The Perception of Hydrophobic Clusters in the Native and Partially Unfolded States
213
For this hydrophobic interaction study it is worth mentioning that an alactalbumin molecule possesses two clusters of aromatic residues. The planes of
these aromatic rings are approximately perpendicular (Fig. 15.1). One of the clusters (Phe-53, Trp-60, Tyr-l03, Trp-104) is situated in the crevice and contains
amino acids belonging to the two structural halves of the protein, the other one
(Phe-31, His-32, Tyr-36, Trp-1l8) is completely situated in the half with the
a-helices (Acharya et al. 1989).
2
Materials and Methods
2.1
Materials
Bovine a-lactalbumin (BLA) is from Sigma (St Louis, Missouri). The protein is
decalcified on a Sephadex G-25 column in 10 mM HCI, lyophilized, and stored at
-20°C until use. Goat a-lactalbumin (GLA) is prepared from fresh milk whey
(Vanderheeren et al. 1998) and stored as described for BLA. In solutions, the concentration of both proteins is determined from the absorption at 280 nm (£280 =
28500 M- I em-I).
The hydrophobic probe 1,1' -bis( 4-anilino-5-naphthalenesulfonate) (bis-ANS)
is from Molecular Probes Inc. (Eugene, Oregon). Its concentration is determined
from its absorption at 385 nm (£385 = 16790 M- I em-I).
All experiments are performed in 10 mM Tris-HCI buffer (pH 7.5) containing
2 mM Ca 2 + or 2 mM EGTA, for Ca 2 +-bound protein and apo-protein (Ca 2 +-free),
respectively. Generally, mixtures of a-lactalbumin and bis-ANS are incubated
overnight at the desired temperature.
2.2
Circular Dichroism
The CD measurements are carried out on a Jasco J-600 spectropolarimeter
(Tokyo, Japan). Cuvettes of 5 (or 10) mm and 1 mm are used for the near-UV and
far-UV regions, respectively. The a-lactalbumin concentration is about 25 IlM.
Circular dichroism signals are monitored as ellipticity changes (expressed as deg
cm 2 dmot l ). Near 220 nm (far-UV) the ellipticity changes are generally dominated by peptide groups in helical structures, while the CD measurements near
270 nm (near-UV) monitor aromatic groups fixed in a specific orientation due to
tertiary structure.
The protein-bis-ANS mixtures for thermal transition studies are preincubated
overnight at the lowest temperature. At each temperature of the transition curve,
the measurements are started 5 minutes after temperature equilibration of the
sample. Evidence that equilibrium states are obtained is presented by the fact that
the ellipticity values are identical in heating and cooling runs, provided they have
not been exposed to more than 70°C for several minutes.
213
For this hydrophobic interaction study it is worth mentioning that an alactalbumin molecule possesses two clusters of aromatic residues. The planes of
these aromatic rings are approximately perpendicular (Fig. 15.1). One of the clusters (Phe-53, Trp-60, Tyr-l03, Trp-104) is situated in the crevice and contains
amino acids belonging to the two structural halves of the protein, the other one
(Phe-31, His-32, Tyr-36, Trp-1l8) is completely situated in the half with the
a-helices (Acharya et al. 1989).
2
Materials and Methods
2.1
Materials
Bovine a-lactalbumin (BLA) is from Sigma (St Louis, Missouri). The protein is
decalcified on a Sephadex G-25 column in 10 mM HCI, lyophilized, and stored at
-20°C until use. Goat a-lactalbumin (GLA) is prepared from fresh milk whey
(Vanderheeren et al. 1998) and stored as described for BLA. In solutions, the concentration of both proteins is determined from the absorption at 280 nm (£280 =
28500 M- I em-I).
The hydrophobic probe 1,1' -bis( 4-anilino-5-naphthalenesulfonate) (bis-ANS)
is from Molecular Probes Inc. (Eugene, Oregon). Its concentration is determined
from its absorption at 385 nm (£385 = 16790 M- I em-I).
All experiments are performed in 10 mM Tris-HCI buffer (pH 7.5) containing
2 mM Ca 2 + or 2 mM EGTA, for Ca 2 +-bound protein and apo-protein (Ca 2 +-free),
respectively. Generally, mixtures of a-lactalbumin and bis-ANS are incubated
overnight at the desired temperature.
2.2
Circular Dichroism
The CD measurements are carried out on a Jasco J-600 spectropolarimeter
(Tokyo, Japan). Cuvettes of 5 (or 10) mm and 1 mm are used for the near-UV and
far-UV regions, respectively. The a-lactalbumin concentration is about 25 IlM.
Circular dichroism signals are monitored as ellipticity changes (expressed as deg
cm 2 dmot l ). Near 220 nm (far-UV) the ellipticity changes are generally dominated by peptide groups in helical structures, while the CD measurements near
270 nm (near-UV) monitor aromatic groups fixed in a specific orientation due to
tertiary structure.
The protein-bis-ANS mixtures for thermal transition studies are preincubated
overnight at the lowest temperature. At each temperature of the transition curve,
the measurements are started 5 minutes after temperature equilibration of the
sample. Evidence that equilibrium states are obtained is presented by the fact that
the ellipticity values are identical in heating and cooling runs, provided they have
not been exposed to more than 70°C for several minutes.