Infrared internal reflection spectrometry of aqueous protein films at the germanium-water interface.

Abstract

For twenty years, infrared spectrometrists have relied on the positions and intensities of the N-H stretching, amide I (primarily C=O stretch), and amide I1 (C-N stretch + NH bend) bands as aids in interpreting the secondary structures of synthetic polypeptides and natural proteins. These three absorption bands provide essentially all of the secondary structural information that can be obtained from the infrared spectrum of a polypeptide ( I ) . Two of the bands however, the N-H stretching band at approximately 3400 cm-l and the amide I band at about 1650 cm-', are effectively obscured in aqueous solution spectra by the -3380 cm-l and 1639 cm-l bands of liquid water. Various efforts to get around this problem have been employed, including the use of D20 as a solvent, employing very thin cells, or using a compensating reference cell (2-4) . T h e thin cell technique (2-4) is inadequate for protein solution spectrometry because of the concomitant requirement of high solute concentrations. DzO solutions pose an additional problem, tha t of hydrogen-deuterium exchange. This results in a change in the intensity of the undeuterated amide TI band, and formation of a deuterated amide I1 band a t the same frequency as the HDO band ( I ) . Internal reflection spectrometry ( 4 ) , using a high refractive index, infrared-transparent prism such as germanium, provides the thin sampling region necessary to avoid the total loss of energy at the water peak maxima. For an internal reflection system, the depth of penetration,'' d,, is defined (41 as the distance into the solution where the evanescent field amplitude decays to e-l of its magnitude at the prism-solution interface. For germanium-water, a t a 45 angle of incidence, this distance is 0.064 XO, where Xo is the in-uacuo wavelength (5, 6). Eighty-four percent of the peak intensity observed in an internal reflection spectrum is derived from absorbing molecules within one d, of the prismsolution interface, and 95% of the observed peak intensity is due to molecules within two d,. Thus, for the amide I peak a t 1650 cm-l, 95% of the band intensity comes from the 7750-A thin region at the germanium-solution interface. This is true for the zero absorption case, but is also applicable for low values of k. Higher values (20.1) of h will depress d, (3) . Using such a thin sampling region in combination with the signal-to-noise enhancing techniques of spectrum-averaging and mathematical smoothing, we have been able to obtain accurate spectra of aqueous proteins by subtraction of the solvent spectrum from that of the solution. Spectrum subtraction was demonstrated by Yang and Low (7) , using a Fourier transform interferometer, for aqueous nitrate and nitrite solutions. but their use of difference spectra was restricted to regions where water bands would not interfere. In this paper, we show that, with the aid of a minicomputer interfaced to a standard dispersive infrared spectrophotometer, the obscured amide I band of a protein can be separated from the overlying water band at 1639 cm-'.