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

Area of Science:

• Immunology
• Biochemistry
• Protein Chemistry

Background:

• Histamine-releasing activity (HRA), a lymphokine, was previously partially purified using Sephadex G-75 and CM-Sepharose 6B chromatography.
• Two distinct peaks of HRA activity were identified in earlier preparations.

Purpose of the Study:

• To analyze purified histamine-releasing activity (HRA) using high-performance liquid chromatography (HPLC).
• To assess the suitability of HPLC for large-scale purification of HRA.

Main Methods:

• Lymphocytes were stimulated with streptokinase to produce HRA.
• Gel-filtration HPLC on a TSK 3000 column separated HRA into two molecular weight fractions.
• Reverse-phase HPLC on a Nucleosil C-8 column analyzed HRA II, identifying cytochrome c as a contaminant.

Main Results:

• HPLC analysis confirmed two HRA activity peaks, with molecular weights of 10,000-20,000 Da and 1300 Da.
• HRA II eluted as a single sharp peak in reverse-phase HPLC.
• Cytochrome c was identified as the primary protein contaminant in HRA preparations.

Conclusions:

• High-performance liquid chromatography is a valuable analytical tool for characterizing HRA.
• HPLC demonstrates potential for the efficient, large-scale purification of HRA.