| Popis: |
The development of the fluorescence detection system (Aviv-FDS) for the AUC allows a single fluorescently labeled species to be quantitatively characterized against a highly concentrated and heterogeneous background. During our use of the FDS to characterize ELP, a novel drug delivery vector (see Lyons et al., Biophys J 104:2009–2021, 2013), in serum, we encountered the Johnston-Ogston (J-O) effect. The J-O effect is a classical anomaly in sedimentation velocity theory and practice describing the nonideal sedimentation properties of a component as a function of high concentrations of other components. We examined the J-O effect using recent advances in AUC hardware, the AU-FDS (AVIV Biomedical), and data analysis methods, DCDT+ and SEDANAL global direct boundary fitting. We empirically quantified the self- and cross-sedimentation nonideality properties of ELP and the two most ubiquitous serum proteins, albumin (∼35–40 mg/ml) and γ-globulins (∼10–15 mg/ml). We have verified and measured the presence of cross-term hydrodynamic nonideality by running SV studies on a fluorescently labeled component (∼100 nM) in a titration experiment with high concentrations of unlabeled components. This has been accounted for through the introduction of a 3 × 3 nonideality matrix of Ks values into SEDANAL. ELP experiments with mixtures of albumin and γ-globulins were also performed in an attempt to recapitulate the J-O behavior of a serum solution. Clearly, other components or effects contribute to the serum J-O effect. Additional experiments with lipids, lipidated serum albumin, and PEG solutions are planned. These studies lay the groundwork for bringing quantitative hydrodynamic analyses into crowded environments and will allow measurement of hydrodynamic and equilibrium macromolecular properties in a physiological state. |
