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.

Design concepts in absorbance optical systems for analytical ultracentrifugation

“…Analytical methods in programs such as UltraScan are continually being developed for MWL data and feature tools to at least partially correct for spectrally correlated noise. 28 A subsequent investigation should consider how optical aberrations could influence results. For instance, defocus will produce a broader intensity distribution of the point image and will appear to broaden the sedimentation boundary.…”

Moving analytical ultracentrifugation software to a good manufacturing practices (GMP) environment

“…This approach follows in the footsteps of the openAUC architecture proposed earlier [ 16 ], and permits direct interfacing with the instrument over ethernet using alternative third-party software solutions like UltraScan. This avenue offers maximum flexibility and fosters development of new and interesting tools that can explore novel features like multi-wavelength experiments [ 19 , 20 , 21 , 22 , 23 , 24 ] and GMP capable data acquisition software, discussed here. Using the network-capable Linux tools provided with the Optima, our laboratory has extended the UltraScan-III AUC analysis software [ 13 , 14 ] and the UltraScan Laboratory Information Management Systems (UltraScan-LIMS) framework [ 15 ] to interface directly with the Optima for experimental design, data acquisition, and remote experiment monitoring.…”

In Vitro Analytical Approaches to Study Plant Ligand-Receptor Interactions

“…Sedimentation starts upon high speed centrifugation and the detector keeps monitoring it along the course of the experiment, typically 16 hours. The detector measures protein absorbance at a single wavelength (190 -800 nm) (Figure 1) (Cole et al, 2008); however, the use of multiple wavelengths is also possible, allowing for spectral deconvolution of interacting and non-interacting components in the sample (Johnson et al, 2018). In an SV experiment, binding affinities can be calculated through ligand titration and by plotting the sedimentation coefficients as a function of the receptor-ligand ratio (Figure 1) (Zhao et al, 2013).…”