Electrochemical Studies of the Effect of Temperature and pH on the Adsorption of α-Lactalbumin at Pt

Abstract

The adsorption behavior of holo- and apo-α-lactalbumin at a Pt/electrolyte interface was studied in an acidic, neutral, and alkaline medium over the temperature range 273 to 353 K, using the cyclic voltammetry technique. It was shown that the surface charge density, resulting from protein adsorption, is directly proportional to the amount of adsorbed protein (surface concentration), indicating that adsorption is accompanied by the transfer of charge. A significant difference in the amount of adsorbed protein (between the two types of protein) was obtained at pH 7 because of the difference in conformation of the molecule in the presence/absence of bound calcium. On the other hand, the resemblance in behavior of the two types of proteins observed at pH 2 and pH 11 suggested that the protein is in its molten globule state and is depleted in calcium at these pHs. The adsorption process was modeled using the Langmuir adsorption isotherm. The values of the Gibbs free energy of adsorption indicated that the protein molecules strongly adsorb onto the Pt surface via chemisorption. The protein expressed the highest affinity toward adsorption at pH 2 and the lowest at pH 11. The adsorption process was found to be endothermic, resulting from the excess energetics required for the breaking of intramolecular interactions relative to those involved in the formation of protein−metal bonds. The adsorption of α-LA onto a Pt surface was found to be an entropically governed process, suggesting structural unfolding of the protein at the electrode surface.

Kinetic and Structural Characterization of Adsorption-induced Unfolding of Bovine α-Lactalbumin

“…The plateau value of 3 Ϯ 0.2 mg/m 2 agrees well with previously reported values. Adsorption of BLA on a platinum surface resulted in a value of 2.9 mg/m 2 (24), and a value of 3.3 mg/m 2 was found for adsorption of BLA on a hydrophobic silicon surface (25). The steep part in the region of low BLA concentration is an indication for high affinity behavior.…”

Biomaterial Interface Investigated by Electrochemical Impedance Spectroscopy

“…Different values for the resistance of the surface functionalized with immunoglobulin G, albumin, fibronectin, heparin, and collagen IV could be determined. [22] In contrast, the diameter of the semicircle in the Nyquist plot and the CTR increase with each adsorption step of polyethylenimine/ albumin multilayers, because the according charge transfer rate of the redox probe Fe(CN) 6 4-/3-decreases. [21] The insulating effect of each additional polyethylenimine /albumin layer impede the electrode access of Fe(CN) 6 4-/3-.…”

“…At the intercept (x → ∞, Z im → 0) the resistance of the solution can be obtained. A straight line in the Nyquist plot at lower frequencies represents a diffusion limited process of ions, or the transfer of redox probes (Co(bpy) 3 3+ [14] Fe(CN) 6 4-/3- [9,12,13,15,21,22,[33][34][35] or ferrocenemethanol [13,15] ) through the surface layer. The according impedance is called the Warburg impedance (W).…”

“…This equivalent circuit involves the CTR (R 1 ) and the diffusive double layer capacitance associated with it. [6,13,14,34,35] The capacitance can be substituted with a CPE. [22,29,37,49] The resistance and capacitance R 1 and C 1 in Figure 3(g) account for an adsorbing cell layer, and R 2 and C 2 stand for the metal oxide layer.…”

“…Interactions of implant surfaces with biological devices, changes in the polarizability of functional groups, the transport of ions or dipoles through polymer layers and mineralization processes, for instance of bone tissue can be assessed. The change of the Gibbs free energy and the entropy due to protein binding to the surface can be calculated [4,6,8] and the affinity of biomolecules to the surface can be estimated. [4] Electrochemical techniques are introduced as biosensors.…”