Albumin-mediated extracellular zinc speciation drives cellular zinc uptake

Abstract

The role of the extracellular medium in influencing metal uptake into cells has not been described quantitatively. In a chemically-defined model system containing albumin, zinc influx into endothelial cells correlates with the extracellular free zinc concentration. Allosteric inhibition of zinc-binding to albumin by free fatty acids increased zinc flux.

Fig. 1. Serum albumin is the principal Zn²⁺ transporter in the extracellular space. Amino acid residues His67, His247 and Asp249 form site A (blue; PDB 5IJF; HSA), the principlal interdomain binding site for Zn²⁺ (red sphere). In the presence of FFAs of sufficient chain length bound at the nearby FFA binding site FA2 (myristate C14:0 shown in black), site A residues disengage, as depicted in the structural overlay (centre), and Zn²⁺ affinity drops dramatically, leading to release of Zn²⁺ from FFA-loaded albumin (tan; PDB 1BJ5; HSA) under physiological conditions.

Fig. 2. ⁶⁶Zn/⁶⁸Zn ratios determined at different extracellular albumin concentrations (0–600 μM; see ESI for full data). Phase I: HUVEC growth medium. Phase II: Physiologically-relevant media (600 μM BSA + 20 μM natural abundance Zn²⁺, no FFA). Phase III: Media containing variable concentration of BSA (0-600 μM) + 20 μM isotopically-enriched ⁶⁸Zn²⁺, with/without FFA supplementation. Cell pellets were collected in a time-dependent manner and analysed by ICP-MS. (a) ⁶⁶Zn/⁶⁸Zn ratios were calculated, plotted and fitted to a mathematical model to derive zinc flux rates. Zinc flux increases with decreasing BSA concentration. (b) Zinc isotopes (⁶⁴Zn, ⁶⁶Zn, ⁶⁷Zn, ⁶⁸Zn, ⁷⁰Zn) were summed up to determine total intracellular zinc (ng × 10⁶ cells). For extracellular [BSA] > 60 μM, intracellular [Zn] remained constant at ca. 50 ng × 10⁶ cells but increased for extracellular [BSA] < 40 μM. See ESI, Tables S2–S5 and S9–S13 for full numerical data and mathematical models.

Fig. 3. Zinc influx depends on the concentration of albumin (a). This can be correlated to free [Zn²⁺] for [BSA] = 40–600 μM (estimated using published stability constants for site A only; see ESI†) (b). The influx rate shows a linear relationship with free [Zn²⁺].

Fig. 4. Isotopic ratios (⁶⁶Zn/⁶⁸Zn) over time for HUVEC cells cultured in presence of 60 μM BSA and either C8:0 (octanoate), C14:0 (myristate) or C16:0 (palmitate) FFAs (300 μM, 5 mol. equiv.). Experimental data (•) are shown with corresponding fitting model for that experimental condition (solid coloured line) alongside the fitting model for 60 μM BSA in the absence of FFAs (dashed black line). The right-hand panel compares the influx rates φ_in for these four conditions. Full numerical data can be found in ESI, Tables S6, S7 and S14–S16.