| Method |
P.591 right column bottom paragraph to p.592 left column top paragraph: "At an aspiration pressure of -2.3 kPa, the pulling force vector acting on an aspirated RBC (red blood cell) tongue and pointing into the micropipette had a higher norm than the colloid osmotic force vector caused by the very high Hb concentration inside the spherical RBC trail (Kelemen et al. 2001) pointing outwards. At this pressure, it was reasonable to assume that most of the cytosolic bulk water had been squeezed out. The cytosolic Hb molecules together with their bound water shell were closely packed, leaving only traces of unbound water within the intermolecular gaps. Researchers have developed a simple geometrical model to estimate the Hb molecular radius, r Hb, under the boundary condition of a circumferential isotropic membrane tension compressing the Hb molecules inside RBCs during aspiration. Researchers considered physiological data as the intracellular Hb concentration in RBCs, C(Hb)=330g/L, molecular weight of tetrameric human Hb, MW(Hb)=64,000 g/Mol, and an average RBC volume of V(RBC) = 88 fL. The molecular Hb radius was calculated using following Eqs. {2a–2c}:
Hb(RBC)=c(Hb)×V(RBC) {2a} N(Hb)=NA×Hb(RBC)/MW(Hb) {2b} rHb=3V(RBC)/4p×N(Hb) {2c}
where NA is Avogadro’s number, Hb(RBC) is the total intracellular Hb content of a single RBC, and where N(Hb) is the number of Hb molecules per RBC. For the calculation of the number of hydration layers per hemoglobin molecule, it was assumed that the amount of 0.35 g H2O/g protein corresponds to one hydration layer. A physiological concentration of hemoglobin at 330 g/L in red blood cells at conditions was assumed. The concentration of hemoglobin in the aspirated cells was calculated using the measured cell volume. The amount of cell water was then estimated by assuming a constant density of the hemoglobin solution. The number of hydration layers was finally obtained by dividing the amount of cellular water per hemoglobin molecule by the assumed value for one hydration layer." |