ABSTRACT
The understanding of lipid bilayer structure and function has been advanced by the application of molecular fluorophores. However, the effects of these probe molecules on the physicochemical properties of membranes being studied are poorly understood. A quartz crystal microbalance with dissipation monitoring instrument was used in this work to investigate the impact of two commonly used fluorescent probes, 1palmitoyl2{12[(7nitro21,3benzoxadiazol4yl)amino]dodecanoyl}snglycero3phosphocholine (NBD-PC) and 1,2dipalmitoylsnglycero3phosphoethanolaminen(lissamine rhodamineBsulfonyl) (Lis-Rhod PE), on the formation and physicochemical properties of a 1palmitoyl2oleoylsnglycero3phosphocholine supported lipid bilayer (POPC-SLB). The interaction of the POPC-SLB and fluorophore-modified POPC-SLB with docosahexaenoic acid, DHA, was evaluated. The incorporation of DHA into the POPC-SLB was observed to significantly decrease in the presence of the Lis-Rhod PE probe compared with the POPC-SLB. In addition, it was observed that the small concentration of DHA incorporated into the POPC:NBD-PC SLB can produce rearrangement processes followed by the lost not only of DHA but also of POPC or NBD-PC molecules or both during the washing step. This work has significant implications for the interpretation of data employing fluorescent reporter molecules within SLBs.
Subject(s)
Docosahexaenoic Acids/metabolism , Fluorescent Dyes/pharmacology , Lipid Bilayers/metabolism , Phosphatidylcholines/metabolism , 4-Chloro-7-nitrobenzofurazan/analogs & derivatives , 4-Chloro-7-nitrobenzofurazan/chemistry , 4-Chloro-7-nitrobenzofurazan/pharmacology , Docosahexaenoic Acids/pharmacokinetics , Fluorescent Dyes/chemistry , Lipid Bilayers/chemistry , Molecular Conformation , Phosphatidylcholines/chemistry , Phosphatidylcholines/pharmacokinetics , Phosphatidylcholines/pharmacology , Quartz Crystal Microbalance Techniques , Rhodamines/chemistry , Rhodamines/pharmacologyABSTRACT
Docosahexaenoic acid (DHA) is the most abundant polyunsaturated omega-3 fatty acid found in mammalian neuronal cell membranes. Although DHA is known to be important for neuronal cell survival, little is know about how DHA interacts with phospholipid bilayers. This study presents a detailed quartz crystal microbalance with dissipation monitoring (QCM-D) analysis of free DHA interactions with individual and mixed phospholipid supported lipid bilayers (SLB). DHA incorporation and subsequent changes to the SLBs viscoelastic properties were observed to be concentration-dependent, influenced by the phospholipid species, the headgroup charge, and the presence or absence of calcium ions. It was observed that 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) SLBs incorporated the greatest amount of DHA concentration, whereas the presence of phospholipids, phosphatidylserine (PS), and phosphatidylinositol (PI) in a POPC SLB significantly reduced DHA incorporation and changed the SLBs physicochemical properties. These observations are hypothesized to be due to a substitution event occurring between DHA and phospholipid species. PS domain formation in POPC/PS 8:2 SLBs was observed in the presence of calcium ions, which favored DHA incorporation to a similar level as for a POPC only SLB. The changes in SLB thickness observed with different DHA concentrations are also presented. This work contributes to an understanding of the physical changes induced in a lipid bilayer as a consequence of its exposure to different DHA concentrations (from 50 to 200 µM). The capacity of DHA to influence the physical properties of SLBs indicates the potential for dietary DHA supplementation to cause changes in cellular membranes in vivo, with subsequent physiological consequences for cell function.
