214
G. V ANDERHEEREN and 1. HAN 55 ENS
2.3
Fluorescence Measurements
The fluorescence measurements are performed with an Aminco SPF-500 spectrofluorimeter (Rochester, New York). The equilibrium binding data for the interaction of bis-ANS with a-lactalbumin is obtained from two fluorescence titration
curves, both at equal pH (pH 7.5), temperature and Ca 2 +-content. First, the limit
fluorescence increase of 1 flM bis-ANS (~Ima,J is determined in the presence of
increasing amounts of protein. Next, this value is used to calculate the concentration of bound bis-ANS from the fluorescence increase on titrating 1 flM protein
with an excess ofbis-ANS. It is assumed that the ~Imax-values obtained from the
former titrations represent the extra fluorescence increase (~I) of 1 flM bound
bis-ANS in the latter one (Vanderheeren and Hanssens 1994).
For both fluorescence titrations the excitation is at 465 nm, which is situated
on the edge of the bis-ANS absorption peak. At this wavelength, even at 400 flM
bis-ANS, the absorbance does not exceed the value of 0.2. The fluorescence emission intensity between 515 and 550 nm is integrated. The background fluorescence of free bis-ANS is subtracted and the resulting fluorescence increase is corrected for inner filter effects.
If tryptophan and bis-ANS are close to each other, they constitute a good fluorescence energy-transfer donor-acceptor pair. For these energy-transfer experiments the excitation is at 290 nm, the absorption maximum for tryptophan. The
fluorescence emission intensity of tryptophan residues between 300 and 420 nm
is measured and corrected for inner filter effects at the emission and excitation
wavelengths.
2.4
Photolabelling of bis-ANS to a-lactalbumin
It has been shown that bis-ANS can be covalently photobound to various proteins
(Gorovits et al. 1995; Seale et al. 1995). For some measurements we needed a
large association ofbis-ANS to native Ca 2 + -bound goat a-lactalbumin with small
free dye concentration. To obtain this, a mixture of 35 flM GLA and 150 flM bisANS in Ca2+ buffer, thermostated at 3rC, is irradiated at a 366 nm wavelength
for 2 hours using a UV 131000 lamp from Desaga (Heidelberg, Germany). Next
the irradiated sample is applied to and eluted from a PD-lO column containing
Sephadex G-25 (Pharmacia, Sweden). The protein with the covalently bound bisANS passes through the column together with the void volume while the free and
non-covalently bound bis-ANS is retarded. In the eluate obtained in this way, on
average 3.3 bis-ANS molecules are covalently bound to 1 Ca 2 + -GLA molecule.
G. V ANDERHEEREN and 1. HAN 55 ENS
2.3
Fluorescence Measurements
The fluorescence measurements are performed with an Aminco SPF-500 spectrofluorimeter (Rochester, New York). The equilibrium binding data for the interaction of bis-ANS with a-lactalbumin is obtained from two fluorescence titration
curves, both at equal pH (pH 7.5), temperature and Ca 2 +-content. First, the limit
fluorescence increase of 1 flM bis-ANS (~Ima,J is determined in the presence of
increasing amounts of protein. Next, this value is used to calculate the concentration of bound bis-ANS from the fluorescence increase on titrating 1 flM protein
with an excess ofbis-ANS. It is assumed that the ~Imax-values obtained from the
former titrations represent the extra fluorescence increase (~I) of 1 flM bound
bis-ANS in the latter one (Vanderheeren and Hanssens 1994).
For both fluorescence titrations the excitation is at 465 nm, which is situated
on the edge of the bis-ANS absorption peak. At this wavelength, even at 400 flM
bis-ANS, the absorbance does not exceed the value of 0.2. The fluorescence emission intensity between 515 and 550 nm is integrated. The background fluorescence of free bis-ANS is subtracted and the resulting fluorescence increase is corrected for inner filter effects.
If tryptophan and bis-ANS are close to each other, they constitute a good fluorescence energy-transfer donor-acceptor pair. For these energy-transfer experiments the excitation is at 290 nm, the absorption maximum for tryptophan. The
fluorescence emission intensity of tryptophan residues between 300 and 420 nm
is measured and corrected for inner filter effects at the emission and excitation
wavelengths.
2.4
Photolabelling of bis-ANS to a-lactalbumin
It has been shown that bis-ANS can be covalently photobound to various proteins
(Gorovits et al. 1995; Seale et al. 1995). For some measurements we needed a
large association ofbis-ANS to native Ca 2 + -bound goat a-lactalbumin with small
free dye concentration. To obtain this, a mixture of 35 flM GLA and 150 flM bisANS in Ca2+ buffer, thermostated at 3rC, is irradiated at a 366 nm wavelength
for 2 hours using a UV 131000 lamp from Desaga (Heidelberg, Germany). Next
the irradiated sample is applied to and eluted from a PD-lO column containing
Sephadex G-25 (Pharmacia, Sweden). The protein with the covalently bound bisANS passes through the column together with the void volume while the free and
non-covalently bound bis-ANS is retarded. In the eluate obtained in this way, on
average 3.3 bis-ANS molecules are covalently bound to 1 Ca 2 + -GLA molecule.