A new procedure for the isolation of dityrosine has been developed. Tyrosine was oxidized by means of incubation both with hydrogen peroxide and horse-radish peroxidase. The reaction mixture was separated by permeation chromatography on Sephadex G-10 being monitored at 280 and 310 nm spectrophotometrically. The dityrosine fraction was freeze-dried and purified on a cation-exchange column (in acidic citrate buffer). The purified fraction was desalted and freeze-dried. The yield was 96 mg of homogenous dityrosine per 1 g of D,L-tyrosine. Some physico-chemical constants of the preparation were measured (optical characteristics with U.V. and I.R. spectra, fluorescence spectra, chromatography on an amino acid analyzer).
The possible presence of dityrosine in elastin derived by two different methods and in structural glycoproteins from aortas of 1 day old chicks, adult rabbits and fetal rabbits was determined by a sensitive spectrofluorimetric procedure. Only chick tissues were found to contain dityrosine, 0.3 residues/100,000 total amino acid residues in aortic elastin and 12-15 residues/100,000 residues in the structural glycoproteins. No dityrosine could be detected in any of the fetal or mature rabbit tissues. However, related fluorescent compounds with different excitation-emission maxima and different elution times were obtained by ion exchange chromatography of structural glycoproteins partially hydrolyzed under alkaline conditions.
A quantitative study of the individual steps in the isolation of elastin from mature and fetal aortas of rabbits was carried out. The proportions of insoluble residue and acetone- and ether-soluble substances were determined. The yields of elastin and other associated components of connective tissue derived from the aortas by two different isolation procedures were compared, i.e. the classical Lansing treatment with hot alkali and the less drastic procedure described by Rasmussen et al. Results were found to be strongly influenced by the method used. The purity of the elastin preparations was evaluated by means of amino acid analyses for relative contents of desmosines and dicarboxylic amino acids. The possible reasons for the differences found are discussed.
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