Body temperature-related structural transitions of monotremal and human hemoglobin

Abstract

In this study, temperature-related structural changes were investigated in human, duck-billed platypus (Ornithorhynchus anatinus, body temperature T(b) = 31-33 degrees C), and echidna (Tachyglossus aculeatus, body temperature T(b) = 32-33 degrees C) hemoglobin using circular dichroism spectroscopy and dynamic light scattering. The average hydrodynamic radius (R(h)) and fractional (normalized) change in the ellipticity (F(obs)) at 222 +/- 2 nm of hemoglobin were measured. The temperature was varied stepwise from 25 degrees C to 45 degrees C. The existence of a structural transition of human hemoglobin at the critical temperature T(c) between 36-37 degrees C was previously shown by micropipette aspiration experiments, viscosimetry, and circular dichroism spectroscopy. Based on light-scattering measurements, this study proves the onset of molecular aggregation at T(c). In two different monotremal hemoglobins (echidna and platypus), the critical transition temperatures were found between 32-33 degrees C, which are close to the species' body temperature T(b). The data suggest that the correlation of the structural transition's critical temperature T(c) and the species' body temperature T(b) is not mere coincidence but, instead, is a more widespread structural phenomenon possibly including many other proteins.

FIGURE 1

Fractional change in ellipticity at 222 ± 2 nm (F_obs) with temperature for oxyhemoglobins obtained from human blood and monotreme blood. Data points were averaged from the original CD data obtained from three to four samples of each type of hemoglobin. The error bars represent standard deviation of the respective fractional changes. Straight lines show approximation of three distinctly linear parts of the graph. Temperature intervals where the transition occurs are marked as T_c (critical temperature) for each species. Gray boxes show physiological temperature range for each species.

FIGURE 2

Original DLS data obtained for human hemoglobin (dots) and the resulting smoothing curve calculated by Epanechnikov kernel (line). A distinct kink is visible at ∼36°C, representing the onset of accelerated particle size increase.

FIGURE 3

Fractional change of particle size with temperature for oxyhemoglobin solutions derived from DLS data. Data points were averaged from the original data obtained from three to four solutions of each type of hemoglobin. The error bars represent the standard deviation of the respective fractional changes. Straight lines show best approximation (R²→1) of two distinctly linear parts of the obtained L-shaped curves. The transition temperature interval may be defined as located close to the intersection point of best-approximating trend lines and is marked as T_c (critical temperature) for each species. Gray boxes show physiological body temperature range for each species.

FIGURE 4

Amino acid sequence alignments of human, platypus, and echidna (minor α-chain marked as echidna-m) HBA and HBB sequences (28) (SwissProt Database). Additionally, a structure genetic matrix was used (29). Asterisks indicate identical residues, dots indicate similar residues, and exclamation marks indicate nonsimilar residues. The gray parts indicate “hot spots” in the Hb molecule where most substitutions take place.