Elsevier

Analytical Biochemistry

Volume 242, Issue 2, 15 November 1996, Pages 202-213
Analytical Biochemistry

Regular Article
Dityrosine: Preparation, Isolation, and Analysis

https://doi.org/10.1006/abio.1996.0454Get rights and content

Abstract

This article describes chromatographic and spectroscopic techniques that have multiple applications in the preparation, isolation, and analysis of dityrosine. A three-step chromatographic procedure facilitates the preparation of 120 mg or more (yield >26% of theoretical maximum) of dityrosine from the enzyme-catalyzed oxidation of tyrosine. DEAE–cellulose chromatography performed in a boric acid–sodium borate buffer removes most of the contaminating pigments. Two-dimensional pH-dependent chromatography on BioGel P-2 separates dityrosine from tyrosine, residual pigments, salts, etc. Elemental analysis indicates that the purified product is ∼92% dityrosine by weight. Fast atom bombardment mass spectrometry and two types of reverse-phase high-performance liquid chromatography (HPLC), monitored in fluorescence and absorbance measurements, verify the purity of the dityrosine. The distinctive pH-dependent chromatography of dityrosine on BioGel P-2, with reversible adsorption to the matrix occurring at pH values less than 3, is useful for the isolation of varying quantities of dityrosine and for analysis per se. Affinity chromatography on immobilized phenyl boronate (Matrex Gel PBA-60) is an alternate method for the isolation and determination of dityrosine, which undergoes specific interactions with the boronate moiety and possible hydrophobic association with the phenyl group. Two new reverse-phase HPLC techniques expedite the analysis of picomole quantities of dityrosine. One employs isocratic elution (92% H2O, 8% acetonitrile, and 0.1% trifluoroacetic acid) of an ODS II Spherisorb column, with both fluorometric and spectrophotometric detection. The other procedure may be performed in conjunction with total amino acid analysis. A rapid gradient program, developed with a Phenomenex Ultracarb 20 column, clearly separates dabsylated dityrosine and tyrosine from other dabsylated amino acids. It is especially useful when dityrosine is a trace component.

References (0)

Cited by (170)

View all citing articles on Scopus

This work was supported by a research grant from the National Institutes of Health (USPHS DK13912).

2

To whom correspondence should be addressed. Fax: (541) 737-0481.

View full text