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Purification of an endogenous digitalislike factor from human plasma for structural analysis
J H Ludens1, M A Clark, D W DuCharme
1Upjohn Laboratories, Kalamazoo, Mich. 49001.
Hypertension (Dallas, Tex. : 1979)
|June 1, 1991
Summary
Researchers purified a naturally occurring digitalislike factor (EDLF) from human plasma. This substance, likely ouabain or an isomer, may regulate the sodium pump and electrolyte balance in humans.
Area of Science:
- Biochemistry
- Physiology
- Analytical Chemistry
Background:
- Previous studies reported the isolation and characterization of an endogenous digitalislike factor (EDLF).
- The precise structure and physiological role of EDLF remained to be fully elucidated due to limited quantities.
Purpose of the Study:
- To develop a robust purification strategy for obtaining sufficient quantities of EDLF for structural analysis.
- To identify the chemical structure of the endogenous inhibitor of Na+,K(+)-ATPase.
Main Methods:
- A multi-step purification process involving XAD-2 extraction, preparative HPLC, enzyme affinity extraction, and semipreparative chromatography was employed.
- Human plasma (300 L) was processed to isolate the target compound.
- Purified EDLF was characterized using chromatographic, mass spectrometric, immunoreactive, and binding assays.
Main Results:
- 31 micrograms (53 nmol) of pure EDLF were successfully isolated from 300 L of human plasma.
- The purification achieved an unprecedented enrichment factor exceeding 0.6 billionfold on a dry weight basis.
- Evidence strongly suggested the purified EDLF is ouabain or a structural isomer.
Conclusions:
- A highly effective purification method was established for isolating endogenous digitalislike factor (EDLF).
- The purified EDLF was identified as ouabain or an isomer, a naturally occurring ligand for the digitalis binding site.
- This finding suggests a potential role for EDLF in modulating the sodium pump and maintaining human electrolyte homeostasis.