Isolation and characterization of the reaction product of 4-diazobenzenesulfonic acid and γ-carboxyglutamic acid

Modification of the assay for measurement of β-carboxyaspartic acid

Satoru Nishimoto, Jingsong Zhao, Chhabil Dass

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

γ-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 γ-carboxyl groups in Gla with two DBS groups. DBS also reacts with another malonic acid derivative, β-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.

Original languageEnglish (US)
Pages (from-to)159-164
Number of pages6
JournalAnalytical Biochemistry
Volume216
Issue number1
DOIs
StatePublished - Jan 1 1994

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Reaction products
Assays
Acids
Proteins
Electrospray ionization
Electrospray Ionization Mass Spectrometry
Mass spectrometry
Derivatives
diazobenzenesulfonic acid
Complex Mixtures
Minerals
Cations
Spectrum Analysis
High Pressure Liquid Chromatography
Spectroscopy

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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title = "Isolation and characterization of the reaction product of 4-diazobenzenesulfonic acid and γ-carboxyglutamic acid: Modification of the assay for measurement of β-carboxyaspartic acid",
abstract = "γ-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 γ-carboxyl groups in Gla with two DBS groups. DBS also reacts with another malonic acid derivative, β-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.",
author = "Satoru Nishimoto and Jingsong Zhao and Chhabil Dass",
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T2 - Modification of the assay for measurement of β-carboxyaspartic acid

AU - Nishimoto, Satoru

AU - Zhao, Jingsong

AU - Dass, Chhabil

PY - 1994/1/1

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N2 - γ-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 γ-carboxyl groups in Gla with two DBS groups. DBS also reacts with another malonic acid derivative, β-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.

AB - γ-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 γ-carboxyl groups in Gla with two DBS groups. DBS also reacts with another malonic acid derivative, β-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.

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