Stacks of close to 100 phospholipid bilayers fully accessible to proteins - An ATR-FTIR-based chemometric analysis on hydrated phospholipid films

A novel method for measuring aqueous solutions of proteins having high affi nity for specific membrane components has been developed from attenuated to tal reflection-Fourier transform infrared (ATR-FTIR) spectra. The method wa s based on the use of a germanium internal reflection element (IRE) which s urface was covered with a PE-biotin film. In order to investigate the poten tialities of this approach, streptavidin in aqueous solutions was recircula ted continuously in a flow-trough mode on the hydrated phospholipid film. P artial least squares (PLS) and principal component regression (PCR) were us ed and compared for establishing a multivariate calibration model for strep tavidin concentrations ranging from 50 to 500 mg l(-1). The best agreement between the known and predicted concentrations of streptavidin by PLS and P CR was obtained when both methods were optimized by selecting the spectral regions having the highest correlation with concentration data. PLS reveale d more accurate results than PCR with a standard error of prediction (SEP) of 18.56 mg l(-1) (7%). The streptavidin amount effectively bound to the PE -biotin film was estimated separately via a univariate regression between A TR-FTIR absorbance and concentration data representing different protein:ph ospholipid ratios. The results demonstrated that the protein was capable of diffusing and attaining all of the available sites in the multilayer PE-bi otin matrix. Such a phospholipid film-based measurement method is a promisi ng tool for developing new sensing methods for analyzing biomolecules in aq ueous solutions. (C) 2001 Elsevier Science B.V. All rights reserved.