Gene action in the human haptoglobins: I. Dissociation into constituent polypeptide chains

Abstract

Haptoglobins were treated at room temperature for 10 minutes with 0.02 m-2-mercaptoethanol (in the presence of 8 m-urea, 0.1 m-boric acid and 0.04 m-sodium hydroxide) to obtain dieulphide cleavage, followed by alkylation with 0.05 m-iodoacetamide. Starch-gel electrophoresis under a wide variety of conditions establishes that the products of these treatments include electrophoretically homogeneous components (hp1Fα, hp1Sα, hp2α etc.) the behaviour of which is related to the genotype at the Hp locus of the individual from whom the haptoglobin was isolated. The Hp genotype has no effect on the electrophoretic behaviour of a second group of reaction products (hpβ). Wide variations in the conditions used for the disulphide cleavage and alkylation establish that the hpα products are not uniquely dependent on the particular reagents and reaction conditions selected. In some circumstances, each hpα product may give rise to more than one electrophoretic zone after reductive cleavage as a consequence of partial splitting of disulphide bridges. A general method for testing for this possibility is described. Evidence is presented that the hp1Fα and hp1Sα components in native haptoglobin contain one internal disulphide bridge and that hp2α contains two such bridges. After reductive cleavage, these bridges are readily re-formed even in the presence of 8 m-urea. The hpα products prepared in the way described are, nevertheless, shown to be fully reduced and stable. It is concluded that the products hp1Fα, hp1Sα and hp2α are undegraded polypeptide chains with no disulphide bonds in their structures, and that the complex differences in native haptoglobins are the consequences of variations in the hpα polypeptides controlled by the Hp locus. The hpβ components are probably derived from a second polypeptide chain not affected by variations in the genotype at the Hp locus.