The A and B fragments of normal type I procollagen have a similar thermal stability to proteinase digestion but are selectively destabilized by structural mutations.

Previous studies demonstrated that the thermal stability of the procollagen triple helix can be assayed by digesting the protein for short periods with high concentrations of trypsin and chymotrypsin. Here we cleaved human type I procollagen or collagen with vertebrate collagenase to generate A fragments from the three-quarter amino termini and B fragments from the one-quarter carboxy termini of the molecules. The thermal stabilities of the fragments were then assayed by rapid trypsin/chymotrypsin digestion. Both fragments were resistant up to 36 degrees C and completely degraded between 37 degrees C and 39 degrees C. In subsequent experiments the same assay was carried out with type I procollagens synthesized by fibroblasts from two patients with lethal variants of osteogenesis imperfecta. With one, the A fragments were selectively destabilized, an observation consistent with previous data indicating that the mutation in the patient produced a deletion of 84 amino acids from the middle of the alpha 1(I) chain. With procollagen synthesized by fibroblasts from the second patient the B fragments were selectively destabilized, an observation consistent with preliminary data indicating a mutation that alters the primary structure of the carboxy-terminal region of the alpha 1(I) chain. Therefore, the procedures described here present a simple and direct method for locating mutations that destabilize the collagen triple helix.