Hemoglobin dynamics in red blood cells: correlation to body temperature

Abstract

A transition in hemoglobin behavior at close to body temperature has been discovered recently by micropipette aspiration experiments on single red blood cells (RBCs) and circular dichroism spectroscopy on hemoglobin solutions. The transition temperature was directly correlated to the body temperatures of a variety of species. In an exploration of the molecular basis for the transition, we present neutron scattering measurements of the temperature dependence of hemoglobin dynamics in whole human RBCs in vivo. The data reveal a change in the geometry of internal protein motions at 36.9 degrees C, at human body temperature. Above that temperature, amino acid side-chain motions occupy larger volumes than expected from normal temperature dependence, indicating partial unfolding of the protein. Global protein diffusion in RBCs was also measured and the findings compared favorably with theoretical predictions for short-time self-diffusion of noncharged hard-sphere colloids. The results demonstrated that changes in molecular dynamics in the picosecond time range and angstrom length scale might well be connected to a macroscopic effect on whole RBCs that occurs at body temperature.

FIGURE 1

(A–C) Quasielastic neutron scattering spectra of hemoglobin in human red blood cells at the different temperatures 16.9°C (A), 31.9°C (B), and 45.9°C (C) at the scattering vector q = 1.6 Å⁻¹. Circles show measured data and the solid line presents the fit over the energy transfer range from −0.75 meV to +0.75 meV. The components correspond to the narrow Lorentzian (dashed line) and the broad Lorentzian (dotted line). The instrumental energy resolution determined by vanadium is indicated in A (dash-dotted line).

FIGURE 2

(A) Half-widths at half-maximum Γ_G of the narrow Lorentzian as a function of q² at T = 16.9°C (squares) and 45.9°C (circles). The line widths reach a plateau at small q², which is interpreted as global diffusion within a confined space. The solid lines are linear fits in the q²-range 1.0−2.56 Å⁻² that are extended to intersect the constant plateaus at smaller q²-values. The linear fits pass through zero (dotted lines). The dashed lines are fits according to a jump-diffusion model in the q²-range 0.49–2.56 Å⁻². (B) Temperature behavior of the apparent translational diffusion coefficients of hemoglobin in red blood cells. Error bars are within the symbols. The dashed line indicates expected normal thermal behavior according to the Stokes-Einstein equation, with where is the viscosity of pure D₂O and R_h = 31.3 Å is the hydrodynamic radius of hemoglobin.

FIGURE 3

Arrhenius plot of the residence time, τ, of the jump-diffusion model for global hemoglobin motion.

FIGURE 4

(A) Variation of the EISF as a function of q at 16.9°C (squares) and 45.9°C (circles).The solid and dashed lines are fits with a model for diffusion in a sphere with a Gaussian distribution of radii. The inset shows the slow decay of the model, which reaches the limiting value of the immobile hydrogen fraction only at high q-values. (B) Gaussian distribution of the sphere radius f(a) at different temperatures.

FIGURE 5

(A) Mean value, â, of the Gaussian distribution as a function of temperature. The dashed line is a linear fit to the values between 16.9°C and 31.9°C and serves as a guide for the eye. (B) Fraction of hydrogen atoms that appear immobile within the instrumental energy resolution. The average value between 16.9°C and 39.9°C is indicated by the dashed line.

FIGURE 6

Half-width at half-maximum values of the broad Lorentzian as a function of q² at T = 16.9°C (squares) and 45.9°C (circles).

FIGURE 7

Apparent global diffusion coefficients of hemoglobin were divided by 1.27 to obtain the translational diffusion coefficients D_trans (circles). The diffusion coefficients at infinite dilution were scaled by a factor 0.56 to yield the theoretical predictions for short-time self-diffusion (squares). The dashed and dotted lines show expected thermal behavior of the respective diffusion coefficients.