Soret spectral and bioinformatic approaches provide evidence for a critical role of the α-subunit in assembly of tetrameric hemoglobin

Soret spectral contributions of the alpha-subunit heme pocket have been evaluated by performing static titrations of apohemoglobin A with CNProtohemin under varied experimental conditions. Increasing the temperature from 5 to 30 degrees C in 0.05 M potassium phosphate buffer, pH 7, resulted in a decreasingly prominent hypsochromic shifts reflecting altered the vinyl-globin interactions. Studies at 10 degrees C in over pH range of 6.7-8.0 revealed a profile for the spectral shifts approximating the side chain pK value (7.4) a histidyl residue. These overall spectral changes correspond to Delta E of <= 7 kJ/mol indicative of electrostatic noncovalent interactions. Further our current molecular modeling studies indicate that the spatial arrangement and critical noncovalent interactions of tyrosine 42 and histidine 45 (aromatic residues unique to the alpha-subunit) make significant contribution to the Soret spectra. Most interestingly, phylogenetic analyses have revealed the presence of a histidyl triad in the alpha-chain of all vertebrates that form heterotetramers.