Abstract
This chapter describes methods of measuring the temperature dependence of the magnetic circular dichroism (MCD) that are necessary to establish the paramagnetic origin of C terms and investigate spin states in heme proteins. In recording a spectrum what is actually measured is the difference in absorption of left and right circularly polarized light (L and RCPL). This value can be expressed in terms of molar absorptivity (or extinction) difference. Certain aspects of MCD spectroscopy make it necessary to pay more careful attention to operating parameters than is usually necessary in absorption spectroscopy. One problem, common to CD as well, is that because very small differences in absorption of L and RCPL are measured, large numbers of photons are needed to obtain good statistics. Consequently, a high-intensity light source and wide slit widths are generally used to improve signal-to-noise levels. Room-temperature MCD measurements can be obtained with the normal rectangular or cylindrical cuvettes used for absorption spectroscopy. Measurements of MCD from room temperature to liquid-nitrogen temperature are sufficient for establishing the paramagnetic origin of intense C terms since this changes the Boltzmann distribution of ground-state populations and MCD intensity by a factor of 3.8.
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