RAMAN-SPECTROSCOPIC STUDY OF THERMALLY AND OR DITHIOTHREITOL INDUCED GELATION OF LYSOZYME

Opaque gels were formed by heating lysozyme solutions (10%, pH 7.2, 50 mM NaCl) containing 10 or 100 mM dithiothreitol (DTT) at 37, 60, 80, or 100-degrees-C but not at 37 or 60-degrees-C in the absence of DTT. Gels formed with 100 mM DTT were soluble in 8 M urea, while gels formed with no or 10 mM DTT at 100-degrees-C were insoluble. Raman spectral analysis indicated increased exposure of aromatic residues and a general decrease in alpha-helical and increase in beta-sheet secondary structure fraction of molecules in gels compared to in solution. Chemical and Raman spectral analyses of sulfhydryl and disulfide groups suggested that reduction or interchange reactions involving intramolecular disulfide bonds of lysozyme were responsible for destabilizing tertiary structure, allowing conformational changes and exposure of hydrophobic groups which led to gel formation. Intermolecular disulfide cross-links were not necessary for gelation but could lead to more stable gels.