Antigenic determinants in human lung elastin peptides

Elastin peptides were fractionated on a SE-Sephadex C-50 column by sequential elution with the following buffers: a) 0. 01 N acetic acid, b) linear gradient from 0. 05 N sodium acetate buffer, pH 4. 0 to 0. 4 N sodium acetate buffer, pH 4. 6, c) 0. 4 N sodium acetate buffer, pH 4. 6, d) 0. 5 N sodium hydroxide. Based on the elution profile, tubes were pooled into 12 fractions. Amino acid analysis of these fractions showed that the first 7 fractions contained mostly nonpolar peptides which lacked desmosine and isodesmosine residues. The other 5 fractions contained more polar peptides with desmosine and isodesmosine. Only polar fractions containing desmosine and isodesmosine proved to be antigenic when assayed by double immunodiffusion test. These antigenic peptide fractions were subjected to further analysis. Separation by gel filtration on Bio-Gel P-150 showed that each of the antigenic fractions consisted of at least 3 polypeptides with apparent molecular weights of 57, 000, 46, 000 and 9, 000. The two higher molecular weight species (polypeptides) were not completely resolved in this system. However, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis all the antigenic fractions gave three well separated bands with apparent molecular weights of 64, 000, 55, 000 and 14, 000. Relative percent protein distribution of these polypeptides was calculated from the SDS-polyacrylamide gel scan profile. The immunoreactivity of these individual polypeptides was further investigated by rocket Immunoelectrophoresis using Coomassie blue staining of the bands. All three bands produced rockets with the antiserum. Quantitative analysis of the rocket areas showed that the 14, 000 dalton polypeptides which accounted for only 2. 6% protein produced a larger rocket area than the 64, 000 and 55, 000 dalton polypeptides which accounted for 82. 9 and 14. 5% protein respectively. These results suggest that the 14, 000 dalton polypeptide has the highest immunoreactivity and the 64, 000 dalton polypeptide the least. Only the 14, 000 dalton molecule contains desmosine and isodesmosine, indicating that elastin antigenicity is not determined solely by the crosslinking amino acids. The results also suggest an uneven distribution of antigenic determinants in the elastin molecule and concentration of the antigenicity in a very small portion of the whole molecule.