Secondary structure of bovine albumin as studied by polarization-sensitive multiplex CARS spectroscopy

The first application of polarization-sensitive multiplex coherent anti-Stokes Raman spectroscopy (MCARS) in the absence of resonance enhancement to the resolution of the secondary structure of a protein in solution is reported. Polarization MCARS spectra of bovine albumin in D2O were obtained in the range 1370 to 1730 cm−1 with the aid of the background suppression technique. The spectra were fitted simultaneously with a single set of parameters (band positions, bandwidths, amplitudes, and depolarization ratios). Polarized Raman spectra simulated with these parameters revealed a good correspondence with the spontaneous Raman spectra measured. The broad amide I′ band was decomposed assuming the three major secondary conformations of protein, of which the contribution of β-sheet structure was found to be negligible. Relative weights of α-helix and random coil conformations agree well with the estimates obtained with Raman and circular dichroism (CD) spectroscopies.