BI-7273 sale Analysis of published albumin X ray
Analysis of published albumin X-ray structures had suggested that an allosteric link may exist, but could not establish whether Zn2+ prevented FFA binding or vice versa. This question was answered through competition experiments monitored by isothermal titration calorimetry (ITC; Fig. 3) for BSA , HSA, and the HSA H67A mutant . ITC provides access to affinity data for both metal ions and FFAs [55,73]. In the absence of FFA, Zn2+ binding to all three proteins under these conditions was exothermic and could be fitted to a “two sequential sites” model (concentrations were too low to capture a third Zn site). From the fitted data (and taking into consideration competition from the Tris buffer), the Kd values for site A on HSA and BSA were 1.7 ± 0.3 μM  and 0.12 ± 0.08 μM , respectively. The corresponding Kd value for the HSA H67A mutant was 9.1 ± 2.9 μM, indicating that the H67A mutation weakened binding to HSA by a factor of approximately 5 when studied by ITC , providing independent confirmation of the location of site A. Similar conclusions were previously reached by equilibrium dialysis . Affinities to site B were over one order of magnitude lower [33,55].
The most significant results from this work concern the clearly adverse effect of the presence of FFA (myristate) on Zn2+ binding (Fig. 3b). Fitting of the data was accomplished by keeping the affinity constant determined for FFA-free HSA, and using the molar fraction of binding site A as a fitting parameter. This demonstrated that with increasing levels of FFA, availability/occupation of site A is progressively diminished (Fig. 4) . In contrast, the stoichiometry of fatty BI-7273 sale binding is not affected by the presence of Zn2+ (Fig. 3c). Hence, and also bearing in mind that most circulating albumin molecules will, on average, have at least 1 FFA molecule bound, whilst only ca. 2% of albumin molecules will carry a Zn2+ ion, FFA levels drive zinc speciation, but not vice versa. Whilst FFA effects on zinc binding are most dramatic at the highest FFA loadings, it is important to realise that effects are already apparent between 0 and 2 mol. equiv. FFA, i.e. in physiologically normal ranges. The possible impact of this albumin-mediated crosstalk between two important signalling networks is outlined in the following section.
Implications of plasma zinc and fatty acid interplay The crosstalk between FFAs and zinc may have far-reaching consequences for normal physiology and in the pathology of diseases associated with fatty acid biochemistry and zinc toxicity and/or cell signalling. Because around 98% of all mobile zinc in plasma are bound to albumin, even small changes in albumin's capacity for zinc binding may have significant consequences. The decrease in zinc binding capacity reflected in Fig. 3b will affect plasma Zn2+ speciation. Indeed, a simple speciation model involving ITC-derived affinity data for HSA sites A and B in the presence and absence of myristate, and a single potential zinc acceptor protein (histidine-rich glycoprotein (HRG); see further below) at physiologically relevant concentrations suggests that FFA-binding dramatically shifts the distribution of Zn2+ in plasma (Fig. 4). The model system is of course quite simplistic, especially regarding the presence of a single acceptor protein, which does not adequately reflect the large number of plasma and cell surface proteins with potential zinc-binding capacities. In the aforementioned model, the minimal proportion of Zn2+ that would be available for interaction with these other proteins is illustrated by the right-hand chart of Fig. 4. Thus, the three major consequences of increases in plasma FFA levels that can be anticipated concern (a) zinc re-distribution to other plasma proteins with Zn-binding ability, (b) export of excess free Zn2+ from plasma, via interaction of free Zn2+ with membrane-bound ZIP transporters , and (c) zinc binding to cell-surface receptors and modulation of their function [9,74]. The latter two processes may occur for any cells in contact with plasma, including immune cells and endothelial cells. Zinc uptake by endothelial cells is the crucial step for re-distribution of Zn2+ from plasma to tissues. Accordingly, our hypothesis predicts that elevated plasma FFA might promote zinc efflux from plasma. Regarding process (c), zinc-sensing receptors are present in a variety of cell types including vascular endothelial cells  and neurons [9,75].