Detailed Raman analysis of protein denaturation using vertical flow method and programmable pump

We have developed a vertical flow (VF) method to improve the generation and collection efficiency of Raman scattering signals. The VF method enhances the Raman signal up to 90 times [Li and Hiramatsu, Anal. Chem. 2019, 91, 9806]. The VF method enabled an online Raman measurement of HPLC eluate (LC-Raman) [Lo and Hiramatsu, Anal. Chem. 2020, 92, 14601]. A programmable pump of the HPLC system is available to gradually change the pH value of the solvent by changing the mixing fraction of two solvents. In this study, we used the programmable pump combined with the Raman spectroscopy to probe the denaturation of bovine serum albumin (BSA) change in the range of pH 7 - 9. The vertical flow method allowed us to record 8300 spectra in 30 min. The obtained 2D Raman data having the spectral and temporal axes was analyzed with singular value decomposition and reconstruction method. We found that four temporal and spectral components predominate the 2D Raman data, namely, (i) the pH-independent Raman signal of BSA, (ii) that due to the denaturation, (iii) the ionization of phosphate in the buffer, and (iv) pH-dependent background. The sufficiently large number of the data points enabled precise determination of the pK(a) values of the denaturation process of BSA in the range of pH 7 - 9 and detailed analysis of the structural change in the denaturation.