DETERMINATION OF THE SECONDARY STRUCTURE OF ISOMERIC FORMS OF HUMAN SERUM-ALBUMIN BY A PARTICULAR FREQUENCY DECONVOLUTION PROCEDURE APPLIEDTO FOURIER-TRANSFORM IR ANALYSIS

A new deconvolution procedure was applied to the analysis of Fourier t ransform inr spectra of human serum albumin secondary structure in the native state and in states denatured by heat and acid treatment. the deconvolution method is based on the use of the Conjugate Gradient Min imization Algorithm, with the addition of suitable constraints directl y obtained by the application to the measured spectrum of the second d erivative operator. This method computes central band frequency, bandw idth, and amplitude of the different spectral components of conformati on-sensitive amide bands. In the specific case, it was applied to anal ysis of the amide I band, and the quantitative determination of the di fferent secondary structures (alpha-helix, beta-sheet, beta-turns, and random) was attempted for all the samples examined. The precision of the quantitative determination depends on the amounts of these structu res present in the protein. The coefficient of variations is <10% for values of aide I component >15%. The accuracy was tested by comparing, by means of linear regression, the results obtained for human serum a lbumin, hemoglobin, alpha-chymotrypsin, and cytochrome c, using our me thod, with those obtained by x-ray crystallograpy and CD; the results obtained by other vibrational spectroscopic approaches were also compa red. The fit standard error between x-ray and ir secondary structure v alues estimated by our method is 2.5% for alpha-helix, 7.16 for beta s tructures, and 5.1% for other (turns and random coils).Quantitative re sults are given for the secondary structures (alpha-helix, turns, and beta-strands) present in the native state (turns and beta-strands up t o now unknown in aqueous solution), together with the percentages of t hese structures and additional ones (random coils and beta-sheets) for med during denaturization. (C) 1996 John Wiley & Sons, Inc.