Experimental errors and their effect on analyzing circular dichroism spectra of proteins

Seven types of error that may interfere with the analysis of protein circular dichroism (CD) spectra for secondary structure are examined. Three of these errors are operational encompassing wavelength synchronization, and proper choice of spectral bandwidth and scan speed. Three are experimental involving intensity adjustments and two sources of baseline shift. The skew baseline shift is analogous to error in CD intensity at short wavelengths due to high sample absorption and low source intensity. The final source of error deals with constrained analyses. We have investigated these types of error to determine how they may be affecting our analysis of protein CD spectra and the role they may play in causing our analyses to fail for some proteins. We find that small errors in the baseline (which are independent of the protein spectrum) will rationalize our poor analyses. Spectroscopists must adopt new standards of precision if sophisticated analyses are to succeed.