An 'Affinity ' Method for Preparing Polypeptides Enriched in the Collagen-Associated Ehrlich Chromogen

The collagen-associated compound which reacts at room temperature with acid dimethylaminobenzaldehyde (Ehrlichs reagent) also reacts in acid with aryl-diaz-onium reagents. We have developed a convenient method for isolating polypep-tides associated with the Ehrlich chromogen from collagen digests utilizing diazotized arylamino-cellulose supports. These peptides, in which the Ehrlich chromogen is 'labeled ' with yellow diazo color, constitute less than 0.5 % of the collagen, and have amino acid patterns similar to those around the cross link regions. The chromogen is not identical with pyridinoline, also thought to be a polyvalent cross link. We described the presence in connective tissues of material that reacted rapidly at room temper-ature with Ehrlich's reagent to produce a cherry red color (7). This material remained associated with collagen fragments which were non-dialyzable after digestion of the tissues with papain, pronase, pepsin, trypsin and collagenase. Its properties were consistent with those of a pyrrole, inter alia. It was suggested that the amino-ketone cross links e.g. dihydroxy lysino-norleucine might have given rise to it, by a Knorr reaction that occurs under very mild aqueous conditions (7). Indeed, the richest source of the Ehrlich chromogen so far examined is the 51 amino acid tripeptide (Col 1)2 XT9 (2) from the cross link region of human leiomyoma collagen (72). We present further ob-servations on the chemical reactivity of the Ehrlich chromogen. It is not identical with pyridinoline, a heterocyclic compound suggested (J) to be derived from 'maturing ' aldimine cross links. We have developed a simple 'affinity ' method, using a solid support, to separate Ehrlich-reactive poly-peptides from those which make up the greater part of the collagenous digest. It promises to simplify greatly the isolation, and the elucidation of the structure and environment in which the Ehrlich chromogen occurs