The effect of cross-links on the mobility of proteins in dodecyl sulphate–polyacrylamide gels

Abstract

The effect of reduction of intramolecular disulphide bridges on the mobility of proteins in 5% (w/v) polyacrylamide gels in the presence of sodium dodecyl sulphate was investigated. A series of polypeptide polymers, containing up to 68 intramolecular disulphide bridges, was prepared by cross-linking proteins of known structure with glutaraldehyde. These model polypeptides were denatured with heat, sodium dodecyl sulphate and urea, and their mobilities in sodium dodecyl sulphate-polyacrylamide gels compared before and after reduction with dithiothreitol. The mobilities of polypeptides containing no cystine were unaffected by reduction. However, reduction generally decreased the mobilities of polypeptides containing cystine; the extent of this decrease depended on the number of cystine residues originally present in the polypeptide polymer, and on the protein from which the latter was derived. In contrast with their higher oligomers, the monomer of lysozyme and the dimer of ribonuclease increased in mobility after reduction. The reduced polypeptide oligomers formed by reaction with glutaraldehyde were generally found to migrate at a rate significantly faster than was expected from their calculated molecular weights. It was concluded that the use of unreduced proteins and protein aggregates for molecular-weight measurements by the sodium dodecyl sulphate-polyacrylamide-gel method may give erroneous estimates of the molecular weight of any protein being investigated.

An Expanded Genetic Code in Candida albicans To Study Protein-Protein Interactions In Vivo

“…The apparent molecular mass of the upper detected dimers is ϳ165 kDa and thus considerably higher than the expected size for two Tup1p monomers (computational calculation of the molecular mass: Tup1p wt, 57.8 kDa; Tup1p with C-terminal V5/His 6 tag, 60.6 kDa). This aberrant migration behavior in a reducing SDS-PAGE might be due to the intermolecular covalent bond that prevents complete unfolding and uniform SDS binding which in turn leads to a reduced electrophoretic mobility, analogous to intramolecular disulfide bonds (60,61). However, depending on the position of the intermolecular bond and the protein, the contrary effect is also possible (62).…”

Human plasma fibrinogen heterogeneity: evidence for an extended carboxyl-terminal sequence in a normal gamma chain variant (gamma').

“…2, gels 3, 4, 11, and 12). The apparent Mr of material occupying this position (82,000-86,000) was lower than the sum of the constituent T chains, an unusual behavior that has been observed by other investigators (33) and which may be related to the presence of covalent interchain crosslinks (34). Because a detailed understanding of this phenomenon was peripheral to the problem at hand, we did not pursue this subject further.…”

The Purification and Properties of 2,4‐Dichlorophenol Hydroxylase from a Strain of Acinetobacter Species

“…Enzyme was cross-linked with glutaraldehyde using a modification of the method of Griffith [37]. Glutaraldehyde (0.50/, w/v; 5 pl) was added to 550 pg enzyme protein in 0.5 ml of 100 mM sodium phosphate buffer, pH 7.0, and left at room temperature for 18 h. The treated enzyme sample was denatured by addition of an equal volume of 100 mM sodium phosphate buffer containing 1 x (w/v) sodium dodecylsulphate and 0.5 x (v/v) 2-mercatptoethanol followed by incubation at room temperature for 2 h.…”