Natural-abundance nitrogen-15 nuclear magnetic resonance spectroscopy of enzymes and other biopolymers is found to be feasible using newly available instrumentation. The long correlation times of such molecules result in short spin-lattice relaxation times, and these in turn allow rapid signal accumulation. The advantages of short T1 values are sometimes offset, however, by unfavorable nuclear Overhauser effects. The dependence of T1 and nuclear Overhauser effects upon correlation time is discussed, and preliminary nitrogen-15 nuclear magnetic resonance results for several biopolymers, including Iysozyme, protamines, pepsin, hemoglobin, vitamin B12, and tRNA, are presented.Nitrogen is widely distributed in molecules of biochemical interest, and nitrogen atoms are often intimately associated with sites of biological activity and major structural features. Nitrogen-15 nuclear magnetic resonance (NMR) spectroscopy is therefore, in principle, a promising tool for the study of biochemical systems, especially in view of the greater range of chemical shifts for nitrogen as compared to hydrogen and carbon. Until the present, however, 15N NMR studies of large molecules at the natural-abundance level have been severely limited by problems of sensitivity. Although 15N has a spin of 'A, and therefore gives rise to sharp resonances, its relative NMR sensitivity for equal numbers of nuclei is only 1.04 X 10-3 times that of the proton. In addition to a small magnetogyric ratio, the natural abundance of 15N is 0.37%. Thus, at the natural-abundance level, the sensitivity of 15N is 3.8 X 10-6 that of the proton. This results in a difference of about 1011 in the time required to obtain a given signal-tonoise ratio at constant field. The sensitivity problem is often exacerbated by problems with nuclear Overhauser effects and relaxation times as will be detailed below.
The effects of changes in the groups attached to the periphery of the porphyrin ring of the heme of various hemoglobin and myoglobins on the environment experienced by the ligand, carbon monoxide, have been studied by observation of the chemical shift of the bound 13CO. The results indicate that the major interaction between bound ligands and substituents around the porphyrin is that transmitted electronically from substituent to ligand. The nature of the protein environment around the ligand and the interaction between the proximal histidine (F8) and the ligand (through the iron atom) impose differences between subunits of hemoglobin and between myoglobins and hemoglobins which are largely, but not entirely, independent of these substituent effects. To assess the influence of protein structure on the chemical shifts of bound ligand, the shifts of 13CO bound to myoglobin and hemoglobins from a wide range of species have also been measured.
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