Quantitative analysis of absorption spectra and application to the characterization of ligand binding curves

The spectrum of a chromophore may change as a result of perturbations in its environment. The spectral changes resulting from the perturbation are often followed by measurements at just one or two wavelengths but it is usually no more difficult to collect entire spectra. The problem comes in analysing the data from such a series of spectra. In this paper we will suggest a simple procedure in which the spectrum observed under any particular set of conditions may be considered to consist of the sum of two distinct spectral forms. The method, which is free of any assumptions regarding the quantitative relationship between the perturbation and the extent of spectral change, defines any given spectrum in terms of an apparent molar fraction of the contributing spectral forms. The variation of this apparent molar fraction provides information from which a quantitative relationship can be developed to describe the dependence of the spectral change on the perturbant. The method is illustrated using the model system of phenol red protonation and is applied to the characterization of the binding of azide ions to cobalt-substituted carbonic anhydrase.