Download Can morphological changes of erythrocytes be driven by hemoglobin?

Can morphological changes of erythrocytes be driven by hemoglobin?
S. G. Gevorkian1,2, A.E. Allahverdyan2, D.S. Gevorgyan3, Wen-Jong Ma1,4 , Chin-Kun Hu1
1
Institute of Physics, Academia Sinica, Nankang, Taipei 11529, Taiwan
Yerevan Physics Institute, Alikhanian Brothers St. 2, Yerevan 375036, Armenia
3
Institute of Fine Organic Chemistry, 26 Azatutian ave., Yerevan 0014, Armenia and
Graduate Institute of Applied Physics, National Chengchi University, Taipei 11605, Taiwan
(Dated: May 18, 2017)
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arXiv:1705.06144v1 [q-bio.BM] 17 May 2017
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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.
PACS numbers: 87.14.E-,87.15.hp,87.15.La
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 [1]. It consists of four globular units linked
into a double-dimer tetrameric structure [1]; see Fig. 1. Each unit can carry one oxygen molecule O2 attached to its
heme group. The oxygen binding ability is cooperative [1]. It decreases upon reducing the pH factor or increasing
the concentration of CO2 [2]. Due to this Bohr’s effect [2] 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 [17, 18].
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 [7]. 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) [13] and to plasticity loss [14]. Morphological changes at
49◦ –50◦ C were studied by scanning electron micrography in [8]. 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 [8]. Nearly 50
% of erythrocytes did not undergo any visible morphological change at 49◦ –50◦ C [8]. 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 [9, 10, 12]. The effect can be suppressed by lowering the
ionic strength, by presence of albumin [9], or (to a large extent) after incorporation of adamantin derivatives into cell
membranes [11]. 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 [3]. These experiments
show that precisely at 49◦ C the hemoglobin releases force [3]. 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 [1, 15]. 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 ≃ 34% [16].
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 [3]. 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 [3]. 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 [3]. Surprisingly, a forcerelease effect was found under heating which is generally supposed to diminish mechanical features of biopolymers.
It was already noted in [4–6] that 49◦ C may indicate on structural changes in hemoglobin, but the important aspect
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of the force release was noted only in [3]. 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 [24]), 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 [3]. Hence the quaternary
structure of hemoglobin partially denaturates, and it is prone to forming spectrin-hemoglobin complexes. Spectrinhemoglobin complexes were studied by various means [19–21]. Their life-time can vary widely [19, 20]. 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 [22, 23]. 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 [25], scenarios of pathological intracellular
polymerization in the process of hemoglobin deoxygenation [26], 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.
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FIG. 1: (Color online) Schematic representation of the hemoglobin-spectrin interaction.
Left figure: Multiprotein complexes in the red cell membrane that are attached to spectrin. Tetrameric band 3 protein is bound
to ankyrin, which binds the quasi-hexagonal spectrin network to erythrocyte membrane. Spectrin consists of and chains. It
stitches the membrane, which is very soft without spectrin. The hemoglobin is depicted with partially unfolded quaternary
structure: two out of four globular sub-units are partially open.
Right figure: possible location of hemoglobin molecules within the spectrin network. In order not to overload the picture, only
few hemoglobin molecules are shown. In reality the hemoglobin concentration is approximately 34 %. Red arrows show the
force that can stitch the erythrocyte.