EC-QC laser spectroscopy for mid-IR transmission measurements of proteins in aqueous solution

ALCARAZ M R; SCHWAIGHOFER A ; BRANDSTETTER M; RAMER, G; KRISTAMENT C; GOICOECHEA H C; LENDL B

ANALYTICAL CHEMISTRY

AMER CHEMICAL SOC

Lugar: Washington; Año: 2015

0003-2700

In this work we report mid-IR transmission measurements of the protein amide I band in aqueous solution at large optical paths. A room-temperature operated tunable external-cavity quantum cascade laser (EC-QCL) allowed applying pathlength of up to 38 µm, which is four times larger than applicable with conventional FTIR spectrometers. To minimize temperature-induced variations caused by background absorption of the 2-vibration of water (HOH-bending) overlapping with the amide I region, a highly-stable temperature control unit was developed. An advanced data processing protocol was established to overcome fluctuations in the fine structure of the emission curve that are inherent to the employed EC-QCL due to its mechanical instabilities. Employing this setup, characteristic spectral features of five well-studied proteins exhibiting different secondary structures could be elucidated at concentrations as low as 2.5 mg ml?1. This concentration range could previously only be accessed by IR measurements in D2O. Mathematical evaluation of the spectral overlap and comparison of second derivative spectra confirm excellent agreement of the QCL transmission measurements with protein spectra acquired by FTIR. This proves the potential of the applied setup to monitor secondary structure changes of proteins in aqueous solution at extended optical path lengths.