The collagen fibril—A model system for studying the staining and fixation of a protein

A collagen fibril is made up of long rod-like molecules regularly D-staggered with respect to one another. This means that (i) its axially projected fine structure, resolvable to ∼ 2 nm in electron micrographs, repeats D-periodically (D = 67 nm), and (ii) the amino acid residues contributing to each element of the fine structure can be inferred from sequence data. Electron-optical data from a fibril D-period can therefore be correlated directly with chemical data. Such correlations confirm the electrostatic nature of the staining reaction when a fibril is positively stained. After negative staining, the principal factor determining the small-scale distribution of stain is local exclusion by ‘bulky’ amino acid side-chains. (‘Bulkiness’ is the average cross-sectional area, or ‘plumpness’, of a side-chain.) A small superimposed positive staining contribution can also be detected. Fixation of collagen by aldehydes and diimidoesters occurs via an initial reaction with lysyl (and hydroxylsyl) side-chains and α-amino groups, followed by secondary cross-linking reactions that differ from fixative to fixative. These secondary reactions determine the nature and abundance of the cross-links and the extent to which they influence subsequent staining behaviour.