A Study On The Structure And Function Of Hemoglobin In Two Phrynocephalus Lizards From Different Altitudes

In the past decades, scientists have found variety of adaptation features and coordination mechanism to high-altitude hypoxia in mammals, birdsand reptiles. As yet, Phrynocephalus erythrurus a native species of Qiangtang plateau (mostly4500-5300m above sea level) is considered to be the highest living reptile in the world. However, the adaptation mechanisms to high altitude in this mysterious species are still unknown.In this study, two species of lizard from the genus Phrynocephalus were collected and used for experiments. Phrynocephalus erythrurus was collected from Tuotuo River (34°13’N,92°13’E), Phrynocephalus przewalskii was collected from Minqin (38°38’N,103°5’E), Gansu province, China. We mainly studied the structure and function of hemoglobin in both species, including hematological parameters, whole blood oxygen dissociation curves and P50values, the expression of α-and β-globin gene in adult and the β-globin gene sequence of the two species.The results showed that hemoglobin content and red blood cell count in Phrynocephalus erythrurus were significantly higher than Phrynocephalus przewalskii (p<0.001), and the mean corpuscular volume of the two species was not significantly different (p>0.05). Whole blood oxygen dissociation curve of Phrynocephalus erythrurus was on the left of curve of Phrynocephalus przewalskii, and the P50value of Phrynocephalus erythrurus was obviously lower than Phrynocephalus przewalskii (p＜0.001). We used reverse phase high-performance liquid chromatography (RP-HPLC) and electrospray ionization mass spectrometry (ESI-MS) to analysis the expression of hemoglobin gene of the two species, the results showed that the expression of four globins was seen in both Phrynocephalus erythrurus and Phrynocephalus przewalskii and the chromatography map and molecular mass (Protein peaks1,2,3and4were15905.2Da,16269.3Da,16063.4Da and15633.2Da in Phrynocephalus erythrurus and15916.0Da,16288.2Da,15633.0Da and16061.3Da in Phrynocephalus przewalskii, respectively) of Hb of the two species were obvious different. We also identified the amino acids substitution sites of the β-chain in two species by the molecular cloning methods. We speculated that both of the two species expression of two α-globin chains and two β-globin chains (named α1,α2, β1and β2, respectively). Alignment analysis showed that amino acid sequence of β1-globin chains in two species was slightly different, there was only one amino acid alternative (β112Ser in Phrynocephalus erythrurus and β112Thr in Phrynocephalus przewalskii). However, amino acid sequence of β2-globin chains was obviously different in two species, there were24amino acids alternative with each other.Finally, we concluded that increasing hemoglobin content and red blood cell count are the strategies for Phrynocephalus erythrurus to adapt to high altitude hypoxia environment, and this response was interpreted as advantageous because blood with more hemoglobin could carry more oxygen to offset the sparser pool of oxygen molecules in hypoxia condition. We suggest that β2-golbin chain was one of the main factors of the Hb of Phrynocephalus erythrurus in the adaption to high altitude hypoxia environment, and detailed mechanism of adaption to high altitude in this species needs further study.