Analysis of transport experiments using pseudo-absorbance data

The measurement of the concentration distribution of a macromolecule across a solution column by absorption optics usually requires optical transmission profiles of both the sample solution and the buffer, measured under identical conditions, to calculate the absorbance as the logarithm of the ratio of reference to sample intensity. For transport experiments, however, where the changes in the local macromolecule concentration with time are measured, a reference buffer intensity is not necessarily required. We demonstrate that the logarithm of the light transmitted through the sample solution, referred to as pseudo-absorbance, can suffice to determine macromolecular transport parameters of interest, with little loss of precision. Local changes in illumination of the sample column or in the detection efficiency of the transmitted light, as well as temporal fluctuations of the light source intensity can be well-described by consideration of time-invariant and radial-invariant signal components in the pseudo-absorbance data, using the systematic noise decomposition techniques developed recently (Schuck, P., and Demeler, B. (1999) Biophys. J. 76, 2288-2296). The practical use of the method is demonstrated with double-sector and single-sector sedimentation velocity experiments, and with analytical electrophoresis experiments. It is shown that pseudo-absorbance analysis can increase the capacity of a sedimentation velocity experiment in ultracentrifugation, and, in general, can considerably simplify the requirements of optical design.