A Rapid Sensitive Assay for the Quantitation of Elastin

Abstract

A rapid, sensitive and direct assay was developed for the quantitation of elastin using reverse phase high performance liquid chromatography. The method is based on the separation of the alkaline hydrolysis-resistant dipeptide valyl-proline, a dipeptide found in high concentrations in elastin. The methodology is applicable to unlabeled elastin samples in the range of 10-100 .mu.g utilizing fluorescamine detection and to hot alkali insoluble as well as soluble forms of elastin. Increased sensitivity was achieved by the use of in vitro radiolabeling of elastin with [3H]proline. This was useful for following synthesis of elastin in cultured cells. Since this technique is not dependent on detection of the elastin-specific crosslink groups, desmosine and isodesmosine, precursor forms of elastin can be measured as well as other forms of elastin deficient in cross-linking. The same chromatographic procedure however, is able to detect these desmosine and isodesmosine crosslinks. In cell cultures under the proper radiolabeling conditions, biosynthesis and cross-linking of elastin can be followed simulataneously in a single chromatographic procedure.