Characterising the secondary structure changes occurring in high density systems of BLG dissolved in aqueous pH 3 buffer

Abstract

This study looks at the influence of reduced levels of hydration as a driving force for transitions in the secondary structure of hydrated proteins. A simple protein-water system was used to study the conditions of typical protein-rich dairy food systems at a fixed pH level, salt content, and temperature. Freeze-dried beta-lactoglobulin (Type A) from bovine milk was dissolved directly into two different buffer systems over a wide range of concentrations between 1 mg/ml (∼54 μM) and 200 mg/ml (∼0.01 M) but at a fixed pH level, pH 3. Circular dichroism (CD), attenuated total reflectance Fourier transform infrared spectroscopy (ATR FTIR), and thioflavin T (ThT) Assay fluorescence spectroscopy were used to measure changes in the secondary structure with respect to protein solution concentration at 20 °C. The findings of all of the techniques indicate that the majority of the secondary structure changes occur within the low protein concentration regime (i.e. <50 mg/ml) before a critical aggregation threshold. Dimerisation, formed by β–sheet cross-linking, is the likeliest mechanism of aggregation. The formation of dimers however counters the current assumption that at pH 3 only monomers exist; rather it seems there is a gradual evolution of the monomeric unfolded state with increasing concentration occurs yielding a β-sheet rich refolded aggregate. Most interesting is the low concentration region (i.e. between 1 mg/ml and 40 mg/ml) where most secondary structural alterations are found to occur; before physical crowding effects are possible. The results indicate that BLG has a limited solubility even in a low concentration regime.