Nanosecond optical rotatory dispersion spectroscopy: application to photolyzed hemoglobin-CO kinetics

A standard technique for static optical rotatory dispersion (ORD) measurements is adapted to the measurement of ORD changes on a nanosecond (ns) time scale, giving approximately a million-fold improvement in time-resolution over conventional instrumentation. The technique described here is similar in principle to a technique recently developed for ns time-resolved circular dichroism (TRCD) spectroscopy, although the time-resolved optical rotatory dispersion (TRORD) technique requires fewer optical components. As with static ORD, TRORD measurements may be interpreted by empirical comparisons or may be transformed, via the Kramers-Kronig relations, to more easily interpreted TRCD spectra. TRORD can offer experimental advantages over TRCD in studying kinetic processes effecting changes in the chiral structures of biological molecules. In particular, the wider dispersion of ORD bands compared with the corresponding CD bands means that ORD information may often be obtained outside of absorption bands, a signal-to-noise advantage for multichannel measurements. Demonstration of the technique by its application to ns TRORD and the transform-calculated TRCD of carboxy-hemoglobin (Hb-CO) after laser photolysis is presented.