QUANTITATION OF THE AREA OF OVERLAP BETWEEN 2ND-DERIVATIVE AMIDE-I INFRARED-SPECTRA TO DETERMINE THE STRUCTURAL SIMILARITY OF A PROTEIN IN DIFFERENT STATES

Maintaining a nativelike structure of protein pharmaceuticals during l yophilization is an important aspect of formulation. infrared spectros copy can be used to evaluate the effectiveness of formulations in prot ecting the secondary structural integrity of proteins in the dried sol id. This necessitates making quantitative comparisons of the overall s imilarity of infrared spectra in the conformationally sensitive amide I region. We initially used the correlation coefficient r, as defined by Prestrelski et al. (Biophys. J. 1993, 65, 661-671), for this quanti tation. Occasionally, we noticed that the r value did not agree with a visual assessment of the spectral similarity. In some cases this was due to an offset in baselines, which led artifactually to an unreasona bly low r value. Conversely, if the spectra were baseline corrected an d there existed a large similarity between peak positions, but differe nces in relative peak heights, the r value would be unreasonably high. Our approach to avoiding these problems is to use area-normalized sec ond-derivative spectra. We have found that quantitating the area of ov erlap between area-normalized spectra provides a reliable, objective m ethod to compare overall spectral similarity. In the current report, w e demonstrate this method with selected protein spectra, which were ta ken from experiments where unfolding was induced by lyophilization or guanidine hydrochloride, and artificial data sets. With this analysis, we document how problems associated with calculation of the correlati on coefficient, r, are avoided.