LC/ESI-MS analysis of two elastin cross-links, desmosine and isodesmosine, and their radiation-induced degradation products.

In this work, the effect of Fenton reaction on two elastin cross-linked amino acids, desmosine (DES) and isodesmosine (IDE), in the absence or presence of different wavelength radiations generated from artificial sources has been evaluated using LC/ESI-MS. Irradiation as well as incubation of DES or IDE solutions in the presence of Fe(2+) and H(2)O(2) resulted in products with m/z 497.1 and 481.1 for [M+H](+). A strongly dose-dependent degradation of both amino acids was observed upon exposure to UVB at doses ranging from 0 to 3 J/cm(2) and a moderate dose-dependent degradation upon exposure to UVA at doses 10 times higher than that of UVB. A significant time-dependent degradation of DES and IDE was also observed upon exposure of these amino acids to a lamp emitting visible light similar to sunlight. Exposure of both amino acids to IR radiation (520 W) for 8 h did not cause significant degradation.

Photolysis and ozonolysis of desmosine and elastolytic peptides.

This paper describes the combined effects of photolysis and ozonolysis upon the main interchain crosslinks of elastin, desmosine and isodesmosine. Photolysis of purified (iso)desmosines in solution leads to the cleavage of the pyridinium rings to give lysine and an analogue of glutaconic aldehyde which is deformylated to a stable compound absorbing at 242 nm. This photoproduct is subsequently fragmented by ozone into glutamic, alpha-aminoadipic and possibly alpha-amino-delta-oxocaproic acids. However the yields of these different compounds are very low because we observed that numerous competing side reactions (polymerisation, recyclisation) accompany the photoozonolytic decomposition of the pyridinium rings of free (iso)desmosines. The results are clearer when reticulated elastolytic peptides are photoozonolysed. The (iso)desmosines, covalently linked in these peptides, are cleaved into lysine, glutamic and alpha-aminoadipic acids (in a ratio 2:1) with yields corresponding to 80-90% of those expected from the decomposition of the (iso)desmosines originally present in the peptide fraction. We have also observed that ozonolysis alone degraded another crosslink present in these peptides, the aldol condensation product, resulting in the production again of glutamic and alpha-aminoadipic acids in amounts consistent with the known concentrations of this particular crosslink in elastin. Finally we noted that the complete photoozonolytic degradation of the (iso)desmosines present in a semi purified reticulated elastolytic fraction resulted in a shift of the size distribution of these peptides toward lower values. It is not certain however that this shift, indicative of a freeing of the polypeptide chains from their original three dimensional network, is due uniquely to the cleavage of the (iso)desmosines. Indeed we have observed that tyrosine and phenylalanine were also degraded during photoozonolysis. Not knowing the mechanism of this degradation it is impossible to rule out the possibility that concomitant cleavages of peptide bonds did occur.

Photolysis of desmosine and isodesmosine by ultraviolet light.

1. Desmosine and isodesmosine were separated by ion-exchange and paper chromatography, after acid hydrolysis of purified elastin from beef ligamentum nuchae. The fractions obtained by ion-exchange chromatography were clearly mixtures of related compounds. The desmosine fraction could be resolved into seven compounds and the isodesmosine into four by paper chromatography. 2. Desmosine was maximally degraded by irradiation at 274 nm and isodesmosine at 285 nm. These wavelengths did not correspond to the absorption maxima of the cross links, but to shoulders of the main absorption peaks. 3. When irradiated at their optimum wavelengths, but at various pH, both desmosine and isodesmosine seemed quite stable at pH greater than 8.5. Between pH 8 and 5, the photolytic rate was maximum and decreased slightly at more acidic pH. Below pH 4.0, one of the products of photolysis was free lysine. 4. In analogy to the mechanism of the photolytic degradation of N-methyl pyridinium chloride, it appears that the (iso)desmosines were degraded via the formation of an open amino aldehyde, which was hydrolysed at acid pH to give free lysine and a substituted glutaconic aldehyde.