Glutaraldehyde-induced D-band pattern of type II collagen fibrils as revealed by negative staining

A study was carried out on the negative staining band patterns (1% phosphotungstic acid (PTA) in phosphate buffer, 0.1 M, pH 7.4) exhibited by type II collagen fibrils after treatment with glutaraldehyde (5% in phosphate buffer 0.1 M, pH 7.4) and a comparison was made with the negative staining patterns of glutaraldehyde-fixed type I collagen fibrils. A characteristic D-band pattern was observed for type II collagen fibrils. The gap/overlap ratio was unusually low, with a 0.40 long gap zone and a 0.60 D overlap zone. This banding displayed eleven major light bands instead of the fifteen bands per period observed in the type I collagen patterns. On comparing the two types of microdensitograms, seven negative peaks (light bands) coincided and among them were the peaks corresponding to the N-terminal and C-terminal telopeptide regions. Although less numerous, the negative peaks of type II fibril traces were broader and more marked than those of type I microdensitograms and this feature accounts for the greater stain exclusion capacity of the type II fibrils. This is consistent with the larger quantity of hydroxylysines in type II collagen and, perhaps, with the more abundant hydroxyprolines.