7] Preparation of isolated chains of human hemoglobin

Abstract

Publisher Summary This chapter illustrates the preparation of isolated chains of human hemoglobin. Human hemoglobins A, A 2 , and F split into their constituent polypeptide chains after treatment with an excess of PMB. The chains so obtained are native in the sense that they contain the heme, reversibly exchange oxygen, and when reconstituted with their partners form tetrametric proteins that have the allosteric properties of normal hemoglobin. Mutant human hemoglobins can also be resolved into the constituent chains by the same procedures. Reports exist that dog, horse, and pig hemoglobin produce new electrophoretic components upon treatment with PMB. Procedures of wider applicability in regard to hemoglobins from different species are based on countercurrent distribution and urea chromatography of heme-free denatured hemoglobins. In addition, the chapter also reviews the splitting of heme-free chains. In these preparations, the chains of hemoglobin are separated after the complete denaturation of the protein. Thus, the splitting mechanism does not depend on the precise location of certain side chains and becomes a general method of wide applicability. The techniques described are standardized on human hemoglobin subunits; it is anticipated that their application to other hemoglobins may require some modification of the experimental conditions.