Histamine-releasing activity. V. Characterization and purification using high-performance liquid chromatography

Michael A. Lett-Brown, David O. Thueson, Diane E. Plank, Lawrence Duffy, J. Andrew Grant

Research output: Contribution to journalArticle

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Abstract

The production of large quantities of the lymphokine(s) histamine-releasing activity (HRA) and its partial purification by Sephadex G-75 and ion-exchange chromatography on carboxymethyl (CM) Sepharose 6B have been detailed (M. A. Lett-Brown, D. O. Thueson, D. E. Plank, M. P. Langford, and J. A. Grant, Cell. Immunol. 87, 434-444, 1984). Two peaks of activity (HRA I and II) were recovered. Preparations of HRA have now been analyzed by high-performance liquid chromatography (HPLC). Thoracic duct lymphocytes stimulated with 200 U/ml streptokinase were used as a source of HRA. Gel-filtration HPLC on a TSK 3000 column separated HRA into two peaks of activity (10,000-20,000 and 1300 Da). Reverse-phase high-performance liquid chromatography using a Nucleosil C-8 column showed that HRA II (the activity eluted at a conductivity of 18-20 mmho on the CM-Sepharose column) eluted as a single sharp peak, the main protein contaminant being cytochrome c, the carrier protein added to enhance the yield of HRA. High-performance liquid chromatography was found to be a useful analytical tool and may be suitable for the large-scale purification of HRA.

Original languageEnglish (US)
Pages (from-to)445-451
Number of pages7
JournalCellular Immunology
Volume87
Issue number2
DOIs
StatePublished - 1984

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Histamine
High Pressure Liquid Chromatography
Sepharose
Thoracic Duct
Streptokinase
Lymphokines
Ion Exchange Chromatography
Reverse-Phase Chromatography
Cytochromes c
Gel Chromatography
Carrier Proteins
Lymphocytes
Proteins

ASJC Scopus subject areas

  • Cell Biology
  • Immunology

Cite this

Histamine-releasing activity. V. Characterization and purification using high-performance liquid chromatography. / Lett-Brown, Michael A.; Thueson, David O.; Plank, Diane E.; Duffy, Lawrence; Grant, J. Andrew.

In: Cellular Immunology, Vol. 87, No. 2, 1984, p. 445-451.

Research output: Contribution to journalArticle

Lett-Brown, Michael A. ; Thueson, David O. ; Plank, Diane E. ; Duffy, Lawrence ; Grant, J. Andrew. / Histamine-releasing activity. V. Characterization and purification using high-performance liquid chromatography. In: Cellular Immunology. 1984 ; Vol. 87, No. 2. pp. 445-451.
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