PH titration of β-lactoglobulin monitored by laser-based Mid-IR transmission spectroscopy coupled to chemometric analysis.

Abstract

A novel external cavity-quantum cascade laser (EC-QCL)-based setup for mid-IR transmission spectroscopy in the amide I and amide II region was employed for monitoring pH-induced changes of protein secondary structure. pH titration of β-lactoglobulin revealed unfolding of the native β-sheet secondary structure occurring at basic pH. Chemometric analysis of the dynamic IR spectra was performed by multivariate curve resolution-alternating least squares (MCR-ALS). Using this approach, spectral and abundance distribution profiles of the conformational transition were obtained. A proper post-processing procedure was implemented allowing to extract information about pure protein spectra and spurious signals that may interfere in the interpretation of the system. This work demonstrates the potential and versatility of the EC-QCL-based IR transmission setup for flow-through applications, benefitting from the high available optical path length.