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Published on: October 1, 2009
Recovery of biologically active enzymes after HPLC separation
M Herold1, G P Rozing, J L Curtis
1Hewlett-Packard GmbH, Analytical Division, Germany.
Biotechniques
|May 1, 1991
Summary
High-performance liquid chromatography (HPLC) effectively recovers enzyme activity. Most enzymes tested showed 90-100% activity recovery, regardless of HPLC instrument materials, ensuring reliable protein analysis.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Chromatography
Background:
- Enzyme activity recovery is critical for accurate protein analysis.
- High-performance liquid chromatography (HPLC) is a common separation technique.
- Potential interactions between HPLC components and sensitive enzymes are a concern.
Purpose of the Study:
- To evaluate the impact of HPLC instruments and column hardware on enzyme activity recovery.
- To determine if stainless steel or titanium flow paths affect enzyme stability.
- To assess the compatibility of various chromatography modes with enzyme activity preservation.
Main Methods:
- Tested eight enzymes (monomeric and oligomeric) with molecular masses from 25,000 to 240,000 Da.
- Utilized three HPLC instruments with stainless steel and titanium flow paths.
- Employed ion-exchange, size-exclusion, and hydrophobic-interaction chromatography with glass and stainless steel hardware.
Main Results:
- Achieved 90-100% activity recovery for most enzymes.
- No significant negative impact on enzyme activity was observed due to HPLC instrument materials (stainless steel, titanium) or column hardware (glass, stainless steel).
- A single instance of significant activity loss (30%) occurred with one specific ion-exchange column and enzyme.
Conclusions:
- HPLC separation is suitable for maintaining enzyme activity.
- The materials used in HPLC instruments and columns do not generally compromise enzyme recovery.
- Careful column selection is important for specific enzyme-column combinations.
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