216
G. V ANDERHEEREN and I. HANSSENS
effectuated. It indicates that the tertiary protein structure is loosened while the
secondary structure elements are largely conserved. By this partial unfolding, the
so called, molten globule state is obtained. Above 25°C, the numeric value of the
ellipticity at 220 nm decreases gradually. The temperature dependence of the
ellipticity is not known. But the fact that the ellipticity decrease is more pronounced in 2 mM EGTA than in 2 mM Ca 2 + proves that the conformation in 2 mM
EGTA changes more as a function of the temperature than in 2 mM Ca 2 +.
In 2 mM Ca 2 + (Fig. 15.2, open squares) the ellipticity changes indicate that up
to 60°C the compact tertiary structure of BLA is conserved. The aromatic groups
determining the ellipticity at 270 nm are kept in frxed orientations and the ellipticity changes at 220 nm indicate that, if any change of secondary structure
occurs at all, it will be small. Between 60 and 75°C the tertiary structure loosens
its compact packing while no pronounced unfolding of the secondary structure
is observed. At 80°C the tertiary structure of Ca 2 + -bound BLA is unfolded. However the ellipticity at 220 nm indicates that the secondary structure of BLA is
more preserved in 2 mM Ca 2 + than in 2 mM EGTA. This observation indicates
that Ca 2 + remains associated to BLA after thermal destabilization of its tertiary
structure. Furthermore, the associated ion stabilizes elements of secondary
structure in the partially unfolded protein. By X-ray diffraction it has been demonstrated that the Ca 2 + -binding loop of (baboon) a-lactalbumin is at the same
time a part of the amino-terminal side of the 31O-helix (residues 76-82) in the [3domain and a part of the carboxyl-terminal side of the a-helix C (residues
86-99) in the a-domain (Acharya et al. 1989). It is clear that at least these elements of the secondary structure will be stabilized by Ca 2 + -binding.
3.3
Determination of the Binding Properties of bis-ANS t~ BLA
The binding properties for the interaction of bis-ANS with apo- and Ca 2 +-BLA
are obtained from two series of fluorescence titration curves, as described in
Materials and Methods. The titrations in the absence of Ca 2 + at 25, 50, 70 and
80°C are presented in Fig.s 15.3 A and B. The corresponding Scatchard plots for
the bis-ANS binding to 1 f!M apo-BLA are presented in Fig. 15.3 C. At 25°C, the
strong fluorescence increase at the start of the titrations of 1 f!M bis-ANS with
apo-GLA and of those of 1 f!M apo-GLA with bis-ANS (Fig.s 15.3 A and B,
squares) is characteristic for the strong binding of fluorophore to the protein.
The limited fluorescence increase obtained in the second titration is nearly twice
that obtained in the frrst titration indicating that two probe molecules bind to
apo-BLA. This is also deduced from the X-intercept in the corresponding Scatchard plot (Fig. 15.3 C, squares). In addition, the linear shape of that plot indicates that the two probe molecules independently bind to apo-BLA with nearly
equal strength. As an a-lactalbumin molecule possesses two distinct aromatic
clusters, it seems reasonable to suppose that one bis-ANS molecule can penetrate
into each cluster. At 55°C (Fig. 15.3 C, down triangles), the Scatchard plot is
hyperbolic. The straight line slopes drawn to the extremities of the curve near the
X- and Y-axes, intersect the X-axis at about 3 and 1 f!M bis-ANS, respectively.
This indicates that one molecule BLA can bind 3 bis-ANS molecules of which one
G. V ANDERHEEREN and I. HANSSENS
effectuated. It indicates that the tertiary protein structure is loosened while the
secondary structure elements are largely conserved. By this partial unfolding, the
so called, molten globule state is obtained. Above 25°C, the numeric value of the
ellipticity at 220 nm decreases gradually. The temperature dependence of the
ellipticity is not known. But the fact that the ellipticity decrease is more pronounced in 2 mM EGTA than in 2 mM Ca 2 + proves that the conformation in 2 mM
EGTA changes more as a function of the temperature than in 2 mM Ca 2 +.
In 2 mM Ca 2 + (Fig. 15.2, open squares) the ellipticity changes indicate that up
to 60°C the compact tertiary structure of BLA is conserved. The aromatic groups
determining the ellipticity at 270 nm are kept in frxed orientations and the ellipticity changes at 220 nm indicate that, if any change of secondary structure
occurs at all, it will be small. Between 60 and 75°C the tertiary structure loosens
its compact packing while no pronounced unfolding of the secondary structure
is observed. At 80°C the tertiary structure of Ca 2 + -bound BLA is unfolded. However the ellipticity at 220 nm indicates that the secondary structure of BLA is
more preserved in 2 mM Ca 2 + than in 2 mM EGTA. This observation indicates
that Ca 2 + remains associated to BLA after thermal destabilization of its tertiary
structure. Furthermore, the associated ion stabilizes elements of secondary
structure in the partially unfolded protein. By X-ray diffraction it has been demonstrated that the Ca 2 + -binding loop of (baboon) a-lactalbumin is at the same
time a part of the amino-terminal side of the 31O-helix (residues 76-82) in the [3domain and a part of the carboxyl-terminal side of the a-helix C (residues
86-99) in the a-domain (Acharya et al. 1989). It is clear that at least these elements of the secondary structure will be stabilized by Ca 2 + -binding.
3.3
Determination of the Binding Properties of bis-ANS t~ BLA
The binding properties for the interaction of bis-ANS with apo- and Ca 2 +-BLA
are obtained from two series of fluorescence titration curves, as described in
Materials and Methods. The titrations in the absence of Ca 2 + at 25, 50, 70 and
80°C are presented in Fig.s 15.3 A and B. The corresponding Scatchard plots for
the bis-ANS binding to 1 f!M apo-BLA are presented in Fig. 15.3 C. At 25°C, the
strong fluorescence increase at the start of the titrations of 1 f!M bis-ANS with
apo-GLA and of those of 1 f!M apo-GLA with bis-ANS (Fig.s 15.3 A and B,
squares) is characteristic for the strong binding of fluorophore to the protein.
The limited fluorescence increase obtained in the second titration is nearly twice
that obtained in the frrst titration indicating that two probe molecules bind to
apo-BLA. This is also deduced from the X-intercept in the corresponding Scatchard plot (Fig. 15.3 C, squares). In addition, the linear shape of that plot indicates that the two probe molecules independently bind to apo-BLA with nearly
equal strength. As an a-lactalbumin molecule possesses two distinct aromatic
clusters, it seems reasonable to suppose that one bis-ANS molecule can penetrate
into each cluster. At 55°C (Fig. 15.3 C, down triangles), the Scatchard plot is
hyperbolic. The straight line slopes drawn to the extremities of the curve near the
X- and Y-axes, intersect the X-axis at about 3 and 1 f!M bis-ANS, respectively.
This indicates that one molecule BLA can bind 3 bis-ANS molecules of which one