Isolation and characterization of the reaction product of 4-diazobenzenesulfonic acid and gamma-carboxyglutamic acid: modification of the assay for measurement of beta-carboxyaspartic acid.

gamma-Carboxyglutamic acid (Gla)-containing proteins are extracellular proteins with enhanced cation or mineral-binding properties. Discovery of new Gla-containing proteins is facilitated by methods that decrease the number of steps and time involved in assaying for Gla. Reaction of 4-diazobenzenesulfonic acid (DBS) and Gla or Gla-containing proteins produces an intensely red-colored product. This method has been used to identify Gla-containing proteins in crude extracts of proteins. The reaction product of Gla and DBS has been purified by reversed-phase HPLC and characterized by uv-visible spectroscopy and electrospray ionization (ESI) mass spectrometry (MS). The red-colored product exhibits an absorption maximum at 530 nm. The ESI-MS data of the colored derivative of Gla are consistent with replacement of the two gamma-carboxyl groups in Gla with two DBS groups. DBS also reacts with another malonic acid derivative, beta-carboxyaspartic acid (Asa). The optimum conditions for colorimetric assay of Asa were established, and the potential of this reaction as an assay for Asa and Asa-containing proteins was studied.