Binding of ascorbic acid and α-tocopherol to bovine serum albumin: a comparative study

Abstract

Binding of ascorbic acid (water-soluble antioxidant) and α-tocopherol (lipid-soluble antioxidant) to bovine serum albumin (BSA) has been studied using isothermal titration calorimetry (ITC), in combination with fluorescence spectroscopy, UV-vis absorption spectroscopy and Fourier transform infrared (FT-IR) spectroscopy. Thermodynamic investigations reveal that ascorbic acid/α-tocopherol binding to BSA is driven by favorable enthalpy and unfavorable entropy, and the major driving forces are hydrogen bonding and van der Waals forces. For ascorbic acid, the interaction is characterized by a high number of binding sites, which suggests that binding occurs by a surface adsorption mechanism that leads to coating of the protein surface. For α-tocopherol, one molecule of α-tocopherol combines with one molecule of BSA and no more α-tocopherol binding to BSA occurs at concentration ranges used in this study. Fluorescence experiments suggest that ascorbic acid has predominantly a "sphere of action" quenching mechanism, whereas, for α-tocopherol, the quenching mechanism is "static quenching" and due to the formation of a ground state complex. Additionally, as shown by the UV-vis absorption, synchronous fluorescence spectroscopy, and FT-IR, ascorbic acid and α-tocopherol may induce conformational and microenvironmental changes of BSA.

Probing the binding mechanisms of α-tocopherol to trypsin and pepsin using isothermal titration calorimetry, spectroscopic, and molecular modeling methods

“…The binding constants of the α-tocopherol-trypsin system and the α-tocopherol-pepsin system are (1.009 ± 0.141) × 10 3 l mol −1 and (1.121 ± 0.107) × 10 3 l mol −1 , respectively. The value of the binding constant of the α-tocopherol-bovine serum albumin (BSA) system obtained by ITC is 6.906 × 10 5 l mol −1 [6], and the binding constant is 4.142 × 10 3 l mol −1 for the α-tocopherol-human serum albumin (HSA) system obtained from spectroscopic methods [7]. They are moderate compared to other strong protein-ligand complexes with binding constants ranging from 10 7 -10 8 l mol −1 [27].…”

Transcriptional Repressor Domain of MBD1 is Intrinsically Disordered and Interacts with its Binding Partners in a Selective Manner

“…The simplest model which explain the independent single site binding where ligand to macromolecule ratio 1:1 is The objective of fitting data is to obtain the value of important parameters which gives better explanation of the data. The fitting procedure is done using single site model based on Wisemen isotherm414243 where V is the volume of the calorimeter cell, Δ H 0 is enthalpy, [L] is ligand concentration, [M] is macromolecule concentration, n is the molar ratio of interacting species, and K is the equilibrium binding constant. To calculate the isobaric heat capacity, ΔC p , the experiment is repeated at three different temperatures such as 288, 298 and 308 K and then ΔH values are plotted against the temperature, and the slope of the plot gives the ΔC p value given that …”

Dissection of the binding ofl-ascorbic acid to trypsin and pepsin using isothermal titration calorimetry, equilibrium microdialysis and spectrofluorimetry

“…1A 1 and A 2 is believed to be due to the The solid line represents the best nonlinear least-squares fit to the independent binding sites model. 13 The binding constants of BSA, trypsin and pepsin with L-ascorbic acid are in the following order of magnitude: L-ascorbic acid-BSA > L-ascorbic acidtrypsin > L-ascorbic acid-pepsin. As the sites available on trypsin/pepsin become progressively occupied during titration, the exothermicity of the peaks decreases and eventually saturates.…”

“…The thermodynamic parameters for the interaction of L-ascorbic acid with trypsin/pepsin obtained from ITC are listed in Table 1. 13 The binding parameters of L-ascorbic acid with trypsin/ pepsin are very important to understand the distribution and function of L-ascorbic acid in the digestive system, since the binding ability of L-ascorbic acid with trypsin/pepsin will provide direct insight into molecular mechanisms concerning the changes in the function of digestive proteases. The exothermicity of the calorimetry peaks in Fig.…”

“…4,5 The health-promoting effects of L-ascorbic acid can be attributed to its biological functions as a cofactor for a number of enzymes, such as hydroxylases involved in collagen synthesis, and as a water soluble antioxidant. [13][14][15] However, to the best of our knowledge, an accurate and full basic data for clarifying the binding mechanisms of L-ascorbic acid to proteases remain unclear. 11,12 The binding study between L-ascorbic acid and serum albumin has been reported previously.…”