Publication Date


Document Type


First Advisor

Erman, James E.

Degree Name

M.S. (Master of Science)

Legacy Department

Department of Chemistry


Hemoglobin; Erythrocytes; Glycera dibranchiata


In the marine annelid Glycera Dibranchiata the physiological functions of oxygen transport and storage are carried out by hemoglobin contained in nucleated coelemic erythrocytes. The low molecular weight or monomer hemoglobin- fraction derived from the red cells was characterized using ion exchange chromatography, analytical isoelectric focusing, uv-vis spectrophotometry and proton nmr spectroscopy . Five monomer hemoglobins and significant non-heme protein were seen in 395nm and 280nm elution profiles of pH 6.8 preparative cation exchange columns eluted with ionic strength gradients. Isocratic elution conditions at the same pH achieved greater resolution and more extensive hemoglobin heterogeneity was suggested. Heme-containing cation exchange fraction I was further fractionated on a DEAF cellulose anion exchange column. Spectrophotometric analysis of the resulting fractions revealed a heme protein whose visible spectrum was characteristic of a cytochrome and presumably nucleic acid component with a strong anionic character and a spectral maximum at 260nm. Analytical isoelectric focusing (IEF) of the monomer fraction confirmed both the protein heterogeneity and the presence of several major hemoglobin variants. On wide range (pH 3.5-9.5) IEF gels the isoelectric order of the hemoglobin bands corresponded to the net charge separation on the cation exchange column with the exception of hemoglobin fraction IV. The fraction IV hemoglobin, which foc-used as a single homogeneous band on wide-range gels, migrated to the same posiiton as the primary hemoglobin of fraction III despite their complete resolution on the iso- cratic cation exchange column. However, the hemoglobin fractions had characteristic proton nmr spectra when oxidized with ferricyanide. Moreover, high resolution nmr is shown to be a reproducible method of distinguishing the monomer variants, and the significance unequivocal assignment of the Glycera monomer hemoglobins has to previous studies is discussed. NMR was further shown to provide an assessment of heme-protein purity. Proton spectra of fraction IV at 200 MHz and 470 MHz indicate a single heme component corroborating the IEF evidence of homogeneity. Preliminary nmr studies of purified fraction IV monomer hemoglobin investigated the pH and temperature dependence of the hyperfine shifted heme methyl resonances. In the pH range studied, two downfield resonances exhibited what appeared to be a coalescence with decreasing pH. All other shifts were essentially constant with pH. While all resonances exhibited Curie behavior, a temperature dependent splitting of one methyl resonance, shown to have an integrated intensity of three protons, suggested the hindered rotation of this group in the hemoglobin molecule. The equilibrium binding of the neutral ligand imidazole was studied by visible difference spectroscopy and nmr spectroscopy. The pH dependence of the association constant was compared to a previous investigation of imidazole binding to sperm whale myoglobin. The discussion of results focused on the different distal histidine residue of the two heme proteins.


Bibliography: pages 162-164


viii, 164 pages




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