Multi‐wavelength Analytical Ultracentrifugation of Human Serum Albumin complexed with Porphyrin

Abstract

Analytical Ultracentrifugation (AUC) is the method of choice to characterize colloidal systems under physiological solution conditions. Recently, a novel multi-wavelength (MWL) detector has been developed which for the first time couples the hydrodynamic separation of colloidal mixtures to spectral deconvolution of interacting and non-interacting solutes present in a mixture. We show here how one can take advantage of the Soret band of porphyrins complexed with different cations to spectrally deconvolute dissimilar optical properties from different molecules. MWL-AUC can baseline-separate even minute changes in hydrodynamic shape and size between HSA-PPIX and apo-HSA. Several sites in Human Serum Albumin (HSA) have been postulated to possess the ability to directly bind metal ions as well as organometallic compounds. This evidence has generated interest in the physiological role of HSA as a metal binding protein but also as a vehicle for synthetic biology uses of the metal binding properties (e.g., synthetic blood and solar energy conversion). One of the most intriguing and potentially useful among these interaction is the ability of HSA to bind heme and other metallo-porphyrins. We 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. Support or Funding Information This work was supported by NIH grant GM120600 and NSF grant NSF-ACI-1339649 (to BD). Supercomputer calculations were performed on Comet at the San Diego Supercomputing Center (support through NSF/XSEDE grant TG-MCB070039N to BD) and on Lonestar-5 at the Texas Advanced Computing Center (supported through UT grant TG457201 to BD). We thank Beckman-Coulter for providing an Optima AUC instrument to conduct these studies, and Eric von Seggern (Beckman Coulter) for helpful discussions to facilitate these experiments. 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) 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. 3D view of a single scan from a simulated, time-synchronized MWL-AUC dataset derived from the iterative 2DSA models. Hydrodynamic properties as a function of wavelength depicting spectral properties This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.