Supersaturated lysozyme solution structure studied by chemical cross-linking

Abstract

Glutaraldehyde cross-linking followed by separation has been used to detect aggregates of chicken egg-white lysozyme (CEWL) in supersaturated solutions. In solutions of varying NaCl content, the number of aggregates was found to be related to the ionic strength of the solution. Separation by SDS-PAGE showed that percentage of dimer in solution ranged from 25.3% for no NaCl to 27.1% at 15% NaCl, and the aggregates larger than dimer increased from 1.9% for no NaCl to 36.8% at 15% NaCl. Conversely, the percentage of monomers decreased from 72.8% without NaCl to 36.1% at 15% NaCl. Molecular weights by capillary electrophoresis (SDS-CE) were found to be multiples of the monomer molecular weights, with the exception of trimer, which indicates a very compact structure. Native separation was accomplished using size-exclusion chromatography (SEC) and gave a lower monomer concentration and higher aggregate concentration than SDS-CE, which is a denaturing separation method. Most noticeably, trimers were absent in the SEC separation. The number of aggregates did not change with increased time between addition of NaCl and addition of cross-linking agent when separated by gel electrophoresis (SDS-PAGE). The results suggest that high ionic strength CEWL solutions are highly aggregated and that denaturing separation methods disrupt cross-linked products.

Structure and kinetics of chemically cross-linked protein gels from small-angle X-ray scattering

“…The PCF is then used, along with eqn (7), to obtain the running coordination number, N(r), in the protein gel (Fig. 5).…”

“…1,2 This bifunctional reagent has been used as a fixative in studies of cell or tissue ultrastructure, 3,4 to stabilise protein crystals for X-ray diffraction, 5,6 and to characterise the quaternary structure of proteins in solution. 7,8 Applications of GA cross-linking in immunochemistry and biotechnology include protein immobilisation on solid carriers, e.g., in affinity chromatography and biosensors, as well as carrier-free immobilisation of enzymes in solution, in amorphous precipitates, or in crystals for use as industrial biocatalysts. [9][10][11][12][13] The present study was motivated by yet another application of protein cross-linking: water NMR studies of biological systems.…”

Elucidation of the mechanism and end products of glutaraldehyde crosslinking reaction by X‐ray structure analysis

“…Glutaraldehyde has been used for several decades as an effective crosslinking agent for many applications including sample fixation for cytochemistry and electron microscopy (Sabatini et al, 1963), immobilization of enzymes and whole cells (Quiocho and Richards, 1966;Schejter and Bareli, 1970;Walt and Agayn, 1994;Khalaf et al, 1996;Lalonde et al, 1997), and protein crystal stabilization for X-ray crystallography (Wang et al, 1998;Lusty, 1999;Hall et al, 2005;Heras and Martin, 2005;Iimura et al, 2005) as well as for biocatalysis (Margolin, 1996;Zelinski and Waldmann, 1997;Haring and Schreier, 1999;Leisola et al, 2001;Margolin and Navia, 2001;Iimura et al, 2005;Roy and Abraham, 2006). Inspite of its wide applicability and high efficiency as a crosslinking agent, the mechanism and chemistry involved in this process is not fully understood (Whipple and Ruta, 1974;Walt and Agayn, 1994).…”