The perturbations of the native state of goat alpha-lactalbumin induced by 1,1'-bis(4-anilino-5-naphthalenesulfonate) are Ca2+-dependent.
In this work we have studied the interaction of the hydrophobic fluorescent probe 1,1'-bis(4-anilino-5-naphthalenesulfonate) (bis-ANS), with the native state of apo- and Ca2+-bound goat alpha-lactalbumin (GLA). In 10 mM Tris-HCl, pH 7.5, at 4 degrees C in 2 mM EGTA as well as at 37 degrees C in 2 mM Ca2+, the native protein is close to its thermal transition. Therefore, it can be expected that in both conditions the protein is equally susceptible to interaction with bis-ANS. Nevertheless, we have observed a number of interesting differences in the interaction of the dye with the apo and Ca2+ form. Native apo-GLA binds two bis-ANS molecules and in the complex with bis-ANS, the far-UV circular dichroism (CD) spectrum of apo-GLA becomes similar to that of the protein in the molten globule state. In contrast, native Ca2+-GLA binds five bis-ANS molecules and the far-UV CD spectrum of native Ca2+-GLA is conserved for the complex. In both cases, the high activation energies observed in kinetic experiments indicate that upon binding, large parts of the protein structure have to be reorganized. The reduced perturbation of the protein structure in the presence of Ca2+ can be attributed to local stabilization effects.
Vanderheeren
G
G
Interdisciplinary Research Center, Katholieke Universiteit Leuven Campus Kortrijk, Kortrijk, Belgium.
Hanssens
I
I
Noyelle
K
K
Van Dael
H
H
Joniau
M
M
D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000737:chemistry; D002942:Circular Dichroism; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000494:pharmacology; D006041:Goats; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D002135:Calcium-Binding Proteins; D005456:Fluorescent Dyes; D014443:Tyrosine; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D014364:Tryptophan; D007768:Lactalbumin; D002118:Calcium