Direct Binding of Ethanol to Bovine Serum Albumin:  A Fluorescent and ¹³C NMR Multiplet Relaxation Study

Abstract

Molecular mechanisms of ethanol interaction with proteins are not well-understood. In the present study, direct interaction of ethanol with hydrophobic binding sites on fatty acid free bovine serum albumin (BSA) was determined using the fluorescent probe 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS), cis-parinaric acid, and 13C NMR. The affinity of ethanol for BSA (Kd) was (5.21 +/- 0.31) x 10(-2) mol. Ethanol (25-200 mmol) competitively inhibited 1,8-ANS binding to BSA in a concentration-dependent manner with a Ki (concentration of ethanol that decreased 1,8-ANS binding by 50%) of 658 mmol. Preincubation of BSA with ethanol significantly decreased cis-parinaric acid binding to BSA, indicating interaction of ethanol with hydrophobic fatty acid-binding site(s) on BSA. Furthermore, ethanol was found to act on three of the five fatty acid-binding sites on BSA. These data indicated selectivity in the interaction of ethanol with hydrophobic sites on BSA. 13C NMR multiplet relaxation was used to characterize the interaction of ethanol with binding sites on BSA. Detailed analysis of [13C]ethanol relaxation data obtained in the presence of increasing BSA concentrations (25-200 mg/mL) led to the conclusion that the ethanol methyl group, as opposed to its hydroxyl group, binds in a hydrophobic pocket(s) on the protein. Ethanol-induced changes in activity of certain proteins may result from direct binding of ethanol to specific hydrophobic binding sites and/or displacement of endogenous ligands from those sites.

Dominant effect of ethanol in thermal destabilization of bovine serum albumin in the presence of sucrose

“…Figure 2 shows the effect of increasing the concentration of ethanol on the thermal stability of BSA. While the apparent T m value of BSA is 70.2 • C, the presence of ethanol destabilizes BSA thereby de- [1]. In this study, it was found that ethanol prevents the binding of cis-parinaric acid to three out of five fatty acid binding sites in BSA.…”

“…Each domain is subdivided into two more domains (A and B) that are predominantly helical and cross-linked by several disulfide bridges [4]. Direct binding of ethanol to the hydrophobic binding sites on fatty acid free bovine serum albumin (BSA) was reported [1]. The study showed that the presence of ethanol inhibited the binding of 1-anilinonaphthalene-8-sulfonic acid (1, 8-ANS) and cis-parinaric acid to BSA.…”

NMR Study on the Low-Affinity Interaction of Human Serum Albumin with Diclofenac Sodium.

“…But till now only 13 C-enriched drug molecules have been used. 23,24) In this paper, we studied the low-affinity interaction between HSA and DCF with NMR technique. 13 position on DCF because this method, while necessitating long accumulation times, has the advantage over 1 H-NMR by affording less complicated spectra.…”

“…10,[17][18][19][20][21][22][23][24] However, in studies on the high-affinity binding between proteins and drug molecules, the application of this spectroscopic method has been limited to small proteins. 17) When the drug molecules are tightly bound to the high-affinity sites of a large protein as HSA, the line-broadening effect makes the drug NMR signal non-observable.…”

Classification of the Binding Modes in Bovine Serum Albumin Using Terminally Substituted Alkane Analogues

“…Though it is established that there are at least two types of ANS binding sites in a BSA molecule, uncertainties still remain as to the properties of each binding site. [5][6][7][8] Inhalation anesthetics and alcohols are hydrophobic molecules and are considered to act by interacting with a number of proteins by hydrophobic interaction. [9][10][11] Therefore, it is important to make clear the binding properties of the hydrophobic molecules to proteins to elucidate the anesthetic action on proteins.…”