Oxygenation properties and isoform diversity of snake hemoglobins 1 2 3

27 Available data suggest that snake hemoglobins (Hbs) are characterized by a combination of unusual 28 structural and functional properties relative to the Hbs of other amniote vertebrates, including 29 oxygenation-linked tetramer-dimer dissociation. However, standardized comparative data are lacking for 30 snake Hbs, and the Hb isoform composition of snake red blood cells has not been systematically 31 characterized. Here we present the results of an integrated analysis of snake Hbs and the underlying α32 and β-type globin genes to characterize (i) Hb isoform composition of definitive erythrocytes, and (ii) the 33 oxygenation properties of isolated isoforms as well as composite hemolysates. We used species from 34 three families as subjects for experimental studies of Hb function: South American rattlesnake, Crotalus 35 durissus (Viperidae); Indian python, Python molurus (Pythonidae); and yellow-bellied sea snake, Pelamis 36 platura (Elapidae). We analyzed allosteric properties of snake Hbs in terms of the Monod-Wyman37 Changeux model and Adair four-step thermodynamic model. Hbs from each of the three species exhibited 38 high intrinsic O2-affinities, low cooperativities, small Bohr factors in the absence of phosphates, and high 39 sensitivities to ATP. Oxygenation properties of the snake Hbs could be explained entirely by allosteric 40 transitions in the quaternary structure of intact tetramers, suggesting that ligation-dependent dissociation 41 of Hb tetramers into αβ dimers is not a universal feature of snake Hbs. Surprisingly, the major Hb isoform 42 of the South American rattlesnake is homologous to the minor HbD of other amniotes and, contrary to the 43 pattern of Hb isoform differentiation in birds and turtles, exhibits a lower O2-affinity than the HbA 44 isoform. 45 46