Metastable polymerization of sickle hemoglobin in droplets

Abstract

Sickle cell disease arises from a genetic mutation of one amino acid in each of the two hemoglobin beta chains, leading to the polymerization of hemoglobin in the red cell upon deoxygenation, and is characterized by vascular crises and tissue damage due to the obstruction of small vessels by sickled cells. It has been an untested assumption that, in red cells that sickle, the growing polymer mass would consume monomers until the thermodynamically well-described monomer solubility was reached. By photolysing droplets of sickle hemoglobin suspended in oil we find that polymerization does not exhaust the available store of monomers, but stops prematurely, leaving the solutions in a supersaturated, metastable state typically 20% above solubility at 37 degrees C, though the particular values depend on the details of the experiment. We propose that polymer growth stops because the growing ends reach the droplet edge, whereas new polymer formation is thwarted by long nucleation times, since the concentration of hemoglobin is lowered by depletion of monomers into the polymers that have formed. This finding suggests a new aspect to the pathophysiology of sickle cell disease; namely, that cells deoxygenated in the microcirculation are not merely undeformable, but will actively wedge themselves tightly against the walls of the microvasculature by a ratchet-like mechanism driven by the supersaturated solution.

Figure 1

A droplet of sickle hemoglobin in castor oil is shown before and after photolysis. The sample of chromatogrpahically purified Hb (in 0.15 M phosphate buffer at pH 7.35; 0.55 M sodium dithionite) has a thickness of 3.7 μm and is observed using a Leitz 32×LWD microscope objective; images are shown at 426 nm in the intensely absorbing Soret band. (Color has been introduced to help distinguish the regions). Laser photolysis by the 488 nm line of an Argon ion laser deoxygenates the COHb, which forms polymers. The rough texture is the result of the combination of turbidity, linear dichroism and non-uniformity of the polymer mass. On the perimeter of the droplet a triangular region has been masked from laser illumination, and remains HbCO, retaining the good optical characteristics of the unpolymerized Hb. Monomers diffuse from that region into the polymerized region as polymerization proceeds, until the final concentration is reached, as shown.

Figure 2

Final concentration as a function of temperature. The solid curve is the solubility of HbS . All samples are in 200–300 μm droplets, in which the masked area is 4–5% of the total, and is placed at the center of the Hb drop. Points connected by dashed lines were polymerized at the higher temperature of the pair, and the temperature then was changed to the lower temperature. At the high temperatures, some symbols occlude others because the concentrations achieved were the same. All temperature-shift experiments involved polymerization at the high temperature, followed by shifting to a lower temperature, and then finally depolymerization. Solo points (all in darker colors) have been polymerized directly to the final temperature. The cross shows a concentration achieved by pressing on the slide, initially gelled at 35°C. Temperatures were regulated by a thermoelectric stage. Samples were observed by an Ealing 15×reflecting objective; spectra were recorded in the Soret region using an Ocean optics 2000 spectrometer. Photolysis was by a Lambda-Pro diode-pumped 532 nm solid state laser.

Figure 3

A droplet shown during collapse of the masked area. As in Figure 1, a triangular edge mask kept an unphotolyzed reservoir of COHbS adjacent to a region polymerized by photolysis. After 71 minutes, the hemoglobin in the reservoir flowed to the perimeter, and we interpret this as due to the outward pressure of the fibers against the surface. A bubble forms first because the edge of the droplet is pinned. Conditions otherwise are as in Figure 1.