Quantitation of pH-induced Aggregation in Binary Protein Mixtures by Dielectric Spectroscopy

Abstract

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 β-lactoglobulin (β-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 β-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.

Appendix Detailed Experimental Protocol

1.1 β-Lg/HENL Experiment

• 1. Cell rinsed once with ethanol followed by three rinses with DI water and then dried in air. Air calibration measurement taken.

• 2. (A) Added 60 μl of DI water to cell. Water calibration measurement taken after stabilization.

• 3. (B) Removed 18 μl. Added 18 μl of 10 mg/ml β-Lg in DI water resulting in 3 mg/ml β-Lg in DI water.

• 4. (C) Added 6 μl of 10 mg/ml HENL in DI water resulting in 0.91 mg/ml HENL and 2.73 mg/ml β-Lg in DI water.

• 5. (D) Removed all liquid from cell by pipettor. Added 60 μl of 5 mM HCl. Cell recalibrated.

• 6. (E) Removed 36 μl. Added 18 μl of 10 mM HCl. Added 18 μl of 10 mg/ml β-Lg in DI water resulting in 3 mg/ml β-Lg in 5 mM HCl.

• 7. (F) Removed 6 μl. Added 6 μl of 10 mM HCl. Added 6 μl of 10 mg/ml HENL in DI water resulting in 0.91 mg/ml HENL and 2.45 mg/ml β-Lg in 5 mM HCl.

• 8. Cell rinsed with and immersed into DI water for storage.

1.2 BSA/HENL Experiment

• 1. Cell rinsed once with ethanol followed by three rinses with DI water and then dried in air. Air calibration measurement taken.

• 2. (G) Added 60 μl of DI water to cell. Water calibration measurement taken.

• 3. (H) Removed 18 μl. Added 18 μl of 10 mg/ml BSA in DI water resulting in 3 mg/ml BSA in DI water.

• 4. (I) Added 6 μl of 10 mg/ml HENL in DI water resulting in 0.91 mg/ml HENL and 2.73 mg/ml BSA in DI water.

• 5. (J) Removed all liquid from cell by pipettor. Added 60 μl of 1 mM NaOH.

• 6. (K) Removed 36 μl. Added 18 μl of 2 mM NaOH. Added 18 μl of 10 mg/ml BSA in DI water resulting in 3 mg/ml BSA in 1 mM NaOH.

• 7. (L) Removed 6 μl. Added 6 μl of 2 mM NaOH. Added 6 μl of 10 mg/ml HENL in DI water resulting in 0.91 mg/ml HENL and 2.45 mg/ml BSA in 1 mM NaOH.

• 8. Cell rinsed with and immersed into DI water for storage.

1.3 Additional Experimental Notes

• 1. The Agilent 4294A Impedance Analyzer had the following settings: measurement parameters $\rightarrow$ Cp-G, sweep type $\rightarrow$ log, sweep direction $\rightarrow$ down, oscillator strength $\rightarrow$ 500 mV, bandwidth $\rightarrow$ 4, start frequency $\rightarrow$ 40 Hz, stop frequency $\rightarrow$ 110 MHz, number of points $\rightarrow$ 601.

• 2. Each time liquid was added into the measurement cell, the entire solution was mixed by repeated actuation of the pipettor for about 10 s.

• 3. Permittivity measurements when adding proteins and mixing were unstable. These sweeps (1–2 per step) were discarded from Figs. 1 and 2.