4TH DERIVATIVE UV-SPECTROSCOPY OF PROTEINS UNDER HIGH-PRESSURE .1. FACTORS AFFECTING THE 4TH DERIVATIVE SPECTRUM OF THE AROMATIC-AMINO-ACIDS

A tunable fourth derivative UV absorbance method based on a variable s pectral shift has been developed and compared to the Savitzky-Golay me thod and the analytical derivative. The parameters of the method were optimised for the analysis of the UV absorbance spectra of the aromati c amino acids to quantify the effect of decreasing solvent polarity on their fourth derivative spectra. The wavelength of the highest maximu m (lambda(max)) (for tyrosine and phenylalanine) or the amplitude of t he highest maximum (A(max)) (for tryptophan), were shown to depend lin early on the dielectric constant of the solvent, ranging from water to cyclohexane. The only effect of pressure in the 1 to 500 MPa range is a small decrease in the fourth derivative amplitude. This method appe ars therefore as a suitable tool to evaluate changes of the dielectric constant in the vicinity of the aromatic amino acids in proteins whic h undergo pressure induced structural changes.