Scanning transmission electron microscopic examination of the hexagonal bilayer structures formed by the reassociation of three of the four subunits of the extracellular hemoglobin of Lumbricus terrestris.

Abstract

A fraction obtained by gel filtration at neutral pH of the extracellular Hb of Lumbricus terrestris dissociated either at pH 9.8 or at pH 4.0, consisting of the three subunits D1 (31 kDa), D2 (37 kDa), and T (50 kDa), was found to produce two peaks when subjected to gel filtration on Superose 6 at pH 7. The first peak, which was eluted at a slightly greater volume than the native Hb, consisted of reassociated hexagonal bilayer structures when examined by scanning transmission electron microscopy. The dimensions of the two reassociated hexagonal bilayer structures were a vertex-to-vertex diameter of 25 nm and a height of 16 nm. The difference in size between the hexagonal bilayer structures and the native Hb is the contribution of subunit M, which consists of a single hemecontaining chain I (16.75 kDa). Although the reassociated hexagonal bilayer structures have overall dimensions smaller than the 30 nm x 20 nm dimensions of the native Hb, the diameters of the central cavities are not substantially altered. Subtraction of the three-dimensional reconstructions of the reassociated hexagonal bilayer structures from those of the native Hb showed that subunit M was primarily localized at the periphery of Lumbricus Hb. The formation of hexagonal bilayer structures in the complete absence of subunit M provides additional support for the "bracelet" model of the quaternary structure ofLumbricus Hb proposed recently by us in which subunits D1 and D2 were assumed to act as linkers for complexes of subunits M and T or to form a "bracelet" decorated with 12 complexes of subunits M and T.

Scanning (atomic) force microscopy imaging of earthworm haemoglobin calibrated with spherical colloidal gold particles

“…5 nm (n ¼ 75, Table 2), which represents approximately a two-fold overestimation as compared with measurements by STEM (30 Ϯ 0 . 8 nm; Kapp et al, 1987).…”

“…The preparation of the earthworm haemoglobin has been described previously (Kapp et al, 1984(Kapp et al, , 1987. For imaging in solution, the haemoglobin molecules (2 mL, 0 .…”

“…1 and 3). The percentage compression was defined as {h STEM -h 1 (or h 2 )}/h STEM , where h STEM is the value for height obtained by scanning transmission electron microscopy (STEM), which we took as 20 nm (Kapp et al, 1987).…”

“…2 MDa with Ϸ200 polypeptide chains. A model of the quaternary structure of two hexagonal tiers of pentagonally shaped subunits has been proposed (Kapp et al, 1982(Kapp et al, , 1984(Kapp et al, , 1987Vinogradov et al, 1986).…”

Three-Dimensional Aberration-Corrected Scanning Transmission Electron Microscopy for Biology

“…Spectacular images of individual atoms, stained DNA, and biological macromolecules were rapidly obtained [63,65]. 3D reconstructions were made through combining data from a set of dark field images [66][67][68], and STEM tomography was recently implemented [69,70].…”