[4] Preparation of derivatives of ferrous and ferric hemoglobin

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This chapter describes the preparation of derivatives of ferrous and ferric hemoglobin. The preparation of the different derivatives can often be done directly from the hemolysate, without any further purification. This is justified by the fact that hemoglobin is the major proteic component of the erythrocytic cytoplasm. In other animal species, especially in fish, the hemoglobin composition consists of several components in comparable amounts, and purification of the individual components is necessary. From a strictly methodological point of view, as a general rule, very laborious procedures should be avoided to keep the protein in the native form; this is particularly true in the case of mutant hemoglobins exhibiting a reduced stability. A hemoglobin solution can be stored aerobically in the cold for a few days without appreciable changes of its properties. It is advisable to keep the protein in a concentrated solution and at neutral or slightly alkaline pH. A more prolonged storage of hemoglobin can be obtained in its deoxygenated form. However, there are technical difficulties in keeping the solution completely oxygen free, and this is an absolute requirement because partially saturated hemoglobin is more susceptible to autoxidation than is the fully oxygenated protein. The method of choice for keeping hemoglobin, as well as many other proteins, is rapid freezing and storage in liquid nitrogen.

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