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Isolation of Sperm Whale Myoglobin by Low Temperature Fractionation with Ethanol and Metallic Ions

Karl D. Hardman 1, Edwin H. Eylar 1, Dilip K. Ray 1, Leonard J. Banaszak 1, and Frank R. N. Gurd 1

From the 1 From the Department of Biochemistry, Indiana University School of Medicine, Indianapolis, Indiana 46207

1. A method is described for the isolation and purification of myoglobin from sperm whale skeletal muscle. The aqueous extract is brought to 25% ethanol by volume at -10 to - 12° and pH 6.5. A fraction precipitated with lead subacetate is discarded. Myoglobin is precipitated from the supernatant solution at pH 7.20 by adding zinc acetate to a concentration of approximately 10 mm. The precipitate is dissolved in the cold by addition of EDTA, dialyzed against EDTA and water, and rendered salt-free by ion exchange resins. Under favorable conditions the myoglobin is almost entirely in the oxygenated form.

2. Removal of impurities and conversion to ferrimyoglobin and deoxygenated ferromyoglobin are described according to several procedures. Purity is assayed by chromatography, electrophoresis, spectrophotometric analysis, and immunochemical analysis.

3. Crystallization of oxymyoglobin is described. Conversion to the ferri form in the crystalline state does not appear to cause disruption of the crystals.

4. Stability of oxymyoglobin prepared by the zinc-ethanol procedure is comparable to that of chromatographically separated preparations. It is much better than that of oxymyoglobin prepared by reduction of ferrimyoglobin by dithionite. The present procedure avoids the rapid autoxidation that occurs during the preparative procedure of Kendrew and Parrish.

5. Absorption and optical rotatory spectra of the highly purified preparations are presented.

6. The microheterogeneity of sperm whale myoglobin described by Edmundson and Hirs is confirmed. Evidence is presented that the different components differ from each other in composition by steps of about the same degree, quite possibly in amide content as suggested by Edmundson and Hirs.

Submitted on August 30, 1965


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