Abstract

FTIR studies of the effects of BPG and IHP binding on the p(SH) stretching vibrations of human deoxyhemoglobin A (deoxyHbA) between 25 and 75{493}C reveal that the effectors stabilize the p(SH) peak of Cysβ93 at 2576 cm -1 at high temperatures (55{493}C or above). In the presence of the effectors, the p(SH) peak of Cys{460}104 at 2558 cm -1 is broadened, and the p(SH) shoulder of Cys{460}112 at 2564 cm -1 fluctuates in intensity with increasing temperature, indicating that the effectors destabilize the {460} 1 β 1 interface of the deoxyHbA tetramer (2{460}2β). At 25{493}C, the Soret CD bands of 0.5 mM effector-bound deoxyHbA are observed at 418 and 432 nm with ellipticities of -2.4 and +28 M -1 cm -1 , respectively. These signals are intensified by 2-5 units at 55{493}C. In contrast, effector-free deoxyHbA loses {598}50% of its Soret CD absorption and {598}10% of its unpolarized Soret absorption on heating to 55{493}C. Effector binding also stabilizes the 287-nm negative CD band and the three-valley CD absorption between 250-270-nm, which reflect changes at the {460} 1 β 1 interface. FTIR-monitored thermal unfolding of 3.0 mM deoxyHbA reveals that loss of the {460}-helical absorption at 1653 cm -1 occurs in three phases. These may be associated with tetramer [arrow right] dimer, dimer [arrow right] monomer and monomer [arrow right] denatured-monomer transitions. In the presence of BPG and IHP, the FTIR denaturation curves are shifted by {598}5{493}C to lower temperature, indicating that effector binding destabilizes the secondary structure of deoxyHbA.