Identification of an intermediate state in the helix-coil degradation of collagen by ultraviolet light.
Differential scanning calorimetry has revealed the presence of a new denaturation endotherm at 32 degrees C following UV irradiation of collagen, compared with 39 degrees C for the native triple helix. Kinetic analyses showed that the new peak was a previously unknown intermediate state in the collagen helix-coil transition induced by UV light, and at least 80% of the total collagen was transformed to random chains via this state. Its rate of formation was increased by hydrogen peroxide and inhibited by free radical scavengers. SDS-polyacrylamide gels showed evidence of competing reactions of cross-linking and random primary chain scission. The cross-linking was evident from initial gelling of the collagen solution, but there was no evidence for a dityrosine cross-link. Primary chain scission was confirmed by end group analysis using fluorescamine. Electron microscopy showed that the segment long spacing crystallites formed from the intermediate state were identical to the native molecules. Clearly, collagen can undergo quite extensive damage by cleavage of peptide bonds without disorganizing the triple helical structure. This leads to the formation of a damaged intermediate state prior to degradation of the molecules to short random chains.
Miles
C A
CA
University of Bristol, Collagen Research Group, School of Veterinary Science, Langford, Bristol BS40 5DU, United Kingdom. chris.miles@bristol.ac.uk
Sionkowska
A
A
Hulin
S L
SL
Sims
T J
TJ
Avery
N C
NC
Bailey
A J
AJ
D000818:Animals; D002152:Calorimetry, Differential Scanning / Q000379:methods; D003094:Collagen / Q000737:chemistry* / Q000528:radiation effects* / Q000648:ultrastructure; D003460:Crystallization; D016166:Free Radical Scavengers / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D017665:Hydroxyl Radical / Q000494:pharmacology; D007700:Kinetics; D008854:Microscopy, Electron; D017433:Protein Structure, Secondary / Q000528:radiation effects*; D051381:Rats; D013623:Tail; D013710:Tendons; D013816:Thermodynamics; D014466:Ultraviolet Rays
D016166:Free Radical Scavengers; D017665:Hydroxyl Radical; D003094:Collagen; D006861:Hydrogen Peroxide