Hemoglobin allosteric studies by vibrational spectroscopy: Fourier transform infrared and resonance Raman approaches

Hemoglobin (Hb) is a topic that has been pursued by scientists for more than a century. One hope behind the fundamental research is to develop blood substitutes based on hemoglobin. Blood donated by humans need be refrigerated, can be contaminated by such diseases as AIDS and Hepatitis, and is often in short supply. Designers of blood substitutes hope to eliminate these problems and develop genetically engineered or chemical products that will be tolerated by people of all blood types.; The projects in this dissertation have focused on fundamental studies of structure and function relationship of hemoglobin by various chemical and biological techniques, such as mutation and isotope labeling. The tools involved are resonance Raman (RR) and Fourier Transform Infrared (FTIR) spectroscopy.; Proximal histidine signals have been identified from microperoxidase −11 following NH/D exchange of the imidazole. UVRR spectroscopy therefore offers promise for monitoring histidine ligation in heme proteins. Heme-bound histidine UVRR bands of hemoglobin and other heme proteins are characterized. The dissertation also presents assignments of tryptophan bands in hemoglobin (Hb) and horseradish peroxidase (HRP).; New UVRR instrument performance characteristics with a kilohertz solid-state laser source are described. Narrow line excitation in the 205–230 nm region is provided by quadrupling a titanium:sapphire laser which is pumped by the second harmonic of a Q-switched YLF laser. Its applications to time-resolved resonance Raman are also presented in detail.; The studies of the hemoglobin allosteric transition from the R to the T state involves a newly developed technique—Step-scan Fourier transform infrared (FTIR) spectroscopy. Hemoglobin ligand dissociation kinetic analysis by step-scan FTIR is reported and intermediates in the allosteric pathway of the R-T transition of Hb are identified.