220
G. VANDERHEEREN and 1. HANSSENS
apo-GLA concentrations is self-association of the protein. By such selfassociation bis-ANS is expected to be further protected from the water phase.
This provokes a further increase of the fluorescence at protein concentrations for
which the probe is already completely bound. The maximum fluorescence
increase of 1 !!M bis-ANS at a low protein concentration (as used in the experiments presented in Fig. 15.5 B, filled squares) is estimated 66 ± 13 and 38 ± 7
(arbitrary units) for apo-GLA and Ca2+-GLA, respectively. These maximum values are used to calculate the concentration of bound bis-ANS from the fluorescence increase observed on titrating 1 !!M GLA with bis-ANS (Fig. 15.5 B). The
titration curve for the titration of 1 !!M native Ca 2 +-GLA with bis-ANS at 37°C
(Fig. 15.5 B, open squares) is slightly S-shaped. This is indicative of positive
cooperativity for the dye binding which is confirmed by the clear concavedownward shape of the according Scatchard plot (Fig. 15.5 C, open squares). On
the other hand, neither the hyperbolic titration curve, nor the linear Scatchard
plot gives any indication of cooperative behavior for the bis-ANS interaction with
native apo-GLA at 4°C (Figs. 15.5 Band C, filled squares). From the abscis intercept of the Scatchard plot the total number of bis-ANS binding sites is found to
be about 5.3 for native Ca 2 + -GLA and 2.6 for native apo-GLA. In order to determine the corresponding degree of cooperativity, the binding data are plotted
according to the Hill equation (Fig. 15.5 D).
log ((n-r)/r) =-m log Kb-m log [bis-ANSl
where Kb is the average binding constant ofbis-ANS to GLA, n is the number of
binding sites, r is the number of sites occupied and m is the Hill coefficient which
expresses the degree of cooperativity. The data of both titrations fit the linear
relationship of the Hill equation. The Hill coefficients determined from the slopes
are 1.08 and 1.67 for apo- and Ca 2 + -GLA, respectively. The values confirm that for
the bis-ANS interaction with native apo-GLA no cooperativity can be detected
(m = 1), whereas for the dye interaction with native Ca 2 + -GLA a positive cooperativity has been found (m > 1). The positive cooperativity as well as the fact that
more dye molecules, as expected from the number of available hydrophobic sites,
bind to Ca 2 +-GLA, may imply that several adsorbed bis-ANS molecules bind at
the same site. A similar behavior has been observed for the interaction of bisANS with tubulin (Ward and Timasheff 1994).
4.4
Conformational Changes Induced by bis-ANS in Native GLA
Information on the conformational changes that occur in the GLA-bis-ANS complexes is gathered from protein ellipticity changes in the far- and near-UV wavelength regions (Figs. 15.6 A and B,respectively).
In the absence ofbis-ANS the far- and near-UV CD-spectra of native apo-GLA
at 4°C and Ca2+ -GLA at 37°C are quite similar, in agreement with their similar
states. In the presence of 60 !!M bis-ANS, the absolute values for the far-UV ellipticity changes of apo-GLA at 4°C (Fig. 15.6 A) are decreased at wavelengths near
225 nm and increased near 208 nm as compared with the native state. Comparable behavior is observed in the molten globule state of GLA, as shown by the far-
G. VANDERHEEREN and 1. HANSSENS
apo-GLA concentrations is self-association of the protein. By such selfassociation bis-ANS is expected to be further protected from the water phase.
This provokes a further increase of the fluorescence at protein concentrations for
which the probe is already completely bound. The maximum fluorescence
increase of 1 !!M bis-ANS at a low protein concentration (as used in the experiments presented in Fig. 15.5 B, filled squares) is estimated 66 ± 13 and 38 ± 7
(arbitrary units) for apo-GLA and Ca2+-GLA, respectively. These maximum values are used to calculate the concentration of bound bis-ANS from the fluorescence increase observed on titrating 1 !!M GLA with bis-ANS (Fig. 15.5 B). The
titration curve for the titration of 1 !!M native Ca 2 +-GLA with bis-ANS at 37°C
(Fig. 15.5 B, open squares) is slightly S-shaped. This is indicative of positive
cooperativity for the dye binding which is confirmed by the clear concavedownward shape of the according Scatchard plot (Fig. 15.5 C, open squares). On
the other hand, neither the hyperbolic titration curve, nor the linear Scatchard
plot gives any indication of cooperative behavior for the bis-ANS interaction with
native apo-GLA at 4°C (Figs. 15.5 Band C, filled squares). From the abscis intercept of the Scatchard plot the total number of bis-ANS binding sites is found to
be about 5.3 for native Ca 2 + -GLA and 2.6 for native apo-GLA. In order to determine the corresponding degree of cooperativity, the binding data are plotted
according to the Hill equation (Fig. 15.5 D).
log ((n-r)/r) =-m log Kb-m log [bis-ANSl
where Kb is the average binding constant ofbis-ANS to GLA, n is the number of
binding sites, r is the number of sites occupied and m is the Hill coefficient which
expresses the degree of cooperativity. The data of both titrations fit the linear
relationship of the Hill equation. The Hill coefficients determined from the slopes
are 1.08 and 1.67 for apo- and Ca 2 + -GLA, respectively. The values confirm that for
the bis-ANS interaction with native apo-GLA no cooperativity can be detected
(m = 1), whereas for the dye interaction with native Ca 2 + -GLA a positive cooperativity has been found (m > 1). The positive cooperativity as well as the fact that
more dye molecules, as expected from the number of available hydrophobic sites,
bind to Ca 2 +-GLA, may imply that several adsorbed bis-ANS molecules bind at
the same site. A similar behavior has been observed for the interaction of bisANS with tubulin (Ward and Timasheff 1994).
4.4
Conformational Changes Induced by bis-ANS in Native GLA
Information on the conformational changes that occur in the GLA-bis-ANS complexes is gathered from protein ellipticity changes in the far- and near-UV wavelength regions (Figs. 15.6 A and B,respectively).
In the absence ofbis-ANS the far- and near-UV CD-spectra of native apo-GLA
at 4°C and Ca2+ -GLA at 37°C are quite similar, in agreement with their similar
states. In the presence of 60 !!M bis-ANS, the absolute values for the far-UV ellipticity changes of apo-GLA at 4°C (Fig. 15.6 A) are decreased at wavelengths near
225 nm and increased near 208 nm as compared with the native state. Comparable behavior is observed in the molten globule state of GLA, as shown by the far-