Calorimetric and binding dissections of HSA upon interaction with bilirubin

• Published in: The Protein Journal Vol. 25; no. 3; pp. 193 - 201
• Main Authors: Moosavi-Movahedi, Z, Safarian, S, Zahedi, M, Sadeghi, M, Saboury, A A, Chamani, J, Bahrami, H, Ashraf-Modarres, A, Moosavi-Movahedi, A A
• Format: Journal Article
• Language: English
• Published: Netherlands Springer Nature B.V 01.04.2006
• Subjects: Bilirubin - chemistry; Binding Sites; Calorimetry; Humans; Serum Albumin - chemistry; Spectrophotometry; Spectrophotometry, Ultraviolet
• ISSN: 1572-3887; 1875-8355; 1573-4943
• DOI: 10.1007/s10930-006-9002-y

The interactions between bilirubin and human serum albumin (HSA) were studied by isothermal titration calorimetry (ITC) and UV-vis spectrophotometry at 27 degrees C in 100 mM phosphate buffer pH 7.4 containing 1 mM EDTA. The biphasic shape of the HSA-bilirubin binding curve depicted the existence of two bilirubin binding sets on the HSA structure which had distinct binding interactions. Each binding set contained one or more bilirubin binding site. The first binding set at subdomain IIA included one binding site and had a more hydrophobic microenvironment than the other two binding sites in the second bilirubin binding set (subdomain IIIA). With our method of analysis, the calculated dissociation constant of the first binding site is 1.28 x 10(-6) M and 4.80 x 10(-4) M for the second and third binding sites. Here, the typical Boltzmann's equation was used with a new approach to calculate the dissociation constants as well as the standard free energy changes for the HSA-bilirubin interactions. Interestingly, our calculations obtained using the Wyman binding potential theory confirmed that our analysis method had been correct (especially for the second binding phase). The molar extinction coefficient determined for the first bound bilirubin molecule depicted that the bilirubin molecules (in low concentrations) should interact with the nonpolar microenvironment of the first high affinity binding site. Binding of the bilirubin molecules to the first binding site was endothermic (deltaH > 0) and occurred through the large increase in the binding entropy established when the hydrophobic bilirubin molecules escaped from their surrounding polar water molecules and into the hydrophobic medium of the first binding site. On the other hand, the calculated molar extinction coefficient illustrated that the microenvironment of the second binding set (especially for the third binding site) was less hydrophobic than the first one but still more hydrophobic than the buffer medium. The binding of the third bilirubin molecule to the HSA molecule was established more through exothermic (electrostatic) interactions.