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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) 2 arXiv:1705.06144v1 [q-bio.BM] 17 May 2017 4 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 2 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. [1] W.A. Eaton, E.R. Henry, J. Hofrichter, and A. Mozzarelli. Is cooperative oxygen binding by hemoglobin really understood? Nature Str. Biol. 6, 351-358 (1999). [2] C. Bohr, K. Hasselbalch, and A. Krogh. About a new biological relation of high importance that the blood carbonic acid tension exercises on its oxygen binding. Skandinavisches Archiv fur Physiologie 16, 402-412 (1904). [3] G. Gevorkian, A. E. Allahverdyan, D. S. Gevorgyan and Chin-Kun Hu. Thermal-induced force release in oxyhemoglobin. Sci. Rep. 5, 13064 (2015). [4] H. Jansson and J. Swenson. Dynamical changes of hemoglobin and its surrounding water during thermal denaturation as studied by quasielastic neutron scattering and temperature modulated differential scanning calorimetry. J. Chem. Phys. 128, 245104 (2008). [5] Y.B. Yan, Q. Wang, H.W. He, and H.M. Zhou. Protein thermal aggregation involves distinct regions: sequential events in the heat-induced unfolding and aggregation of hemoglobin. Biophys. J. 86, 1682-1690 (2004). 3 [6] G.M. Artmann et al. Circular dichroism spectra of human hemoglobin reveal a reversible structural transition at body temperature. Eur. Biophys. J. 33, 490-496 (2004). [7] M. Schultze. Anat. Entw. Mech. 1, 1 (1865). [8] W.T. Coakley et al. J. Therm. Biol. 4, 85 (1979). [9] W.T. Coakley et al. Effects of ionic strength, serum protein and surface charge on membrane movements and vesicle production in heated erythrocytes. Biochimica et Biophysica Acta-Biomembranes, 602, 355-375 (1980). [10] J.F. Brandts, L. Erickson, K. Lysko, A.T. Schwartz, and R.D. Taverna, Biochemistry, 16, 3450 (1977). [11] A. Herrmann et al.. Biochim. Biophys. Acta, 812, 277-285 (1985). [12] I.T. Ivanov and L.C. Benov. J. Therm. Biol. 17, 381-389 (1992). [13] I.M. Harris, J.M. McAllister, and T.A.J. Prankerd. Clin. Sci. 16, 223 (1957). [14] P. Teitel. Nature 206, 409 (1965). [15] G.M. Fermi, M.F. Perutz, B. Shaanan, R. Fourme. The crystal structure of human deoxyhaemoglobin at 1.74 Å resolution. J. Mol. Biol. 175, 159 (1984). [16] K. Trincher, Biology and information, elements of biological thermodynamics (Consultants Bureau, NY, 1965). [17] M.V. Fok, N.B. Arkhangelskii, A.R. Zaritskii, G.A. Prokopenko, and V.V, Faddeev. The role of erythrocyte membrane in oxygen delivery to tissues. Preprint of Moscow Physical Institute, number 41, 1985. [18] B.R. Wood, L. Hammer, and D. McNaughton. Resonance Raman spectroscopy provides evidence of heme ordering within the functional erythrocyte. Vibrational spectroscopy 38, 71-78 (2005). [19] L. M. Snyder et al., J. Clin. Invest. 76, 1971-1977 (1985). [20] P. Datta, S.B. Chakrabarty, A. Chakrabarty, and A. Chakrabarti, Blood Cell Mol. Dis. 30, 248253 (2003). [21] A. Basu and A. Chakrabarti, Journal of Proteomics 128, 469-475 (2015). [22] R.I. Weed, C.F Reed, G. Berg, Journal of Clinical Investigation 42, 581 (1963). [23] E. A. Moffatt and E. H. Harris, J. Basic Eng. 94, 363-367 (1972). [24] S.R. Goodman, I.S. Zagon. The neural cell spectrin skeleton: a review. American Journal of Physiology-Cell Physiology 250, C347-C360 (1986). [25] H. Chu, A. Breite, P. Ciraolo, R.C. Franco, and Ph.S. Low. Characterization of the deoxyhemoglobin binding site on human erythrocyte band 3: implications for O2 regulation of erythrocyte properties. Blood. 110, 140 (2007). [26] C. Dong, R.C. Chadwick, and A.N. Schechter. Influence of sickle hemoglobin polymerization and membrane properties on deformability of sickle erythrocytes in the microcirculation. Biophysical Journal. 63, 774-783 (1992). [27] H. Turlier et al. Equilibrium physics breakdown reveals the active nature of red blood cell flickering. Nature Physics. 12, 513-521 (2016). 4 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.