Effects of high pressure and low temperature on β-lactoglobulin unfolding and aggregation

Abstract

A solution of β-lactoglobulin (β-LG) isolate (2% protein, w/w, in 10 mM Bis-TRIS buffer, pH 7; 40 g cylindrical sample) was processed for about 15 min. at 300 MPa in the range +5/+36°C, in water (+25°C) or in a propanediol/water mix (+25, +4 or −2°C) as pressure transmitting medium (PTM). Sample temperature was monitored with T thermocouples located in the sample cylinder. Pressure-induced unfolding of β-LG was significantly lower after processing in PTM at +4 or −2°C (sample at +10/+15°C or +5/+10°C, respectively) than at +25°C (sample at +25/+36°C). Indeed, the residual enthalpy of thermal denaturation (Δ H td) of the pressurized β-LG solutions, measured by differential scanning calorimetry 1–2 h after pressure release, was close to 60, 53, 83 or 86% of that of native β-LG, when pressurization was carried out in water at +25°C or in propanediol/water at +25, +4 or −2°C, respectively. β-LG aggregates (36–500 kDa) were detected by gel permeation chromatography when pressurization was carried out at +25°C (in water or in propanediol/water) but not at lower temperatures. The same β-LG isolate solution was also frozen by pressure release through immersion in propanediol/water at −29°C, pressurization to 300 MPa, cooling the sample to −16°C (at 300 MPa) in 13 min, and releasing pressure in 3–4 s. This sequence induced instant and uniform ice nucleation throughout the sample. Freezing was then completed to −20°C at 0.1 MPa, followed by immediate thawing in water at +20°C. β-LG processed in this manner retained 93% of its native enthalpy (Δ H td), indicating only slight protein unfolding. Chromatography revealed the absence of protein aggregation. Freezing β-LG isolate solutions (2 or 10% protein, w/w, in the same buffer) at atmospheric pressure in still air at −20 or −35°C, followed by frozen storage, also did not cause any unfolding or aggregation. Conformation changes in pure β-LG (1 mg/ml of 10 mM Bis-TRIS or of 50 mM phosphate buffer, pH 7.0) induced at 200 MPa by low or sub-zero temperatures without ice formation were monitored by UV spectroscopy under pressure. Decreasing the temperature of the β-LG solution under 200 MPa from +20 to +4°C, then to −15°C, led to marked ‘blue shifts’ in the 4th derivative UV spectra, revealing that tyrosine and tryptophan residues became significantly more exposed to an aqueous microenvironment at low temperatures. These shifts may be interpreted as different β-LG conformations existing under various P/ T conditions: native conformation at atmospheric pressure/+20°C; pressure-denatured state at 200 MPa/+20°C; fully hydrated, cold denatured state at 200 MPa/−15°C. This could explain why pressure up to 300 MPa induced less protein unfolding or aggregation at a low or sub-zero temperature than at +25/+36°C.