A new mapping technique for collagen chains

A new, highly sensitive method for peptide mapping of collagen chains has been developed which utilizes a modification of the radioiodination technique in polyacrylamide gels described by Elder et al. (1977b). Optimal conditions include the use of the Bolton-Hunter reagent to produce 125I-labeled collagen with the enzyme proteinase K, prior to resolution of the cleavage products by two-dimensional electrophoresis and chromatography. Unambiguous results were obtained by restricting comparison among collagens to a given set which was radioiodinated using the same procedure, i.e., in solution, or in a dried or hydrated gel. Iodination of collagens in solution, followed by proteinase K digestion, resulted in highly reproducible maps which were free of background contamination and which permitted characterization of chains with a defined mobility of SDS-PAGE after the iodination procedure. This technique has provided additional evidence that the alpha 1, alpha 2, and alpha 3 chains of type V collagen are structurally unique. In addition, relationships among several fragments from pepsin-treated type IV collagen, which consists of two distinct chains, could be further elucidated.