An improved method for the purification of rat liver-type fatty acid binding protein from Escherichia coli

Velkov, Tony; Chuang, Sara; Prankerd, Richard J.; Sakellaris, Haralambos Harry; Porter, Christopher John; Scanlon, M.J.

doi:10.1016/j.pep.2005.04.006

Cited by 13 publications

(18 citation statements)

References 33 publications

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“…Chem. 29,2010rat L-FABP in which a 2:1 binding stoichiometry was also observed with apparent dissociation constants of 1.7 mM and 15.5 mM for the high-and low-affinity sites, respectively [23]. A competitive fluorescence displacement assay was employed in order to determine apparent inhibition constants (K i ) for the interaction of L-FABP with PFCAs five to nine carbon atoms in length.…”

Section: Experimental Determination Of the Binding Nature Of Pfcas Tomentioning

confidence: 99%

“…Isothermal titration calorimetry (ITC) has been used to determine the binding stoichiometry and affinity of various ligands for L-FABP [23,29,30]. This technique allows for direct monitoring of the interaction between the ligand and L-FABP and thus eliminates the ambiguity associated with competitive Figure 4, provides greater detail of the stoichiometry than does the fluorescence displacement assay.…”

Section: Experimental Determination Of the Binding Nature Of Pfcas Tomentioning

confidence: 99%

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Experimental characterization of the mechanism of perfluorocarboxylic acids' liver protein bioaccumulation: The key role of the neutral species

Woodcroft

Ellis

Rafferty

et al. 2010

Environmental Toxicology and Chemistry

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Perfluorocarboxylic acids (PFCAs) of chain length greater than seven carbon atoms bioconcentrate in the livers of fish. However, a mechanistic cause for the empirically observed increase in the bioconcentration potential of PFCAs as a function of chain length has yet to be determined. To this end, recombinant rat liver fatty acid-binding protein (L-FABP) was purified, and its interaction with PFCAs was characterized in an aqueous system at pH 7.4. Relative binding affinities of L-FABP with PFCAs of carbon chain lengths of five to nine were established fluorimetrically. The energetics, mechanism, and stoichiometry of the interaction of perfluorooctanoic acid (PFOA) with L-FABP were examined further by isothermal titration calorimetry (ITC) and electrospray ionization combined with tandem mass spectrometry (ESI-MS/MS). Perfluorooctanoic acid was shown to bind to L-FABP with an affinity approximately an order of magnitude less than the natural ligand, oleic acid, and to have at least 3:1 PFOA:L-FABP stoichiometry. Two distinct modes of PFOA binding to L-FABP were observed by ESI-MS/MS analysis; in both cases, PFOA binds solely as the neutral species under typical physiological pH and aqueous concentrations of the anion. A comparison of their chemical and physical properties with other well-studied biologically relevant chemicals showed that accumulation of PFCAs in proteins as the neutral species is predictable. For example, the interaction of PFOA with L-FABP is almost identical to that of the acidic ionizing drugs ketolac, ibuprofen, and warfarin that show specificity to protein partitioning with a magnitude that is proportional to the K(OW) (octanol-water partitioning) of the neutral species. The experimental results suggest that routine pharmacochemical models may be applicable to predicting the protein-based bioaccumulation of long-chain PFCAs.

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Section: Experimental Determination Of the Binding Nature Of Pfcas Tomentioning

confidence: 99%

Section: Experimental Determination Of the Binding Nature Of Pfcas Tomentioning

confidence: 99%

Experimental characterization of the mechanism of perfluorocarboxylic acids' liver protein bioaccumulation: The key role of the neutral species

Woodcroft

Ellis

Rafferty

et al. 2010

Environmental Toxicology and Chemistry

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“…1 and Table 1, respectively. All proteins were expressed in E. coli BL21 cells as previously described [19][20][21]. In brief, human I-BABP was purified by HIC coupled to an anion exchange step as previously described [21].…”

Section: Expression and Purification Of Recombinant Proteinsmentioning

confidence: 99%

“…All proteins were expressed in E. coli BL21 cells as previously described [19][20][21]. In brief, human I-BABP was purified by HIC coupled to an anion exchange step as previously described [21]. Additionally, in order to isolate protein that had not been subjected to HIC, a separate I-BABP purification was performed by a method previously described by the Ira Ropson laboratory [19].…”

Section: Expression and Purification Of Recombinant Proteinsmentioning

confidence: 99%

A protocol for the combined sub-fractionation and delipidation of lipid binding proteins using hydrophobic interaction chromatography

Velkov¹,

Lim²,

Capuano³

et al. 2008

Journal of Chromatography B

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“…In particular, drugs with structural similarities to fatty acid, such as the presence of a hydrophobic moiety and a free carboxylic acid group, appear to bind to I-FABP and L-FABP with higher affinity (low micromolar) (22)(23)(24)(25)(26)(27)(28). Compounds found to bind FABPs in vitro include NSAIDS (fenamates, propionic acid derivatives, indole acids and heteroacyl acetic acid derivatives) and fibrates, both of which commonly exhibit a free carboxylic acid group, and others such as certain steroids and benzodiazepines which do not have a free carboxylic acid group (22,23,26,27).…”

Section: Introductionmentioning

confidence: 99%

Fatty Acid Binding Proteins: Potential Chaperones of Cytosolic Drug Transport in the Enterocyte?

et al. 2011

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Consistent with their role in the cellular transport of endogenous lipophilic substrates, FABPs appear to facilitate the intracellular disposition of drug molecules that bind FABPs in vitro. Drug binding to FABPs in the enterocyte may also attenuate gut wall metabolism in a manner analogous to the reduction in hepatic extraction mediated by drug binding to plasma proteins in the systemic circulation.

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An improved method for the purification of rat liver-type fatty acid binding protein from Escherichia coli

Cited by 13 publications

References 33 publications

Experimental characterization of the mechanism of perfluorocarboxylic acids' liver protein bioaccumulation: The key role of the neutral species

Experimental characterization of the mechanism of perfluorocarboxylic acids' liver protein bioaccumulation: The key role of the neutral species

A protocol for the combined sub-fractionation and delipidation of lipid binding proteins using hydrophobic interaction chromatography

Fatty Acid Binding Proteins: Potential Chaperones of Cytosolic Drug Transport in the Enterocyte?

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