title: UV resonance raman spectra of ligand binding intermediates of sol-gel encapsulated hemoglobin.
abstract: We report for the first time specific conformational changes for a homogeneous population of ligand-bound adult deoxy human hemoglobin A (HbA) generated by introducing CO into a sample of deoxy-HbA with the effector, inositol hexaphosphate, encapsulated in a porous sol-gel. The preparation of ligand-bound deoxy-HbA results from the speed of ligand diffusion relative to globin conformational dynamics within the sol-gel (1). The ultraviolet resonance Raman (UVRR) difference spectra obtained reveal that E helix motion is initiated upon ligand binding, as signaled by the appearance of an alpha14beta15 Trp W3 band difference at 1559 cm(-1). The subsequent appearance of Tyr (Y8a and Y9a) and W3 (1549 cm(-1)) UVRR difference bands suggest conformational shifts for the penultimate Tyralpha140 on the F helix, the "switch" region Tyralpha42, and the "hinge" region Trpbeta37. The UVRR results expose a sequence of conformational steps leading up to the ligation-induced T to R quaternary structure transition as opposed to a single, concerted switch. More generally, this report demonstrates that sol-gel encapsulation of proteins can be used to study a sequence of specific conformational events triggered by substrate binding because the traditional limitation of substrate diffusion times is overcome.
authors.affiliation: Department of Physiology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors.forename: L J
authors.initials: LJ
authors.lastname: Juszczak
authors.forename: J M
authors.initials: JM
authors.lastname: Friedman
mesh_terms: D000328:Adult; D001665:Binding Sites; D002263:Carboxyhemoglobin / Q000737:chemistry*; D005782:Gels; D006441:Hemoglobin A / Q000737:chemistry*; D006801:Humans; D008024:Ligands; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman / Q000379:methods
chemical_list: D005782:Gels; D008024:Ligands; D006441:Hemoglobin A; D002263:Carboxyhemoglobin