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Evaluation of Particle Beam Fourier Transform Infrared Spectrometry for the Analysis of Globular Proteins: Conformation of β-Lactoglobulin and Lysozyme

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Abstract:

The efficacy of the particle beam LC/FT-IR interface, in its development as a tool for the determination of dynamic protein structure from experiments such as HPLC separations and folding/refolding intermediate analysis, is presented here. The particle beam apparatus can be used to desolvate proteins rapidly in preparation of IR measurements. Several experiments have been designed to determine whether the operation of the particle beam apparatus causes alteration to the complex structural features of globular proteins, and whether it produces a solid-state spectrum representative of protein solution structure. It is shown here that the structural integrity of β-lactoglobulin is maintained when nebulized, desolvated from solution, and deposited onto the IR-substrate. Since enzyme activity is dependent upon the maintenance of higher-order structure, a complementary series of spectrophotometric-activity experiments with lysozyme collected from the particle beam were performed to determine the state of the tertiary and quaternary structures. The lysozyme particle beam deposit not only produced a secondary structure estimate similar to that of solution; it also retained its biological activity. It is demonstrated that the particle beam can induce structural changes in proteins with a carrier-liquid concentration gradient; this characteristic is useful for band assignment.

Keywords: Desolvation; Lysozyme; Micrococcus lysodeikticus; Particle beam LC/FT-IR spectrometry; β-Lactoglobulin (βLG)

Document Type: Research Article

DOI: http://dx.doi.org/10.1366/0003702944027390

Affiliations: Department of Chemistry, University of Georgia, Athens, Georgia 30602-2556

Publication date: October 1, 1994

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