Fatty acid-specific fluorescent probes and their use in resolving mixtures of unbound free fatty acids in equilibrium with albumin

Abstract

We report the first measurements for profiling mixtures of unbound free fatty acids. Measurements utilized fluorescent probes with distinctly different response profiles for different free fatty acids (FFA). These probes were constructed by labeling site-specific mutants of the rat intestinal fatty acid binding protein (rI-FABP) with acrylodan. The probes were produced and screened by high-throughput methods, and from more than 30 000 such probes we selected six that together have sufficient specificity and sensitivity for resolving the profile of unbound FFA (FFAu) in mixtures of different FFAu. We developed analytical methods to determine the FFAu profile from the fluorescence (ratio) response of the different probes and used these methods to determine FFAu profiles for mixtures of arachidonate, linoleate, oleate, palmitate, and stearate in equilibrium with bovine serum albumin (BSA). Measurements were performed using mixtures with a range of total FFAu concentrations, including 0.9 nM, which is similar to normal plasma levels. We also measured single FFA binding isotherms for BSA and found that binding was described well by six to seven sites with the same binding constants (Kd). The Kd values for the FFA (4-38 nM) were inversely related to the aqueous solubility of the FFA. We constructed a model with these parameters to predict the FFAu profile in equilibrium with BSA and found excellent agreement between the profiles measured using the FFA probes and those calculated with this model. These results should lead to a better understanding of albumin's role in buffering FFAu and to profiling FFAu in intra- and extracellular biological fluids.

Figure 1. Fluorescence spectra of FFA-specific probes

The set of spectra for each probe represent titrations with increasing concentrations (in μM inserts) of the FFA to which they best respond. (A):L1P8H2 with OA, (B): L2P22G6 with PA (C): L10P7A4 with AA (D):L11P7B3 with SA (E):L13P7B4 with OA and (F):L18P5G12 with LA. For all spectra the excitation wavelength was 375 nm and the probe concentration was 1 μM.

Figure 2. The probe response profiles

Profiles for each probe are represented as the fractional change in R for 1 nM [FFA_u] calculated using the calibration constants in Table 2. For each profile the responses correspond from left to right for the most (POA) to least (SA) soluble FFA. POA – red, LNA – green, AA – blue, LA – cyan, OA – pink, PA – yellow, and SA – brown.

Figure 3. BSA binding isotherms for 5 different FFA

Measured FA bound/total BSA values are shown as solid square symbols and the line through these data represent best fits of the single-class albumin binding model. These results are representative of at least two separate titrations for each FFA.