The first CD spectrum of a polypeptide was reported by Holzwarth (14) in 1962 with the spectrum of poly-L-glutamic acid. The advantage of CD over the related and then common technique of optical rotatory dispersion (ORD) is that it has the simplifying property of vanishing except at the absorption wavelength of an electronic transition of an optically active chromophore. This greatly aided the interpretation of the spectrum with respect to protein conformation.

The classical description of optical rotation starts by defining unpolarized light waves as plane waves which are randomly oriented with respect to the direction of propagation. The electric component of the plane wave can be described by the following equation:

and the magnetic component by:

where E0 and H0 are the amplitudes of the waves. (wt - nkz) describes the phase of the wave, with w = 2pv (circular frequency), k = 2p/l and n is the refractive index. ve and vh are the electric and magnetic vectors.

As light passes through a medium, aside from absorption it may also be refracted, which alters its velocity. The velocity of light through a medium (v) is related to its velocity in a vacuum (c) by the following equation:

where n is the refractive index. The refractive index is also a function of frequency. If an incident light beam is composed of several frequencies, their velocities will vary and dispersion will occur.

A plane polarized light wave is composed of two vector components, El, left circularly polarized light (lcpl) and Er, right circularly polarized light (rcpl). Both lcpl and rcpl can be thought of as rotating in opposite directions along the axis of propagation. If El and Er rotate at different velocities however, the resultant vector E, while still plane polarized, is rotated through an angle.

Circular dichroism is the result of differential absorption between El (lcpl) and Er (rcpl). The resultant vector E traces out an ellipse rather than a circle, which is the case when El = Er.(see Figure 1) This ellipticity Q, is normally expressed as molar ellipticity:

where Q is the observed ellipticity in degrees, c is the solute concentration in moles per liter and d is the cell path length in cm, or the following equation may be used:

The quantum mechanical interpretation of optical rotatory power is given by the Rosenfeld (15) equation:

Y0 and Ym are the wavefunctions for the ground and excited states respectively of the electronic transitions. m is the electric transition moment operator and m is the magnetic transition moment operator. The Im refers to the imaginary part of the quantity in brackets. From the Rosenfeld equation it can be seen that the relative orientation of the electronic and magnetic transition dipole moments are related to the rotatory strength (Rm0) by the cosine of the angle between them. The rotatory strength, analogous to the dipole strength in absorbance spectroscopy, may therefore vanish. In contrast to absorbance spectroscopy it can be seen that while a transition may be magnetically allowed and electronically forbidden, and thus very weak in absorbance spectroscopy, it may contribute significantly to the rotatory strength in CD spectroscopy. The n-p* transition of the peptide chromophore with its large magnetic transition dipole moment is an important example.

There are three mechanisms which give rise to optical rotatory power in an asymmetric molecule. The first mechanism is the one-electron mechanism of Condon, Altar and Eyring (16) where the interacting electric and magnetic transition dipole moments are on the same chromophore. The asymmetric electrostatic environment of the neighboring atoms act as a perturbing field to break down the symmetry of the chromophore and allow the two transitions to mix.

The second mechanism is the coupled oscillator mechanism of Kuhn (17), Kirkwood (18) and Moffitt (19) where the electronic transitions of two chromophores of an asymmetric molecule couple to produce a magnetic moment resulting in optical activity.

The third mechanism is the m-m mechanism of Schellman. (20,21) In this mechanism, a magnetically allowed transition on one chromophore is coupled to an electronic transition on another chromophore via an electric quadrupole associated with the magnetically allowed transition. The two chromophores must be in close proximity to one another.

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