotting chromatography and has immunological activity still. The test collects 0.3g human myoglobin by using routine biochemistry method, and the recovery ratio is 30%.And it lay the foundations for producing the monoclonal antibody of the human myoglobin.