Transient CD measurements at submillisecond time resolution—application to studies of temperature-jump relaxation of equilibria of chiral biomolecules

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Abstract
A new system for the measurement of transient circular dichroism (CD) has been developed, with improved bandwidth, optical aperture, and source ratioing electronics, and applied to the study of temperature‐jump relaxation processes of chiral complexes of ligands with biological molecules. Transient changes in circular dichroism and optical transmission are measured simultaneously. The optimal CD resolution is 2×10−6 (66×10−6 degrees ellipticity) at 300 μsec time constant and optimal absorbance resolution is ±4×10−5. The noise spectrum of the light source is important in determining the resolution achievable in either channel, and broadband stabilization of the mercury arc source is necessary. The potential of low‐noise laser sources has also been demonstrated. Careful optical alignment reduces spurious signals to the order of the CD resolution. A theoretical treatment of the CD resolution has been evaluated specifically as a function of sample absorbance and available light power. Resolution is determined by detector shot noise, electronic system noise, and hence, time constant (1/bandwidth). Theoretical resolution for photon‐noise‐limited sources is approached in experimental measurements. At high light power the CD resolution is a slowly varying function of total sample absorbance, including both dichroic and nondichroic absorbing species. This property allows relaxation times to be determined over sufficient concentration range for analysis of temperature‐jump (TJ) relaxation effects. Finally we indicate the additional spectroscopic features characterizing simple equilibria which may be derived from observation of the transient circular dichroism in TJ relaxation experiments.