Influence of Prosthetic Groups on Protein Folding and Subunit Assembly

Abstract

Abstract Separated, heme-free, α- and β-globins have been prepared from the heme-containing α and β subunits of human hemoglobin. It is shown that the α- and β-globins are different in molecular weight, secondary structure, sulfhydryl reactivity, and a number of other properties from the heme-containing α and β subunits to which the isolated globin chains can be reconstituted and from which they derive. Moreover, the following have been demonstrated. (a) The separated globin chains are folded very differently from one another, although the basic folding features of the parent subunits are highly similar. (b) Removal of heme from the isolated β chain results in a β-globin with an s20,w value of 2.7, corresponding to a dimer. The heme-containing chain is tetrameric. The dimeric form of β-globin occurs in 20 mm as well as 0.1 m phosphate (or Tris) and over a range of pH values from 6.7 to 8. (c) Removal of heme from the isolated α chain results in an α-globin of markedly increased sedimentation coefficient. The value of the sedimentation coefficient is dependent on the concentration of buffer in which the initial globin precipitate is dissolved and against which it is dialyzed. When the initial phosphate concentration is 0.1 m, the s20,w value is 2.5 (corresponding to dimer). At 20 mm phosphate, the value is 6.6 (possibly corresponding to octamer). The heme-containing subunit has an s20,w value of 1.9 (corresponding to monomer, predominantly). (d) Sulfhydryl reactivity is sharply reduced in both isolated globin chains, in contrast to complete accessibility of sulfhydryl in the heme-containing separated subunits. In 15% sodium dodecyl sulfate, all sulfhydryl groups in the globin chains react with Ellman's reagent. (e) In the absence of heme, at accessible temperature (4°) and low phosphate concentration, the isolated, soluble α- and β-globin chains do not measurably combine. Under these conditions apohemoglobin (αβ-globin) occurs as a dimer. An analysis of secondary structure in terms of primary sequence reveals that pros thetic group binding has quite different effects on the two globin chains and that this represents a major influence on the attainment of native structure of hemoglobin.