Some functional and structural properties of Bufus paracnemis and Pipa pipae hemoglobins

1. Hemoglobin from terrestrial and an aquatic amphibia Bufo paracnemis and Pipa pipae respectively both living in the same region (Belém, Pará) were compared. 2. The number of hemolysate components were determined by starch-gel electrophoresis and CMC-chromatography. P. pipae hemoglobin presented 4 components and B. paracnemis 2, all of anodic mobility. 3. Functional properties of the hemoglobins (oxygen affinity. Bohr effect, carbon monoxide equilibrium) were determined and compared. 4. The differences found in the functional properties were correlated with the different habitat: aquatic or terrestrial for the amphibia. 5. The study of the oxygen functional properties of the hemoglobin showed in the stripped proteins of P. pipae in the presence of ATP, an oxygen affinity (P50 = 7.24 mmHg) that of B. paracnemis at the same pH (P50 = 15.84 mmHg). At high pH the P50 values are different being 15.84 mmHg for the terrestrial frog and 5 mmHg for P. pipae haemoglobin both at pH 8. In addition Bohr effect was noted only in P. pipae hemolysate in the presence of ATP. 6. The CO-equilibrium affinity constant in the presence of ATP are similar in both frogs, of about, log C50 = -6.9. The ratio Pco/Po2 for B. paracnemis hemoglobin was about 300 whereas for that of P. pipae was about 100 only. 7. The kinetic study of reactive sulfhydryl groups in both frog hemoglobin with 4-PDS (specific SH group reagent was used and shown high pseudo-first order constant for B. paracnemis hemoglobin) (k' = 0.46/min) either in presence or not of ATP, compared for that of P. pipae where values were K' = 0.003/min in the stripped protein and k' = 0.014/min in the presence of ATP. 8. Denaturation kinetic studies of the hemoglobin with sodium benzoate was performed and the results were compared with that of Rana catesbeiana, i.e. the pseudo-first order of the hemoglobin denaturation reaction are low for the aquatic P. pipae adult although for the B. paracnemis high molecular resistant was also noted. For P. pipae hemoglobin, nevertheless with ATP such resistance becomes higher. This does not occur with B. paracnemis hemoglobins.