Interaction between Two Sulfate-Conjugated Uremic Toxins, p-Cresyl Sulfate and Indoxyl Sulfate, during Binding with Human Serum Albumin

Recently, p-cresyl sulfate (PCS) has been identified as a protein-bound uremic toxin. Moreover, the serum-free concentration of PCS, which is associated with its efficacy of hemodialysis, appears to be a good predictor of survival in chronic kidney disease (CKD). We previously found that PCS interacts with indoxyl sulfate (IS), another sulfate-conjugated uremic toxin, during renal excretion via a common transporter. The purpose of this study was to further investigate the interaction between PCS and IS on the binding to human serum albumin (HSA). Here, we used ultrafiltration to show that there is only one high-affinity binding site for PCS, with a binding constant on the order of 10(5) M-1 (i.e., comparable to that of IS). However, a binding constant of the low-affinity binding site for PCS is 2.5-fold greater than that for IS. Displacement of a fluorescence probe showed that PCS mainly binds to site II, which is the high-affinity site for PCS, on HSA. This finding was further supported by experiments using mutant HSA (R410A/Y411A) that displayed reduced site II ligand binding. A Klotz analysis showed that there could be competitive inhibition between PCS and IS on HSA binding. A similar interaction between PCS and IS on HSA was also observed under the conditions mimicking CKD stage 4 to 5. The present study suggests that competitive interactions between PCS and IS in both HSA binding and the renal excretion process could contribute to fluctuations in their free serum concentrations in patients with CKD.