Can morphological changes of erythrocytes be driven by hemoglobin?

TL;DR

This paper proposes that thermally induced force-release in hemoglobin can explain the morphological changes observed in erythrocytes at high temperatures.

Contribution

It introduces a novel explanation linking hemoglobin's thermally induced force-release to erythrocyte morphological changes.

Findings

Erythrocytes change shape at 49°C due to internal forces.

Hemoglobin's thermally induced force-release correlates with these morphological changes.

The study provides a new perspective on erythrocyte biomechanics.

Abstract

At 49 C erythrocytes undergo morphological changes due to an internal force, but the origin of the force that drives changes is not clear. Here we point out that our recent experiments on thermally induced force-release in hemoglobin can provide an explanation for the morphological changes of erythrocytes.

Full text

Subject terms: erythrocyte, morphological changes, hemoglobin, force-release

It is well-known since 1865 that at 49∘ C erythrocytes undergo morphological changes (vesiculation and deformation) due to an internal force. This effect is employed in medicine, but the origin of the force that drives morphological changes is not clear. Here we point out that our recent experiments on thermally induced force-release in hemoglobin can provide an explanation for the morphological changes of erythrocytes.

The oxygen transport in our organisms is carried out by hemoglobin eaton . It consists of four globular units linked into a double-dimer tetrameric structure eaton ; see Fig. 1. Each unit can carry one oxygen molecule ${\rm O_{2}}$ attached to its heme group. The oxygen binding ability is cooperative eaton . It decreases upon reducing the pH factor or increasing the concentration of ${\rm CO}_{2}$ bohr . Due to this Bohr’s effect bohr a tissue with a stronger need of oxygen receives it more.

Hemoglobin is densely packed in erythrocyte. In contrast to other cells, erythrocytes do not have a nucleus for the purpose of greater storage. The orientation of hemoglobin molecules in erythrocytes is not random fok ; fok2 .

Erythrocyte is known to change its physical features after thermal treatment at 49∘–50∘ C. This was discovered in 1865 via detecting a rich spectrum of erythrocyte morphological changes at and above 49∘–50∘ C schultze . The effect is routinely employed for studying the spleen enlargement, because when thermally treated and radioactively tagged erythrocytes are immersed back to blood, they are trapped in the spleen. This trapping was prescribed both to changing the form of erythrocyte (from disc to sphere) harris and to plasticity loss romania . Morphological changes at 49∘–50∘ C were studied by scanning electron micrography in coakley . It was found that thermal effects at 49∘–50∘ C depends rather weakly on heating rate (provided that this rate is sufficiently slow, i.e. slower than 0.75 C per second) and that morphological changes proceed via two major scenarios. Either the biconcave erythrocyte form changes to a rosette shape with well-established protuberances, or the erythrocyte fragments into several parts coakley . Nearly 50 % of erythrocytes did not undergo any visible morphological change at 49∘–50∘ C coakley . The effect of morphological changes was prescribed to denaturation of spectrin, a cytoskeletal protein that stitches the intracellular side of the plasma membrane in eukaryotic cells including erythrocyte spectrin ; coakley2 ; ivanov . The effect can be suppressed by lowering the ionic strength, by presence of albumin coakley2 , or (to a large extent) after incorporation of adamantin derivatives into cell membranes herrmann . However, the detailed mechanism of the morphological change is not yet understood; in particular this concerns the physical part of the problem, i.e. the origin of the force that drives vesiculation and deformation.

We carried out micromechanical experiments on crystals of horse and human hemoglobin hem . These experiments show that precisely at 49∘ C the hemoglobin releases force hem . The main advantage of using biopolymer crystals is that there is a possibility of displaying those motions of the macromolecule that can have only transient character in the solution eaton ; perutz3 . These motion are controlled by the water content and intermolecular contacts, which in their turn are regulated by the crystal syngony. Thus the solid state hemoglobin is close to its in vivo state in mammal erythrocytes, where the hemoglobin is densely packed with concentration $\simeq 34\%$ trincher .

In its partially unfolded state—i.e. for a temperature higher than the physiological temperatures, but lower than the unfolding temperature—the hemoglobin responds to heating by a sudden release of force and a subsequent jump of the Young’s modulus hem . The detailed structure of this effect is different for human and horse hemoglobin, but the temperature where the effect takes place is equal to 49∘ C for both types of hemoglobin hem . This temperature does not depend on the hydration level (in contrast to denaturation temperatures) and also on the solvating level. We argued that the effect relates to certain slowly relaxing (mechanical) degrees of freedom of the quaternary structure of hemoglobin that accumulate energy during heating and then suddenly release it at 49∘ C hem . Surprisingly, a force-release effect was found under heating which is generally supposed to diminish mechanical features of biopolymers. It was already noted in swed ; yan ; artmann that 49∘ C may indicate on structural changes in hemoglobin, but the important aspect of the force release was noted only in hem . Such an effect is absent in the thermal response of myoglobin. Myoglobin also binds and unbinds oxygen, but does so without a sizable cooperativity. This relates to its function: myoglobin is a depot (not transporter) of oxygen in muscles.

Here we conjecture that the driving force for the morphological transitions of erythrocytes at 49∘-50∘ C does come from hemoglobin. The spectrin denaturation still does play a role in those morphological changes, e.g. because it transfers the force over the erythrocyte membrane; see Fig. 1 for a schematic representation. Spectrin denaturation alone cannot explain morphological changes, since (normally) denaturation does not relate to force release. Also, the spectrin is found in membranes of other cells that do not carry hemoglobin (e.g. neurons neuron_spectrin ), but no high-temperature morphological effects are known for them.

We cite the following arguments in support of this conjecture:

– The onset of morphological changes is at 49∘ C, and precisely at this same temperature the hemoglobin releases force. The temperature does not depend on the type of hemoglobin (this was checked for human and horse hemoglobins), and it also does not depend on hydration and solvation levels.

– Before the force release, the internal friction of hemoglobin shows a sizable increase hem . Hence the quaternary structure of hemoglobin partially denaturates, and it is prone to forming spectrin-hemoglobin complexes. Spectrin-hemoglobin complexes were studied by various means hmspectrin2 ; hmspectrin1 ; hmspectrin0 . Their life-time can vary widely hmspectrin1 ; hmspectrin0 . It is also known that spectrin-hemoglobin complexes do not seriously alter the hemoglobin oxygenation.

If this conjecture is confirmed by further experiments, it may mean that the interaction between hemoglobin and spectrin is relevant for oxygen carrying function of erythrocytes. We note that dependencies of erythrocyte membrane features on the hemoglobin content were suggested several times, but no experimental support was so far found on them contra1 ; contra2 . However, these experiments did not study the thermal features at 49∘ C, which is the main focus of this contribution.

Once our conjecture looks for the origin of a mechanic force, it can be relevant for mechanic explanation of several important aspects of hemoglobin, including hemoglobin binding on spectrin 24 , scenarios of pathological intracellular polymerization in the process of hemoglobin deoxygenation 25 , and non-equilibrium dynamics of hemoglobin 27 . For the first case our conjecture can explain how the influence is transferred from the hemoglobin to erythrocyte. In the second case it may prevent the intracellular polymerization. For the third case, the force may be involved in generating the non-equilibrium potential.

Acknowledgements

This work was supported in part by Grant MOST 105-2112-M-001 -004.

Author contributions

SGS designed research, performed research, analyzed data.

AEA analyzed data, wrote the paper.

DSG performed research, analyzed data.

WJM analyzed data, wrote the paper.

CKH analyzed data, wrote the paper.