Confocal Raman Microscopy of Protein Adsorbed in Chromatographic Particles

Abstract

Confocal Raman microscopy is a nondestructive analytical technique that combines the chemical information from vibrational spectroscopy with the spatial resolution of confocal microscopy. It was applied, for the first time, to measure conformation and distribution of protein adsorbed in wetted chromatographic particles. Monoclonal antibody was loaded into the Fractogel EMD SO(3) (M) cation exchanger at 2 mS/cm or 10 mS/cm. Amide I and III frequencies in the Raman spectrum of the adsorbed protein suggest that there are no detectable changes of the original β-sheet conformation in the chromatographic particles. Protein depth profile measurements indicate that, when the conductivity is increased from 2 mS/cm to 10 mS/cm, there is a change in mass transport mechanism for protein adsorption, from the shrinking-core model to the homogeneous-diffusion model. In this study, the use of confocal Raman microscopy to measure protein distribution in chromatographic particles fundamentally agrees with previous confocal laser scanning microscopic investigations, but confocal Raman spectroscopy enjoys additional advantages: use of unlabeled protein to eliminate fluorescent labeling, ability for characterization of protein secondary structure, and ability for spectral normalization to provide a nondestructive experimental approach to correct light attenuation effects caused by refractive index (RI) mismatching in semiopaque chromatographic particles.

Advanced characterization of immobilized enzymes as heterogeneous biocatalysts

“…Like FTIR, Raman spectroscopy is in principle well suitable for the analysis of proteins in solid samples, be it that dried proteins or frozen protein solution are investigated [21,108,[119][120][121] ( Table 3). Background signals in solid-supported enzyme preparations can be unexpectedly high however [122].…”

“…Raman spectra are well resolved and signal quality is high when metal nanoparticles of 10-100 nm size fabricated from colloidal silver or gold are used [123][124][125]. The application of Raman spectroscopy to the characterization of immobilized proteins is increasing [37,120,[125][126][127][128][129]. Loss of secondary protein structure on immobilization was analyzed [120,129].…”

“…The application of Raman spectroscopy to the characterization of immobilized proteins is increasing [37,120,[125][126][127][128][129]. Loss of secondary protein structure on immobilization was analyzed [120,129]. Orientation of enzymes on solid surface was determined and attachment via covalent bonds was also elucidated [130].…”

How enzymes are adsorbed on soil solid phase and factors limiting its activity: A Review

“…Raman spectroscopy is also potentially a strong technique to analyse enzyme structure in dried or frozen solution samples (Thyparambil et al, 2015;Xiao et al, 2012). There is increasing use of Raman spectroscopy to characterize immobilized enzyme.…”

“…There is increasing use of Raman spectroscopy to characterize immobilized enzyme. (Ash and Vincent, 2012;Xiao et al, 2012).…”