Pressure-Induced Denaturation of Monomer β-Lactoglobulin Is Partially Irreversible:  Comparison of Monomer Form (Highly Acidic pH) with Dimer Form (Neutral pH)

Abstract

This study was conducted to assess the effect of high hydrostatic pressure on monomer beta-lactoglobulin (BLg) at acid pH by fluorescence spectroscopy under pressure and by circular dichroism (CD) and (1)H NMR spectroscopies after release of pressure. The intrinsic (tryptophan) fluorescence measurement and the study of 8-anilinonaphthalene-1-sulfonate (ANS) binding to BLg indicated that at pH 2.0 the recovery of center of spectral mass or ANS fluorescence was almost complete upon pressure release. No difference in (1)H NMR spectra was observed between pressurized and unpressurized BLg. In addition, NMR detection of the H/D exchange of aromatic protein indicated that the conformation of the vicinity of tryptophan residues could be refolded almost completely after release of pressure. These results seemingly confirm that the pressure-induced denaturation of BLg at pH 2.0 is reversible. However, cis-parinaric acid binding ability of pressurized BLg was largely lost, although its retinol binding ability was the same as its unpressurized one. Furthermore, CD spectra of the far-UV region and 2D NMR spectra evidently revealed the difference in the conformation of the molecule between unpressurized and pressurized BLg. These results are interpreted as an existence of partially fragile structure in the BLg molecule by high pressure.

Interactions of milk proteins during heat and high hydrostatic pressure treatments — A Review

“…Dufour, Hui Bon Hoa, & Haertle, 1994;Valente-Mesquita et al, 1998). In contrast, cis-parinaric acid (cPA) binding to pressurized β-LG was lost at both pHs (Dufour et al, 1994;Ikeuchi et al, 2001). Stapelfeldt and Skibsted (1999) found a decrease in the fluorescence quantum yield of cPA when it was pressurized in the presence of β-LG.…”

“…At higher pressures, β-LG forms larger aggregates, most probably because of the lack of the hydrophobic effect that stabilizes the helical and sheet structures of β-LG by exposing CysH121. Ikeuchi et al (2001) monitored in situ intrinsic fluorescence and 8-anilino-1-naphthalenesulfonate (ANS) binding by fluorescence of β-LG under pressure (0-400 MPa) at pH 2 or pH 7. β-LG was irreversibly denatured at pH 7 whereas it was reversibly denatured at pH 2. This irreversibility was attributed to novel intermolecular disulfide bonds through SH/S-S interchange reactions, as previously discussed Valente-Mesquita et al, 1998).…”

Immunoreactivity and digestibility of high-pressure-treated whey proteins

“…The structural changes undergone by b-Lg under HP are partially reversible (see Lo´pez-Fandin˜o, 2006 for review). The refolding ability of b-Lg depends on the pressure, temperature and pH of the treatment, being more favoured at 200 than 400 MPa, at 37 than 20 1C, and at acid than neutral pH values (Belloque, Chico´n, & Lo´pez-Fandin˜o, 2007;Ikeuchi et al, 2001). In general terms, the situation is similar to what occurs during thermal denaturation of b-Lg; however, there are important differences between the effects of both treatments that lead to different structures and product characteristics (see Considine, Patel, Anema, Singh, & Creamer, 2007 for review).…”

Effects of high pressure on functionality of whey protein concentrate and whey protein isolate

“…One of the important aspects of pressure treatment is that food can be processed with minimal effect on the natural colour, flavour, taste and texture with little or no loss of vitamins [44]. High pressure has also been used as an effective technique of altering the surface functional properties of water-soluble proteins [25,35]. Pressure acts as a physicochemical parameter that alters the balance of intramolecular and solvent-protein interactions.…”