Confocal Raman Microscopy of Protein Adsorbed in Chromatographic Particles

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 SO₃ (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

Anhydrous Monoalkylguanidines in Aprotic and Nonpolar Solvents: Models for Deprotonated Arginine Side Chains in Membrane Environments

In this study, the synthesis of crystalline dodecylguanidine free base and its spectroscopic characterization in nonpolar environments are described. IR as well as ¹H and ¹⁵N NMR spectra of the free base dissolved in aprotic solvents are substantially different from the previously reported spectra of arginine, or other monoalkylguanidinium compounds, at high hydroxide concentrations. The current results provide improved modeling for the spectroscopic signals that would be expected from a deprotonated arginine in a nonpolar environment. On the basis of our spectra of the authentic dodecylguanidine free base, addition of large amounts of aqueous hydroxide to arginine or other monoalklyguanidinium salts does not deprotonate them. Instead, hydroxide addition leads to the formation of a guanidinium hydroxide complex, with a dissociation constant near ∼500 mM that accounts for the established arginine p<i>K</i> value of ∼13.7. We also report a method for synthesizing a compound containing both phenol and free-base guanidine groups, linked by a dodecyl chain that should be generalizable to other hydrocarbon linkers. Such alkyl-guanidine and phenolyl-alkyl-guanidine compounds can serve as small-molecule models for the conserved arginine–tyrosine groupings that have been observed in crystallographic structures of both microbial rhodopsins and G-protein-coupled receptors