Rapid micro-isolation of mammalian oxymyoglobin for biophysical studies

A rapid, single-step procedure is described for the isolation of mammalian oxymyoglobin for physical studies. The procedure employs a high-capacity, reusable, Hg-resin through which the myoglobin is filtered. The mammalian myoglobins for which sequences have been reported lack reactive cysteine. The column binds hemoglobin and other sulfhydryl-containing proteins, and retards othe proteins. The procedure requires less than twenty minutes for the isolation of oxymyoglobin from homogenized muscle tissue. Virtually no metmyoglobin is formed. The yields are 2-3 times greater than by other methods. The procedure can easily be scaled down for isolation of myoglobin from a few milligrams of muscle. Identical rate constants for oxygen association, dissociation, and CO association were measured by laser photolysis for beef heart myoglobin prepared by this procedure and that of Yamazaki et al. (Yamazaki, I., Yokota, K. and Shikama, K. (1964) J. Biol. Chem. 239, 4151). Gel electrophoresis indicates that the preparation is at least 95% pure.