"GA-banding": a new terminology and a study of the glutaraldehyde-induced band pattern of type I collagen fibrils

The negative staining D-band patterns of glutaraldehyde-reacted collagen fibrils were compared to those of fresh collagen fibrils. Negative staining was obtained by using 1% phosphotungstic acid (PTA) diluted in phosphate buffer 0.1 M, pH 7.4. The stain was dripped onto grids where native type I collagen fibrils, isolated from bovine dermis, were collected. Ultrastructural pictures were digitized to form microdensitometric traces. The glutaraldehyde-induced patterns showed fifteen light bands (micrographs) or negative peaks (microdensitograms), whose D-locations were constant and characteristic. In order to make this ultrastructural feature a precise reference parameter, these bands were called "GA-bands" and numbered. When comparing this averaged microdensitogram with that of negatively stained fresh fibrils, peak "GA1" and peak "GA7" were observed to correspond to peak "X2" (known as N-terminal telopeptide region) and peak "X3" (known as C-terminal telopeptide region) respectively, while there was no correspondence between the other peaks of the two traces. It means that the regions where preexistent crosslinks exist are unaffected by interaction with glutaraldehyde, while in the other regions, where new glutaraldehyde-crosslinks occur, the band pattern modifies. The unchanged D-location of peaks "GA1" and "GA7" leads to the conclusion that the D-shortening induced by glutaraldehyde is not due to shifting of tropocollagen molecules but to changes in their orientation with respect to fibril long axis or in secondary-tertiary structure of collagen.