Influence of binding of sodium dodecyl sulfate, all-trans-retinol, and 8-anilino-1-naphthalenesulfonate on the high-pressure-induced unfolding and aggregation of beta-lactoglobulin B.
Bovine beta-lactoglobulin B (beta-LG) is susceptible to pressure treatment, which unfolds it, allowing thiol-catalyzed disulfide bond interchange to occur, facilitating intermolecular bonding (both noncovalent and disulfide). In the present study, beta-LG was mixed with sodium dodecyl sulfate (SDS), all-trans-retinol (retinol), or 8-anilino-1-naphthalenesulfonate (ANS) on a 1:1.1 molar basis, and aliquots were held at pressures between 50 and 800 MPa for 30 min at pH 7.2 and 20 degrees C. Polyacrylamide gel electrophoresis (PAGE) showed that beta-LG alone (control) was converted into a non-native monomer and a series of dimers, trimers, etc., at pressures beyond 100 MPa; SDS inhibited the formation of non-native species up to 200 MPa, and neither retinol nor ANS inhibited the formation of the non-native species as effectively as SDS. At pressures beyond 350 MPa, SDS ceased to have any inhibitory effect, but both ANS and retinol showed significant inhibition. The near- and far-UV CD patterns and the ANS fluorescent data were consistent with the PAGE data, but the retinol fluorescent data did not show sufficient change to interpret. The results suggested that there were three discernible structural stages. In Stage I (0.1-150 MPa), the native structure is stable; in Stage II (200-450 MPa), the native monomer is reversibly interchanging with non-native monomers and disulfide-bonded dimers; and in Stage III (>500 MPa), the free CysH in non-native monomer and dimer interacts with -S-S- bonds to produce high molecular weight aggregates of beta-LG. SDS inhibited the Stage I to Stage II transition at 200 MPa, and ANS and retinol inhibited the Stage II to Stage III transition at 600 MPa.
Considine
Thérèse
T
Riddet Centre, Massey University, Private Bag 11 222, Palmerston North, New Zealand.
Singh
Harjinder
H
Patel
Hasmukh A
HA
Creamer
Lawrence K
LK
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D007782:Lactoglobulins / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D011108:Polymers / Q000378:metabolism; D011312:Pressure; D017510:Protein Folding; D012967:Sodium Dodecyl Sulfate / Q000378:metabolism*; D014801:Vitamin A / Q000378:metabolism*
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D007782:Lactoglobulins; D008024:Ligands; D011108:Polymers; D014801:Vitamin A; D012967:Sodium Dodecyl Sulfate; C027132:1-anilino-8-naphthalenesulfonate