Quantitative high-performance affinity chromatography: evaluation of use for analyzing peptide and protein...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Chromatography

Background:

• Macromolecular interactions are crucial in biological systems.
• Characterizing these interactions often requires sensitive and accurate analytical methods.
• High-performance affinity chromatography (HPAC) offers potential for microscale analysis.

Purpose of the Study:

• To evaluate analytical high-performance affinity chromatography as a microscale method for characterizing macromolecular interactions.
• To determine equilibrium dissociation constants for hormone-protein binding.
• To identify suitable pressure-resistant affinity supports for HPAC.

Main Methods:

• Utilized Arg8-vasopressin and bovine neurophysin II immobilized on novel affinity supports.
• Employed zonal elution of labeled and unlabeled peptide hormone.
• Extended theoretical treatment of analytical affinity chromatography.
• Used ultraviolet absorbance for continuous elution monitoring.

Main Results:

• Successfully determined equilibrium dissociation constants for hormone binding to immobilized neurophysin II.
• Achieved quantitative recovery of microamounts of hormone (0.05-15 micrograms) within 20-30 minutes.
• Demonstrated good agreement between measured constants and solution-based values.
• Identified silica-, agarose-, and glass-based matrices as effective pressure-resistant affinity supports.

Conclusions:

• Analytical HPAC is a viable microscale method for characterizing macromolecular interactions.
• The developed system accurately quantifies hormone-protein binding constants.
• Several new affinity supports are suitable for HPAC applications in high-performance liquid chromatography.