ABSTRACT
Demonstrated herein is the possibility of using the accessibility of tryptophan (Trp) residues in immunoglobulin M (IgM) upon modification with Koshland reagent (2-hydroxy-5-nitrobenzyl bromide) as an index of the conformational changeability of IgM. Of fourteen Trp's in the native IgM (per HL-region) only one appeared to be most accessible, evidently Trp312 in the mu-chain. Irreversible acidic and thermal conformational transitions in IgM increase the number of accessible Trp's approximately two-fold. Following partial enzymatic deglycosylation of IgM, deep scission of mannose in particular, all Trp's become inaccessible. Modification of the most accessible Trp increases 2-3 fold the number of tyrosine residues readily accessible upon nitration with tetranitromethane. Modification of four trp's using spin-label method data causes a sharp reduction of the mobility of the C mu 3 domain and a simultaneous decrease in the solubility of modified IgM.
Subject(s)
Immunoglobulin M/chemistry , Tryptophan/chemistry , 2-Hydroxy-5-nitrobenzyl Bromide , Electron Spin Resonance Spectroscopy , Glycoside Hydrolases , Protein Conformation , SolubilityABSTRACT
A method is proposed for spin-labelling using 2,2,6,6-tetramethyl-4-aminopiperidine-1-oxyl, the N-acetylneuraminic acid residue within the Fab-region oligosaccharide of immunoglobulin M (IgM). The selectivity of spin-labelling was achieved by the controlled periodate oxidation of the oligosaccharide groups of the IgM prior to the spin-labelling. The spin-labelled IgM prepared in this way may be useful for the studies of dynamics of the IgM structure, particularly upon antigen binding.
