| Abstract
| - Ochratoxin A (OTA), a fungal metabolite of strains of Penicillium and Aspergillus, binds in its dianion formto Sudlow site I of human serum albumin (HSA) with high affinity. In this study, isothermal calorimetry(ITC) is used to study the binding of OTA and its O-methyl derivative (MOA). Calculations of the equilibriumgeometry of the monoanion and dianion of OTA reveal only small structural changes among the lowestenergy conformers. The ITC data show the binding of MOA, which lacks the phenolic proton of OTA, isaccompanied by the uptake of a proton from the surrounding solvent. At pH 7.13, the binding of OTA isaccompanied by uptake of 0.43 ± 0.15 protons from the solvent. At this pH, the monoanion (0.54) anddianion (0.46) forms of OTA are both present in solution. However, the pKa of the phenolic group of OTAdecreases by more than three units upon protein binding, and so all available OTA is bound to the protein asthe dianion. To account for the ITC data, a model is proposed in which the proton is provided by the phenolicmoiety of OTA in the case of initial binding of the monoanion, and a proton is taken up from the surroundingsolvent for initial binding of the dianion. The binding constant of MOA is 2 orders of magnitude smaller thanthat of OTA, indicating the ion pair between the phenoxide group of OTA and the protonated amino acid isa major contributor to the high binding affinity of OTA to HSA. To identify the specific amino acid involved,the binding of OTA to bovine, rat, and porcine serum albumins was examined. Deprotonation of the monoanionof OTA occurred upon binding to all species. Assuming the amino acid is conserved between species andtaking into account crystal structures of ligands bound to site I of HSA and their ability to displace OTAfrom HSA, either R218 or R257 is involved in the ion pairing with OTA. These two amino acids sit acrossthe binding cavity from one other in site I.
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