Nucleation and growth of fibres and gel formation in sickle cell haemoglobin

Abstract

Deoxygenated sickle haemoglobin polymerizes into long 210-A diameter fibres that distort and decrease the deformability of red blood cells, and cause sickle cell disease. The fibres consist of seven intertwined double strands. They can form birefringent nematic liquid crystals (tactoids) and spherulites. Rheologically, the system behaves as a gel. The equilibria show a phase separation and a solubility. The reaction kinetics show a delay time, are then roughly exponential and are highly dependent on concentration and temperature, and accord with the double nucleation model. But these conclusions are derived from macroscopic data, without direct observation of individual fibres. We have now used non-invasive video-enhanced differential interference contrast (DIC) and dark-field microscopy to observe nucleation, growth and interaction of sickle deoxyhaemoglobin fibres in real time. The fibres originate both from centres that produce many radially distributed fibres and on the surface of pre-existing fibres, from which they then branch. The resulting network is cross-linked and dynamic in that it is flexible and continues to grow and cross-link. Our results support most aspects of the double nucleation model.

Growth and form of spherulites

“…TABLE II have modeled the effects of these dynamic heterogeneities by appropriately reducing χ, the ratio of the rotational to translational mobilities. Ultimately, this growth front nucleation (otherwise termed 'symphathetic' [24], 'double' [25][26][27][28], or 'secondary nucleation' [63][64][65]) randomizes the local crystallographic orientation, leading at long times to structures having an isotropic (spherical and circular in 3D and 2D, respectively) average large-scale structure. The variety of spherulites derives from the variability in the crystallographic symmetries of the parent crystal, the rate at which thermal fluctuations cause the crystallization front to branch, constraints on the orientation of newly formed grains and ordinary side-branching initiating from the growing dendritic tips.…”

“…Magill, and others preceding him [23], have emphasized that a critically large viscosity, characteristic of high supercooling, seems to be required for spherulites to form. The occurrence of "secondary" nucleation at the growth front (similar to "sympathetic" nucleation observed during solid state precipitation [24] or "double nucleation" in the biological literature [25][26][27][28]) has also been emphasized as an essential feature of spherulite formation in polymeric fluids [29]. Random lamellar branching with preferred crystallographic misfit is also expected to play an important role [1,15].…”

Electrochemical modulation of sickle cell haemoglobin polymerisation

“…The polymers then grew rapidly producing a resulting network which was highly branched and cross-linked to give a gel. Branching, arising from the thick regions o f the fibre, was thinner than the parent fibre, whilst, the images o f the gel showed that it was a dynamic and flexible entity [42]. increase.…”

“…The growth rates of the first and second phase o f the bi-phasic relationship w ere calculated to be 67 (± 8) nm s'1 and 40 (± 5) nm s"1 respectively and the average growth rate at this section of the Pt coil electrode was calculated to be 54 (± 9) nm s'1. Table 3.1 shows the average growth rate o f HbS fibres formed free in solution [42] compared with the fibre growth rate at a Pt coil electrode (figure 3.9).…”

“…Samuel et al [42] described the observation o f the nucleation, growth and interaction o f HbS fibres in real time using non-invasive video-enhanced differential interference contrast (DIC) and dark-field microscopy. This provided, for the first time, the direct visualisation of the polymerisation process in detail.…”