Determination of the secondary structure of isomeric forms of human serum albumin by a particular frequency deconvolution procedure applied to Fourier transform IR analysis.

A new deconvolution procedure was applied to the analysis of Fourier transform ir 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 Minimization Algorithm, with the addition of suitable constraints directly obtained by the application to the measured spectrum of the second derivative operator. This method computes central band frequency, bandwidth, and amplitude of the different spectral components of conformation-sensitive amide bands. In the specific case, it was applied to analysis of the amide I band, and the quantitative determination of the different 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 structures present in the protein. The coefficient of variation is < 10% for values of amide I component > 15%. The accuracy was tested by comparing, by means of linear regression, the results obtained for human serum albumin, hemoglobin, alpha-chymotrypsin, and cytochrome c, using our method, with those obtained by x-ray crystallography and CD; the results obtained by other vibrational spectroscopic approaches were also compared. The fit standard error between x-ray and ir secondary structure values estimated by our method is 2.5% for alpha-helix, 7.16% for beta structures, and 5.1% for other structures (turns and random coils). Quantitative results are given for the secondary structures (alpha-helix, turns, and beta-strands) present in the native state (turns and beta-strands up to now unknown in aqueous solution), together with the percentages of these structures and additional ones (random coils and beta-sheets) formed during denaturization.