[18] Ligand binding and conformational changes measured by time-resolved absorption spectroscopy

This chapter describes the performance of a time-resolved absorption spectrometer with a time resolution of 10 nsec. The spectrometer used in this chapter is capable of collecting spectral data over selected wavelength intervals throughout the visible and near-ultraviolet (UV) spectral region and provides performance and stability comparable to conventional double-beam recording spectrophotometers. The chapter also focuses on some techniques that have been developed to determine accurately the fraction of hemes photolyzed and to utilize this information in kinetic analyses. These techniques are based on characterizing the distribution of orientations of the photoproduct molecules generated by partial photolysis of the sample with a linearly polarized excitation pulse. The qualitative features of the photoproduct progress curves provide a sampling of the power and potential of this procedure for analysis of partial photolysis data. The curves produced by such analyses can be described much more quantitatively.