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

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Abstract
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.degree. C and completely degraded between 37.degree. C and 39.degree. C. In subsequent experiments the same assay was carried out and 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.