A tri state mechanism for oxygen release in fish hemoglobin: Using Barbus sharpeyi as a model

Abstract

Hemoglobin is a porphyrin containing protein with an α2β2 tetrameric structure and like other porphyrin compounds shows spectral behavior of species specific characteristics. Researchers tend to relate bands in the hemoglobin spectra to certain structural and/or functional features. Given the fact th↔at hemoglobin is the main oxygen carrier in animals functioning through the Oxy↔Deoxy equilibrium, the determination of oxy and deoxy conformations of hemoglobins of different animals may shed light on their oxygen binding properties. Absorption spectra at 280 and 373nm have been widely used to quantitate the formation of hemoglobin deoxy conformation. In the present work, however, we used an optical density ratio of OD373/OD280 as an index for deoxy formation. This ratio was determined for Barbus sharpeyi and human hemoglobins at different SDS concentrations, pH levels and temperatures to compare them from a structure-function point of view. Our data showed that under low concentrations of SDS (<2mM) Barbus sharpeyi hemoglobin folds in a tri-state pattern while human hemoglobin folds through a two-state phenomenon. This finding indicates that in contrast to those of other non aquatic animals, the hemoglobin of Barbus sharpeyi has a loosely folded tetrameric structure with remarkably more oxygen affinity.

Keywords: Barbus sharpeyi; Hemoglobin; Tri State Mechanism.

Figure 1

UV spectra of BS and human hemoglobin obtained in phosphate buffer (pH 7 and 50 mM at 37°C)

Figure 2

The far CD spectra of BS and human hemoglobin (0.2 mg/ml Hemoglobin dissolved in 50 mM phosphate buffer at pH 7.3 and 37°C

Figure 3

thermal denaturation of fish and human hemoglobin monitored by far-UV CD spectroscopy and presented as θ222(T) curve in 50 mM phosphate buffer, pH 7.3

Figure 4

CD spectra of human hemoglobin (curve a) and BS hemoglobin (curve b) in the absence and presence of 1.5 mM SDS

Figure 5

a) the fluctuation of $\frac{{\mathit{OD}}_{373}}{{\mathit{OD}}_{280}}$ ratios obtained for BS and human hemoglobin samples in phosphate buffer (50mM and pH7 at 37°C), b) The plot of calculated $\frac{∆\mathit{ratio}}{∆\left[\mathit{SDS}\right]}$ from the of graph a against SDS concentration.

Figure 6

the plot of $\frac{{\mathit{OD}}_{373}}{{\mathit{OD}}_{280}}$ ratios obtained at different pH for BS and human hemoglobin