Fluorescent‐labeled crosslinks in collagen: Pyrenebutyrylhydrazine

The aldehydes present in acid‐soluble type I collagen react with pyrenebutyrylhydrazine to form various types of complexes under different reaction conditions. These complexes exhibit one or more of three different pyrene fluorescence bands: monomer, excimer, and aggregate fluorescence. Collagen, whose aldehydes have been reduced with NaBH 4 , does not react with this fluorescent hydrazine, confirming that the hydrazine reacts specifically with aldehyde groups to form hydrazones. The absence of a reaction with pepsin‐treated collagen also shows that the fluorescent labels are primarily in the nonhelical terminal telopeptides. Upon dialysis, the pyrene label bound to a saturated aldehyde in an α‐chain is lost; whereas that bound to an unsaturated aldehyde remains on the protein. The pyrene monomer fluorescence in the β‐chain of old collagen is stronger than that of young collagen. The formation of the pyrene excimer fluorescence implies the proximity of two pyrene molecules, probably attached to two adjacent aldehydes. Upon changing from acidic to neutral pH, both excimer and aggregate fluorescence bands disappear within a few seconds, revealing a very rapid alteration at the telopeptides.