Quantitation of pH-induced aggregation in binary protein mixtures by dielectric spectroscopy.

This paper presents a quantitative approach for measuring pH-controlled protein aggregation using dielectric spectroscopy. The technique is demonstrated through two aggregation experiments, the first between ß-lactoglobulin (ß-Lg) and hen lysozyme (HENL) and the second between bovine serum albumin (BSA) and HENL. In both experiments, the formation of aggregates is strongly dependent on the solution pH and is clearly indicated by a decrease in the measured permittivity when the second protein is added. A quantifiable lower-bound on the ratio of proteins involved in the aggregation process is obtained from the permittivity spectra. Lower-bound aggregation ratios of 83 % for ß-Lg/HENL at pH 6.0 and 48 % for BSA/HENL at pH 9.2 were consistent with turbidity measurements made on the same solutions.

Increased bandwidth for dielectric spectroscopy of proteins through electrode surface preparation.

Dielectric spectroscopy measurements of liquids are often limited by electrode polarization. The influence of surface polishing and deposition of the conducting polymer polypyrrole/polystyrenesulfonate (PPy/PSS) on the polarization impedance is investigated. A quantitative description of the electrode polarization contribution to the real-valued permittivity spectrum is derived. This description explains the origin of the ω(-const). (const.>1) dependency commonly observed in permittivity measurements. Electrode surface roughness is correlated with both the magnitude and phase of the constant phase element. Generally, rougher electrodes have better performance, and an order of magnitude bandwidth improvement is achieved using PPy/PSS electrodes.

Influence of pK(a) shifts on the calculated dipole moments of proteins.

The protein dipole moment is a low-resolution parameter that characterizes the second-order charge organization of a biomolecule. Theoretical approaches to calculate protein dipole moments rely on pK(a) values, which are either computed individually for each ionizable residue or obtained from model compounds. The influence of pK(a) shifts are evaluated first by comparing calculated and measured dipole moments of ß-lactoglobulin. Second, calculations are made on a dataset of 66 proteins from the Protein Data Bank, and average differences are determined between dipole moments calculated with model pK(a)s, pK(a)s derived using a Poisson-Boltzmann approach, and empirically-calculated pK(a)s. Dipole moment predictions that neglect pK(a) shifts are consistently larger than predictions in which they are included. The importance of pK(a) shifts are observed to vary with protein size, internal permittivity, and solution pH.

Note: electrode polarization of Galinstan electrodes for liquid impedance spectroscopy.

Electrode polarization is a significant obstacle in the impedance measurements of ionic liquids. An atomically smooth electrode surface could potentially reduce unwanted impedance contributions from electrode polarization. Liquid metal electrodes were formed by adhering Galinstan to acrylic plates in a parallel-plate capacitor arrangement. Electrode polarization was compared to a similar cell with stainless steel electrodes. The impedance of salt and protein solutions (ß-lactoglobulin) was measured from 40 Hz to 110 MHz. Because of oxide layer formation, the performance of the Galinstan electrode is significantly different than the theoretical ideal.

Method for estimating the internal permittivity of proteins using dielectric spectroscopy.

Protein charge organization is dependent on the low-permittivity region in the hydrophobic core of the molecule. We suggest a novel approach to estimate the dielectric constant of this region by comparing measured and simulated first- and second-order charge moments. Here, the dipole moment is measured as a function of pH using dielectric spectroscopy. The results are compared to dipole moments based on Poisson-Boltzmann estimates of pK(a) shifts calculated from structures in the Protein Data Bank. Structures are additionally refined using CHARMM molecular dynamics simulations. The best estimate for the internal permittivity is found by minimizing the root-mean-square residual between measured and predicted charge moments. Using the protein ß-lactoglobulin, a core dielectric constant in the range of 6-7 is estimated.

Dielectric spectroscopy of molecular interactions based on the avidin-biotin complex.

Dielectric spectroscopy is used to probe the electrical properties of biomolecules dissolved in liquids. A 40 µl cell is constructed out of acrylic with polished, stainless steel electrodes. Experiments are performed on avidin and biotin-labeled BSA, showing characteristics of aggregation. Experiments with avidin and biotin demonstrate shifts in dielectric relaxation of the avidin associated with changes in the dipole moment and size of the molecule due to biotin binding. These shifts are analyzed in the context of biomolecular changes. These experiments demonstrate the utility of impedance spectroscopy to detect changes due to small molecules binding to proteins.

Temperature-stable parallel-plate dielectric cell for broadband liquid impedance measurements.

A liquid impedance cell for broadband impedance measurements up to 110 MHz is presented. The design incorporates temperature control and minimizes parasitic capacitance and inductance. The cell is simple to fabricate and uses chemically resistant materials, stainless steel, and Teflon. This dielectric cell can be used in a variety of liquid measurements, particularly those related to impedance measurements of biological objects in solution. Temperature control is illustrated in measurements of the permittivity of deionized water from 5 to 55 °C. Numerical fitting procedures employed on the relaxation curves indicate good agreement with previous studies on beta-lactoglobulin and hen lysozyme. Titration capability is demonstrated through dielectric titration of hen lysozyme and beta-lactoglobulin.