Multi-wavelength analytical ultracentrifugation of human serum albumin complexed with porphyrin

Abstract

The new Beckman Coulter Optima AUC instrument, which features multi-wavelength detection that couples the hydrodynamic separation of colloidal mixtures to spectral deconvolution of interacting and non-interacting solutes present in a mixture, was used to analyze the composition of human serum albumin (HSA) bound to metallo-protoporphyrin. We present new methods implemented in UltraScan that permit Optima AUC-derived multi-wavelength data to be spectrally decomposed in the same fashion as has been made possible for the Cölfen detector earlier. We demonstrate this approach by spectrally separating sedimentation velocity experimental data from mixtures of apo-HSA and HSA complexed to different metallo-protoporphyrins. We further demonstrate how multi-wavelength AUC can accurately recover percentages of metallo-protoporphyrin-bound HSA and apo-HSA from mixtures and how multi-wavelength AUC permits the calculation of molar extinction coefficients for porphyrins bound to HSA. The presented method has broad applicability to other complex systems where mixtures of molecules with different spectral properties need to be characterized.

Keywords: Human serum albumin; Multi-wavelength analytical ultracentrifugation; Porphyrin; Spectral analysis; UltraScan.

Figure 1

Left: Overlay of normalized spectra for apo-HSA (black), HSA complexed with porphyrin (PPIX) bound to either Zn (blue) or Sn (red); center: HSA complexed to porphyrin bound to Sn (red) and difference spectrum when the HSA absorbance is subtracted (black); right: HSA complexed to porphyrin bound to Zn (blue) and difference spectrum when the HSA absorbance is subtracted(black)

Figure 2

Weight-averaged meniscus offset positions from six measured menisci as a function of wavelength (blue circles), fitted with a 5th-order polynomial (red line). Green, cyan, blue and magenta lines: Variation of meniscus positions for 4 separate samples measured after chromatic aberration correction. All measured values are within the radial resolution of the Optima AUC

Figure 3

Wavelength selection for HSA complexed to porphyrin bound to Sn (blue) and Zn (red). Selected wavelength are optimally distributed along the spectrum to capture unique chromophore information between 240–430 nm

Figure 4

PCSA-Monte Carlo analysis of decomposed multi-wavelength SV data for four different mixtures of apo-HSA (red) and HSA-PPIX (blue) with increasing concentration of apo-HSA from left to right. Top row: HSA-PPIX-Sn, bottom row:HSA-PPIX-Zn