Structural and functional characterization of Delphinus delphis hemoglobin system

Structural analysis of the hemoglobin (Hb) system of Delphinus delphis revealed a high globin multiplicity: HPLC-electrospray ionization-mass spectrometry (ESI-MS) analysis evidenced three major beta (beta 1 16,022 Da, beta 2 16,036 Da, beta 3 16,036 Da, labeled according to their progressive elution times) and two major alpha globins (alpha 1 15,345 Da, alpha 2 15,329 Da). ESI-tandem mass and nucleotide sequence analyses showed that beta 2 globin differs from beta 1 for the substitution Val126 -> Leu, while beta 3 globin differs from beta 2 for the isobaric substitution Lys65 -> Gln. The alpha 2 globin differs from the alpha 1 for the substitution Ser15 -> Ala. Anion-exchange chromatography allowed the separation of two Hb fractions and HPLC-ESI-MS analysis revealed that the fraction with higher pI (HbI) contained beta 1, beta 2 and both the alpha globins, and the fraction with lower pI (HbII) contained beta 3 and both the alpha globins. Both D. delphis Hb fractions displayed a lower intrinsic oxygen affinity, a decreased effect of 2,3-BPG and a reduced cooperativity with respect to human HbA(0), with HbII showing the more pronounced differences. With respect to HbA(0), either the substitution Pro beta 5 -> Gly or the Pro beta 5 -> Ala is present in all the cetacean beta globins sequenced so far, and it has been hypothesized that position 5 of beta globins may have a role in the interaction with 2,3-BPG. Regarding the particularly lowered cooperativity of HbII, it is interesting to observe that the variant human HbA, characterized by the substitution Lys beta 65 -> Gln (HbJ-Cairo) has a decreased cooperativity with respect to HbA(0).