The Oxygenation Properties Of Hemoglobin In Myospalax Baileyi And The Underlying Molecular Mechanisms

Myospalax baileyi?The plateau zokor?is a one of subterranean rodents native to Qinghai-Tibet Plateau.Belonging to genus Myospalax family of Spalacidae,they spend nearly entire life underground in sealed burrows with hypoxia-hypercapnia.Many studies have been done to explore the adaptation mechanism of the plateau zokor to hypoxia-hypercapnia environment from the aspects of morphology,physiology,biochemistry and molecular biology,etc.Hemoglobin transports oxygen and carbon dioxide in the vertebrate blood and plays an important role in the adaptation of animals to extreme environments.To explore the adaptation mechanism of the plateau zokor hemoglobin to hypoxia-hypercapnia environment,an oxygen dissociation curve measuring apparatus has been developed according to the?modified diffusion chamber?method.Then the O₂ equilibrium of the purified hemoglobin were performed at different pH,temperature and CO₂ content in the absence?stripped?and present of Cl^-and DPG?two major anion allosteric effectors of Hb-O₂ affinity in the red cells?with mouse hemoglobin as control.The Hb-O₂ binding properties of the plateau zokor were compared with mouse hemoglobin,including its sensitivities to Cl^-,DPG,high CO₂,temperature and their Bohr effects.In addition,the structural model of deoxyhemoglobin in the plateau zokor was constructed by homology modeling using the crystal structure of mouse deoxyhemoglobin?3HRW.pdb?as template.Then molecular dynamics simulations were performed on templates and models to investigate the structural characteristics of the plateau zokor hemoglobin.Combined with O₂ binding properties of the plateau zokor hemoglobin,the molecular underpinnings of the adaptation of Hb-O₂ binding properties to hypoxia-hypercapnia were studied.The results of Hb-O₂ binding properties showed that the plateau zokor hemoglobin has remarkably high intrinsic oxygen affinity,and the P50 of the plateau zokor hemoglobin are smaller than mice under all the same experimental conditions.Under conditions close to normal body temperature and pH,ie 37°C and pH 7.6,the plateau zokor hemoglobin is less sensitive to Cl^-and DPG than mouse under the same conditions.The presence of 5%CO₂ further reduced the sensitivity of these two kinds of hemoglobins to Cl^-and DPG.However,at low temperature?25??and low pH,the plateau zokor hemoglobin has a high anion sensitivity similar to that of mouse hemoglobin.The stripped Hb of the plateau zokor is slightly less sensitive to 5%CO₂ than mouse,while the plateau zokor hemoglobin is more sensitive to 5%CO₂ than mouse hemoglobin in near-physiological conditions?both Cl^-and DPG were presented?.The Bohr effects of hemoglobin in the plateau zokor and mouse are less than that in other mammal hemoglobin under similar experimental conditions,and there are no significant differences between them.Therefore,the plateau zokor hemoglobin is insensitive to pH changes.Although the intrinsic temperature sensitivity of the plateau zokor hemoglobin is higher than that of mouse,it's still similar to that of animals with low temperature sensitivity,and the hemoglobin of the plateau zokor has significant low temperature sensitivity when both Cl^-and DPG were presented.Based on the results of Hb-O₂ binding properties of the plateau zokor,we suggested that the elevated Hb-O₂ affinities are mainly attributed to its remarkably high intrinsic Hb-O₂ affinity combined with a relative suppressed anion sensitivities?under normal temperature,pH,and high CO₂ conditions?.High sensitivities to anion allosteric effectors at low temperature and low pH accelerate the releasing of O₂ in peripheral tissues of the body.The relatively high CO₂ effects?both Cl^-and DPG were present?elevate the O₂ releasing capacity and CO₂ carrying capacity of the plateau zokor hemoglobin in metabolic tissues where CO₂ content is higher.The low temperature sensitivity of the plateau zokor hemoglobin ensures steadily delivering O₂ to the cold peripheral tissues,while reducing the loss of heat in the body.Although the low Bohr effect of the plateau zokor hemoglobin is not conducive to O₂ releasing in aerobic tissues,the adaptive changes in other O₂-binding properties mentioned above and the increased microvessel density in metabolic tissues are act as the trade off of high intrinsic Hb-O₂ affinity to accelerate O₂unloading.The main molecular underpinnings of the intrinsic high Hb-O₂ affinity in the plateau zokor is the decrease of hydrogen bonds between?1?1 and?2?2 semirigid dimers interface.Specifically,amino acid substitution of the 131Ser?Asn on the?2 chain weakens the connection between the two semirigid dimers,thus reducing the space resistance of T/R quaternary structural transition.These two effects work together making the plateau zokor has remarkably high intrinsic Hb-O₂affinity.Furthermore,131 postion of?subunit is the key Cl^-binding site?between 131Ser and1Val?,substitution at this site may be the reason of lower Cl^-sensitivity of the plateau zokor hemoglobin compared with mouse hemoglobin at normal temperature and pH.Simultaneously,this mutation can promote the binding of CO₂ to N-terminal Val,thus leads to the high sensitivity of plateau zokor hemoglobin to CO₂.With regard to the suppressing sensitivity to DPG,it may be attributed to the?4Thr?Ser and?5Asp?Gly substitutions near the DPG binding site at?chains.Another mechanism of the high Hb-O₂ affinity of the plateau zokor may be the stabilization of dimer internal structure caused by the substitution of nonpolar Ala to polarity Asn at?111 and the substitution of Gly?Ala at?115,which generate an additional hydrogen bond in interdimer??1?1and?2?2?and increase the hydrogen bonds between?and?chain in each dimer.Lastly,compared with mouse hemoglobin,the amino acids line the opening of heme pockets of the?1,?2and?2 chains of plateau zokor hemoglobin have higher flexibility,which may be beneficial to O₂entering and leaving heme pockets.This may be another molecular mechanism of high intrinsic Hb-O₂ affinity of the plateau zokor.