7.5.1 Basic principles
Nuclear magnetic resonance (NMR) is the spectroscopic method used to observe nuclear-spin reorientation in an applied magnetic field. Various applications of NMR in biochemistry include structural identification of biomolecules, chemistry of individual groups in macromolecules, structural and dynamic information of biomacromolecules, metabolic studies, as well as kinetic and association constants of ligand bindings to macro-molecules (Evans, 1995; Roberts, 1993; Stassinopoulou, 1994). The technique monitors the absorption of energy associated with transition of nuclei between two nuclear magnetic energy levels. The NMR phenomenon is observable because certain nuclei behave like tiny spinning bar magnets. The spinning nucleus generates a magnetic field and thus has an associated magnetic moment, which interacts with the applied field. Most important among such nuclei in biomacromolecular applications are and 13C having nuclear spin values (I) of 1/2 (Table 7.9). The corresponding NMR, namely proton magnetic resonance (PMR) and 13C-magnetic resonance (CMR) will be considered in this section.
The sensitivity of the 13C nucleus is 1.6% that of for equal numbers of nuclei in the same magnetic field. By taking into account of its natural abundance of only 1.1%, this reduces the relative sensitivity of 13C as being roughly 1.8 x 10-4 that of *H. However, CMR has several advantages. First, in most cases, the chemical shifts of 13C occur over a much broader range than chemical shifts of 1H. The large range of 13C chemical shifts permits separate visualization of many individual nuclei in various monomers of biomacromolecules. Second, CMR spectra are expected to be first-order, i.e. no 13C-13C spin-spin couplings would normally be detected because 13C is only 1.1% abundant so that most neighboring nuclei are nonmagnetic 12C. Third, 13C can be inserted into a molecule at a specific locus to replace 12C. In this way, a site-specific magnetic probe is obtained that does not perturb the structure. However, 13C shifts are much less sensitive to the environment than are 1H shifts. Therefore it is more difficult to explore conforma-tional changes and effects of ligand interactions.
Nuclei with a net magnetic dipole such as 1H and 13C will orient the dipole axis in an external magnetic field in certain quantized orientations. The number of possible orientations is given by 2I + 1. If a nucleus with I = 1/2 is placed in a uniform magnetic field, it may take up one of two orientations with respect to the field (the external magnetic field H defines the z-axis). Those may be considered as a low-energy orientation in which the nuclear magnet is aligned with the field (having quantum numbers ms = +1/2), and those referred to as a high-energy orientation in which the magnet is aligned against the field (having quantum numbers ms = -1/2). The transition between these two energy states can be brought about by the absorption of suitable electromagnetic radiation of energy.
The nuclear magnetization has direction (i.e. vector). The component, Mz, which is defined to be along the applied magnetic field (Ho) direction, and the components Mx and My, at right angles to Ho. The Mxy components arise because the spins do not align perfectly along Ho. At equilibrium, the spins are randomly distributed and the net Mxy = 0. On application of a rotating radiofrequency field with frequency at or near precession frequency, the
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