Structure of haptoglobin and the haptoglobin-hemoglobin complex by electron microscopy

Abstract

The human serum protein, haptoglobin, forms a stable, irreversible complex with hemoglobin. Haptoglobin is composed of two H chains, which are connected via two smaller L chains to give a protein of 85,000 M r. In the complex, each H chain binds an αβ dimer of hemoglobin for a total molecular weight of 150,000. The scanning transmission electron microscope has been used to derive new information about the shape and structure of haptoglobin and hemoglobin, and about their relative orientation in the complex. The micrographs of negatively stained images show that haptoglobin has the shape of a barbell with two spherical head groups, which are the H chains. These are connected by a thin filament with a central knob, which corresponds to the L chains. The overall length of the molecule is about 124(± 8) Å and the interhead distance is 87(± 7) Å. In the haptoglobin-hemoglobin complex, the head groups are ellipsoidal and under optimal staining conditions bilobal. Thus, the αβ dimers are binding to the H chains, but off the long axis of the barbell by 127 ° in a trans configuration. This angle considerably restricts the region on the surface of the H chain structure that can contain the hemoglobin binding site. The interhead group distance for complex is 116.5(± 6.3) Å or 30 Å greater than for haptoglobin. The N terminus of the β chain was located on the trans off-axis configured barbell structure of complex by using a hemoglobin that was crosslinked between the αβ dimers in the region of the β N terminus. The distances and angles that are measured on the micrographs for the native and crosslinked complex molecules permit the directions of two of the αβ dimer ellipsoid axes to be assigned. Taken together, these data provide an approximate relative orientation for the binding of the αβ dimer to the H chain of haptoglobin.