Multi-wavelength analytical ultracentrifugation of human serum albumin complexed with porphyrin
Challenge
The Analytical Ultracentrifugation (AUC) required an
enhanced mode of separation based on distinct optical properties.
The Cölfen detector was constrained by its limited
sensitivity in the UV spectrum.
The Optima AUC's method of capturing wavelengths
sequentially, rather than simultaneously, posed complexities in multi-wavelength data
analysis.
The inherently poor solubility of protoporphyrins in
water led them to form non-reactive aggregates.
Difficulties arose in quantifying the molar
extinction coefficients and in distinguishing the proportion of Human Serum Albumin (HSA)
complexed in aqueous solutions.
Solution
Integrated multi-wavelength detection capabilities into
the AUC.
Leveraged the Optima AUC instrument, which exhibited
enhanced sensitivity in the low UV range.
Adapted the UltraScan method to accommodate and decipher
data derived from Optima AUC's multi-wavelength process.
Perfected the sample preparation process to improve
solubility and ensure primary bonding between porphyrins and apo-HSA.
Employed MWL-AUC combined with UV/visible
spectral analysis to isolate extinction signals, providing a clearer perspective on
protein-porphyrin interactions.
Conclusion
Multi-wavelength SV experiments can effectively quantify
distinct components in mixtures with similar hydrodynamic properties based on unique
absorbance spectra.
These data help determine extinction coefficients for
species hard to obtain in pure form.
UltraScan's new AUC analysis tools enhance the analysis
of multi-wavelength data from the Beckman Coulter Optima AUC instrument.
Multi-wavelength AUC is ideal for
characterizing complex mixtures with unique absorbance spectra components.
