Julius A. Allen - Delaware State University
Co-Author(s): Hacene Boukari, Delaware State University, Dover, DE
Macromolecular crowding, the presence of relatively high concentration of macromolecules in a solution, alters significantly the chemical and physical properties of a molecule or macromolecule. Examples are structure, shape, conformational stability, binding of small molecules, enzymatic activity, and aggregation. It occurs since high concentrations of macromolecules (e.g. polymers, proteins, DNA) reduce the volume of water available for other molecules, resulting in increase of their effective concentrations. In this study we demonstrate how fluorescence spectroscopy and fluorescence correlation spectroscopy (FCS) can be applied to probe the effect of polymeric Polyethylene Glycol (PEG) solutions (up 800 mg/ml) on the behavior of Alexa488 fluorophores. Our focus is on assessing changes of the translational diffusion of Alexa488 within PEG solutions. PEG is a linear polymer that is molecularly formed by adjoining strands of ethylene glycol. PEG is one of the few water-soluble, biocompatible polymer that is approved by FDA for use in food industry, pharmaceutical products, and medicine. Alexa488 is a bright, photostable fluorophore that be readily excited with a 488-nm laser line and with emission around 520 nm. We used ISS ALBA spectrometer to carry out fluorescence correlation spectroscopy (FCS) measurements on these samples of PEG and Alexa488 mixtures prepared at different PEG concentrations. Analysis of the data yields changes of the translational diffusion of Alexa488 in these solutions. We find that measured FCS correlations of Alexa488 show systematic and uniform shift to longer delay times, indicating slowing down of Alexa488 diffusion. More interestingly, changes of the apparent diffusion coefficient appear exponential with increase of PEG concentration. Although this result is consistent with slowing down of the translation of Alexa488 due to friction with PEG polymers, it cannot be accounted for by the increase of the bulk viscosity of the PEG solution as would be suggested by the Stokes-Einstein relation. Alternatively, we introduce the entropic model proposed by de Gennes and his collaborators for the interpretation of the data. The results of this work will be the basis for future studies of the effects of PEG on biological molecules such as proteins and DNA.
Funder Acknowledgement(s): NSF CREST-8763, DoD W911NF-14-1-0454, NNSA DE-NA0002683, NIH Delaware INBRE P20 RR016472
Faculty Advisor: Hacene Boukari, firstname.lastname@example.org
Role: Sample preparation, collection of FCS data