Protein concentration is not an absolute prerequisite for the determination of secondary structure from circular dichroism spectra: a new scaling method.

We present here a simple and rapid method to extract good estimates of protein secondary structure content from circular dichroism (CD) spectra without any prior knowledge of the sample concentration. The method involves two steps: first, a single-wavelength normalization procedure and, second, the application for each secondary structure of a quadratic model based on one or two wavelength intensities. These quadratic models were derived by a cross-validation analysis of a new protein CD spectrum database. Tested on CD spectra of proteins at different concentrations, the normalization was shown to render the method virtually independent of the sample concentration. Further tests on CD spectra not recorded in our laboratory showed that our quadratic models are of general applicability. Even though the success of the present approach is less than that for currently available methods, its simplicity and the fact that the concentration is not needed may be very attractive for the study of small amounts of membrane proteins or peptides for which an accurate concentration determination might be very difficult or impossible to obtain.