Abstract
The relative unknown conformational stability of monovalent bulks of influenza virus haemagglutinin (HA) from three different strains (B/Guangdong, A/New Caledonia and A/Panama) was investigated with fluorescence and circular dichroism (CD) spectroscopy. Various stress conditions (concentration of denaturant, freeze-thawing, pH and temperature) affected the spectroscopic properties of the haemagglutinin proteins differently. Unfolding experiments revealed a poor stability of Guangdong haemagglutinin (GD-HA) in comparison with New Caledonia (NC-HA) and Panama haemagglutinin (P-HA). Freeze-thawing altered the secondary and tertiary structure of Guangdong haemagglutinin and only the tertiary structure of Panama haemagglutinin. From pH 4.6-9.2 the tertiary structures of Guangdong, New Caledonia and Panama haemagglutinin were all affected to a different extent. The secondary structure was only altered at low pH. Incubation of haemagglutinin at 60°C resulted in denaturation of the protein and a dramatic change of the fluorescence spectrum, indicative of oxidised tryptophan (Trp). In conclusion, fluorescence and circular dichroism spectroscopy are highly suitable techniques to monitor the stability of haemagglutinin in a straightforward and fast way.
| Original language | English |
|---|---|
| Pages (from-to) | 65-75 |
| Journal | European Journal of Pharmaceutical Sciences |
| Volume | 23 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - Sept 2004 |
| MoE publication type | A1 Journal article-refereed |
Funding
The authors are grateful to Gideon Kersten (Unit Research and Development, Netherlands Vaccine Institute (NVI)) for his help concerning the fluorescence measurements and to Suzanne Hermeling (Department of Pharmaceutics, Utrecht University) for her assistance during the CD measurements.
Keywords
- Circular dichroism spectroscopy
- Fluorescence spectroscopy
- Influenza virus haemagglutinin
- Physicochemical characterisation
- Stability
