Conformational changes of alpha-lactalbumin induced by the stepwise reduction of its disulfide bridges: the effect of the disulfide bridges on the structural stability of the protein in sodium dodecyl sulfate solution.
Four disulfide bridges of bovine alpha-lactalbumin (alpha-lact) were selectively reduced to obtain its derivatives with three, two, and zero disulfide bridges (designated as 3SS, 2SS, and 0SS alpha-lact, respectively). The original helicity was almost maintained in 3SS alpha-lact missing only the Cys6-Cys120 bridge. Upon the reduction of both Cys28-Cys111 and Cys6-Cys120 bridges, various changes occurred in the protein. In particular, the maximum fluorescence of 1-anilinonaphthalene-8-sulfonic acid was observed in this stage. Upon the reduction of all disulfide bridges, the hydrophobic box of the protein, formed by Trp60, Ile95, Tyr103, and Trp104, was disrupted and an internal helical structure was destroyed. The conformation of each derivative was examined mainly in a solution of sodium dodecyl sulfate. In the surfactant solution, the helicity increased from 33% to 37% in 3SS alpha-lact, from 26% to 31% in 2SS alpha-lact, and from 18% to 37% in 0SS alpha-lact, as against from 34% to 44% in intact alpha-lact. On the other hand, the tryptophan fluorescence of each derivative was affected in very low surfactant concentrations, suggesting that the tertiary structure considerably changed prior to the secondary structural change in the surfactant solution.
Takeda
K
K
Department of Applied Chemistry, Okayama University of Science, Japan.
Ogawa
K
K
Ohara
M
M
Hamada
S
S
Moriyama
Y
Y
D000817:Anilino Naphthalenesulfonates; D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D003902:Detergents; D004220:Disulfides / Q000737:chemistry*; D005456:Fluorescent Dyes; D019791:Guanidine; D006146:Guanidines; D007768:Lactalbumin / Q000737:chemistry*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012967:Sodium Dodecyl Sulfate; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D000817:Anilino Naphthalenesulfonates; D003902:Detergents; D004220:Disulfides; D005456:Fluorescent Dyes; D006146:Guanidines; D012967:Sodium Dodecyl Sulfate; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan; D007768:Lactalbumin; D019791:Guanidine