FTIR Analysis Of Cytochrome b s In H 2 O And D 2 O

Abstract

The application of infrared spectroscopy to biological samples has been made possible largely by the advent of FTIR. The advances in instrumentation which have allowed this application are well documented, however, there are still some barriers to the full utilization of this technique for biological samples such as proteins. The general objectives are to obtain spectra in the 1700 - 1600 cm -1 region, where the Amide I band is located. This broad, generally featureless, band is a complex composite of bands which are characteristic of specific types of secondary structure in the protein 1. The problems to be overcome fall into several categories. The need to work in aqueous solutions, where water has a very large absorption band at 1650 cm4, has been circumvented to a large extent by working in D20, although it is still necessary to employ short path length cells, as the absorbance of the H-O-D overlaps both the Amide I and Amide II regions. In spite of the similarity of D20 and H2O, there are still advantages to obtaining spectra in both solvents as in D20 the accessible N-H groups in the peptide bonds will undergo H->D exchange. A comparison between spectra obtained in the two solvents will therefore give information on solvent accessibility of the peptide bonds in the different types of secondary structure and will also assist in assigning bands to particular secondary structures. Coupled with this requirement of short path length cells is the tendency of proteins to denature on surfaces. This requires that demountable cells be used and these cells, especially with 6 - 50 μm spacers, have a variability in path length from sample to sample, and especially from sample to the solvent blank which makes subtraction of the solvent blank very difficult. This problem of solvent subtraction is most acute when H2O is the solvent as here the high solvent absorbance is coupled with the smaller, and more variable, path length. Once the sample spectrum minus the solvent has been obtained the broad Amide I band has to be subjected to some type of enhancement procedure, usually Fourier self-deconvolution2, in order to resolve the bands due to particular types of protein secondary structure. Both the solvent subtraction and the deconvolution are somewhat subjective and open to criticism. In this report we demonstrate that reproducible deconvolved spectra can be obtained in both D20 and H2O.