Spectroscopic methods are used in the structural characterization of biomolecules (Bell, 1981; Campbell and Dwek, 1984; Greve et al., 1999; Hammes, 2005). These methods are usually rapid and noninvasive, require small amount of samples, and can be adapted for analytical purposes. Spectroscopy is defined as the study of the interaction of electromagnetic radiation with matter, excluding chemical effects (photochemistry refers to the interaction with chemical effects). The electromagnetic spectrum covers a wide range of wavelengths (Figure 7.1).
The interaction of electromagnetic radiation with matter gives rise to scattering, absorption or emission of the radiation. Scattering is usually detected by measuring the intensity of radiation at some angle 0 to the incident wave (turbidity refers to measuring the reduced transmitted light at 0 = 0). Electrons are the usual scatterers in molecules, while nuclei scatter neutrons. If the scattered radiation has the same frequency as the incident radiation, the scattering is said to be elastic (conservation of energy), otherwise the scattering is inelastic (changes in frequency). Three cases will be considered:
1. Refraction and reflection: Refraction results when light is scattered in the same direction as that of the incident light. The wavelength (X) of the incident radiation is much greater than the dimension of large arrays of essentially rigid particles, e.g. crystals, very little scattering is observed other than 0 = 0. Reflection then results when light is scattered in the direction opposite to that of the incident light.
2. Diffraction: When the wavelength of the incident radiation is much less than the dimension of arrays in the crystals, three-dimensional interference patterns usually called diffraction patterns are generated. The diffraction pattern gives information about the lattice and the constituent molecules of the array.
3. Solutions: When the dimension of a particle in solution is much less than X, scattering observed at 0 = 0 is related to the concentration and size of the particle. When the dimension of the scatter is greater than X, angular dependent scattering can be measured to provide information about the size and shape of the particle.
Absorption is usually measured by varying the frequency (or wavelength) of the applied radiation. The frequency dependence of absorption arises because energy is
Biomacromolecules, by C. Stan Tsai Copyright © 2007 John Wiley & Sons, Inc.
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X_rays vis microwave (NMR)
Figure 7.1 Electromagnetic radiation and its corresponding spectra absorbed by transition induced between different energy states of the molecules in the sample. Each molecule is associated with many types of energies of which the most important are:
Etotal Eelectronic + Evibration + Erotation + Etranslation + Eelectron spin orientation + Enuclear spin orientation
Each of these energies is quantized into energy levels that are characteristic states of the molecule. The ground state is defined as the state of the lowest energy and states of higher energy are called excited states, that is, they are said to be degenerate if two or more states of the molecule have the same energy. A molecule will absorb radiation only when the frequency (v) of the radiation is related to the energy difference (AE) between two energy levels by the equation:
AE = hv, where h is Planck's constant (6.67 x 10-27ergs). The frequency is related to the wavelength by v = c/^, in which c is velocity of light in vacuum (3 x 1010 cm s-1). Another factor (selection rule) for predicting the transition probability is that there must be a charge displacement between one energy state to another. Only those components of the electromagnetic radiation that are in the same direction as the transition dipole moment will cause transitions. If the angle between the direction of the applied wave and the direction of the transition moment is 0, then the effective value of the transition moment is proportional to cos 0 and the transition probability is proportional to cos2 0. Two types of charge displacements during the transition are electric transition dipole (|ie) and magnetic transition dipole (in). The applied electromagnetic radiation interacts with these dipoles to cause changes in energy states; the electric component interacts with | e and the magnetic component interacts with | m, and the larger the transition dipole moment, the larger will be the transition probability.
The selection rules help to predict the probability of a transition but are not always strictly followed. If the transition obeys the rules it is allowed, otherwise it is forbidden. A molecule can become excited in a variety of ways, corresponding to absorption in different regions of the spectrum. Thus certain properties of the radiation that emerges from the sample are measured. The fraction of the incident radiation absorbed or dissipated by the sample is measured in optical (ultraviolet and visible) absorption spectroscopy and some modes of nuclear magnetic resonance spectrometry (NMR). Because the relative positions of the energy levels depend characteristically on the molecular structure, absorption spectra provide subtle tools for structural investigation.
Emission of radiation is measured at some angle 0 to the incident beam as a molecule changes from an excited energy state to a lower energy state. A molecule can change its energy from a higher excited state to lower one by three processes:
1. Simulated emission: A light amplification by stimulated emission of radiation (LASER) operates in systems where a nonequilibrium distribution of energies is created by a pump that induces transition to a higher excited state. As a result, the emission of some radiation is made to stimulate a cascade of emission. This emission will stop when the equilibrium to the population of energy states is returned.
2. Thermal (radiationless) emission: The common way for a molecule to return to a lower energy state is by the liberation of heat via collision, vibration and molecular motion in the intermolecular and intramolecular de-excitation processes.
3. Spontaneous emission: The molecule acts an oscillator and radiates its energy hv without any other interaction with its environment.
The measurement of the emitted radiation at a wavelength other than that used for the excitation form the basis for fluorescence, phosphorescence and Raman scattering spectroscopies.
In addition to the intensity, other properties such as polarization are concerned in optical rotatory dispersion and circular dichroism. The various processes give rise to different spectroscopic methods, as summarized in the Table 7.1. Various spectra (UV, IR, NMR and MS) of simple biomolecules can be accessed from Spectral Database Systems (SDBS) of the National Institute of Material and Chemical Research, Japan at http://www.sist.go.jp/RIODB/SDBS/menu-e.html.
Several factors must be considered for a particular biomacromolecular structure application that will affect the choice of spectroscopic methods. These include structural resolution necessary, chemical nature of biomacromolecule (protein, nucleic acid, or glycan), amount/concentration of biopolymer available, sample preparation (solid or solution), solvents of interest, and desired structure information (secondary or tertiary structure). Structural resolution varies considerably for the various spectroscopic methods, with X-ray diffraction and NMR providing atomic resolution (high resolution) and ultraviolet (UV) absorption revealing merely information about the polarity of the chromophore's environment (low resolution). X-ray studies require crystals while NMR experiments prefer solutions in deuterated solvent. Solvent preferences can affect the choice of spec-troscopic method as, for example, infrared (IR) encounters strong interference from water, while optical rotatory dispersion (ORD) and circular dichroism (CD) do not. Some of the commonly used spectroscopic methods in structural analyses of biomacromolecules will be discussed.
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