The reactive sulfhydryl group on Cys β93 in human adult hemoglobin (HbA) has been the focus of many studies because of its importance both as a site for synthetic manipulation and as a possible binding site for nitric oxide (NO) in vivo. Despite the interest in this site and the known functional alterations associated with manipulation of this site, there is still considerable uncertainty as to the conformational basis for these effects. UV resonance Raman (UVRR) spectroscopy is used in this study to evaluate the conformational consequences of chemically modifying the Cys β93 sulfhydryl group of both the deoxy and CO-saturated derivatives of HbA using different maleimide and mixed disulfide reagents. Included among the maleimide reagents are NEM (n-ethylmaleimide) and several poly(ethylene glycol) (PEG)-linked maleimides. The PEG-based reagents include both different sizes of PEG chains (PEG2000, -5000, and -20000) and different linkers between the PEG and the maleimide. Thus, the effect on the conformation of both linker chemistry and PEG size is evaluated. The spectroscopic results reveal minimal perturbation of the global structure of deoxyHbA for the mixed disulfide modification. In contrast, maleimide-based modifications of HbA perturb the deoxy T state of HbA by "loosening" the contacts associated with the switch region of the T state α1β2 interface but do not modify the hinge region of this interface. When the NEM-modified HbA is also subjected to enzymatic treatment to remove the C-terminal Arg α141 (yielding NESdes-ArgHb), the resulting deoxy derivative exhibits the spectroscopic features associated with a deoxy R state species. All of the CO-saturated derivatives exhibit spectra that are characteristic of the fully liganded R structure. The deoxy and CO derivatives of HbA that have been decorated on the surface with large PEG chains linked to the maleimide-modified sulfhydryl through a short linker group all show a general intensity enhancement of the tyrosine and tryptophan bands in the UVRR spectrum. It is proposed that this effect arises from the osmotic impact of a large, close PEG molecule enveloping the surface of the protein.
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