TY - GEN
T1 - Detailed Raman analysis of protein denaturation using vertical flow method and programmable pump
AU - Lo, Yu Hao
AU - Hiramatsu, Hirotsugu
N1 - Publisher Copyright:
© COPYRIGHT SPIE. Downloading of the abstract is permitted for personal use only.
PY - 2022
Y1 - 2022
N2 - 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 pKa values of the denaturation process of BSA in the range of pH 7-9 and detailed analysis of the structural change in the denaturation.
AB - 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 pKa values of the denaturation process of BSA in the range of pH 7-9 and detailed analysis of the structural change in the denaturation.
KW - HPLC pump
KW - Non-resonance Raman spectroscopy
KW - Vertical flow method
KW - pH titration
UR - http://www.scopus.com/inward/record.url?scp=85129788908&partnerID=8YFLogxK
U2 - 10.1117/12.2609284
DO - 10.1117/12.2609284
M3 - Conference contribution
AN - SCOPUS:85129788908
T3 - Progress in Biomedical Optics and Imaging - Proceedings of SPIE
BT - Biomedical Vibrational Spectroscopy 2022
A2 - Huang, Zhiwei
PB - SPIE
T2 - Biomedical Vibrational Spectroscopy 2022: Advances in Research and Industry
Y2 - 20 February 2022 through 24 February 2022
ER -