5.2.1.
Preparation of porcine p-IactoglobuIinThe lyophilised whey provided by D. Gallagher (Department of Food Chemistry, University College Cork, Ireland) was prepared by the acid precipitation of caseins from skim milk at pH 4.5 (Gallagher et aI., 1 996). Porcine P-lg was purified from the lyophilised acid whey using the procedure of Dalgalarrondo et al. ( 1 992).
OH BHT C - OH 11 o R o C-OH I1 o Palmitic Acid Retinol or Vitamin A cis-Parinaric Acid R = H, CHa ANS
Figure 5 . 1 . 1 . 1 . The structures of ligands referred to in the text. BHT, butylated hydroxytoluene; ANS, 1 ,8-anilinonaphthalene sulphonate.
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The porcine {3-lg was further purified by Gavin Manderson (New Zealand Dairy Research Institute, Palmerston North, New Zealand) using the following procedure. The porcine {3-lg sample was concentrated fourfold by stirred cell ultrafiltration, using apparatus supplied by Amicon, Inc. (Beverly, MA, USA). It was purified further by size-exclusion chromatography on a Superdex 75 column (50 mm x 600 mm) using the Pharmacia FPLC system (see Section 4.1 .2.3). The column was equilibrated with pH 6.7 phosphate buffer (26 mM sodium phosphate and 68 mM NaCl) prior to chromatography. After checking the purity of each fraction by alkaline-PAGE (see Section 3 .2. 1 .4), selected fractions were pooled and stored frozen at -21 QC.
5.2.2. Measurement protocols Sample treatments
Frozen solutions of porcine or bovine {3-lg were thawed at 4 QC, dialysed against pH 6.7 phosphate buffer (26 mM sodium phosphate and 68 mM NaCI) or pH 7.7 Tris-phosphate buffer ( 1 3 mM Tris, 1 3 mM sodium phosphate and 68 mM NaCl) and then filtered using a membrane filters (0.45 !lm, Millipore Corporation, Bedford, MA, USA). The concentration of protein was determined using 280 nm absorbance and extinction coefficients of 5.65 ( 10.0 mg/mL of porcine {3-lg) (Kessler and Brew, 1 970) and 9.6 (10.0 mglmL of bovine {3-lg) (Bell and Mckenzie, 1 967) so that they could be diluted to appropriate concentrations with buffer for each set of experiments.
Retinol, PnA and palmitic acid were made to 1 mg/mL in boiled and degassed ethanol and stored under oxygen-free nitrogen in the dark. On occasions, equimolar concentrations of BHT were mixed into these solutions. This reagent does not compete with ANS, PnA or retinol for binding to bovine {3-lg (unpublished result, L. K. Creamer, 1 995). However, it does provide some protection against oxidative degradation of retinol and PnA.
CD measurements
The porcine and bovine {3-1g concentrations were adjusted to 1 . 0 mg/mL with pH 6.7 phosphate buffer. For investigation of ligand binding, the molar ratio {3- 19:1igand was kept between 1 .0: 1 . 1 and 1 .0: 1 .2 throughout the experiment. The {3-lg solutions were placed in a water-jacketed 1 0 mm path length CD cell (Jasco,
9 1
Ishikawa-cho, Hachioji city, Tokyo, Japan) which was connected t o a Neslab model RTE-lOO water bath (Neslab Instruments Inc., Newington, NH, USA). Each f3-lg solution was held at 20 DC for 20 min prior to measuring the spectrum. Because of the difficulty in obtaining good temperature control in the short path length cells, far-UV CD measurements were not made.
The temperature of the water bath was then increased to 40 DC and held for 20 min, and then another spectrum was run. This procedure was repeated using 20 or 1 0
D C intervals until a temperature o f 80 D C was reached. The water bath temperature was then decreased by 20 DC increments to 20 DC. A calibration run was done to detennine the relationship between cell contents and water bath temperatures, after the water bath had been calibrated against a secondary temperature standard (Manderson,
1 998). The baseline correction is described in Section 3 .2.2.
Ligand fluorescence spectroscopy
Fluorescence measurements were made on a 3.0 mL volume of the unheated 1 .0 mglmL stock solution of porcine P-Ig or bovine P-Ig B was put into a 1 0 mm x 1 0 mm fluorimeter cell. The cell was then placed in the temperature-controlled cell holder of a Perkin-Elmer MPF-2A fluorescence spectrophotometer (Norwalk, CT, USA), which was connected to the Neslab water bath, and given 20 min to attain thennal equilibrium at a water bath temperature setting of 20 DC. Measurements were then made using excitation and emission slit widths of 8 nm, at a scan speed of 25 nrn/min.
For ANS fluorescence measurement, an aliquot (60 ilL) of ANS ( 1 .4 1 mM) was added to the protein solution in the fluorimeter cell and mixed by inversion, after which the fluorescence spectrum of the ANS was detennined. After ANS addition, the mixture was excited at 370 nm and the emission was scanned from 375 to about 520 nm.
For retinol fluorescence measurement, the molar ratio f3-1g:ligand was kept between 1 .0: 1 .0 and 1 .0: 1 .2 and the fluorescence spectrum of the retinol was detennined as above. The mixture was excited at 350 nm and the emission was scanned from 3 1 0 to about 5 1 0 nm.
The temperature of the water bath was increased to 40 DC and then increased stepwise by 4 DC increments to 88 DC; after each increase, the fluorescence emission
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spectrum was recorded as described above. After measurements had been made at 88 °C, the temperature of the water bath was decreased stepwise by 1 0 °C increments to 20 °C and, after each decrease and a 20 min holding period, the emission spectrum was recorded as described above. During all holding periods, the �-lg solution was shielded from the light source of the fluorimeter.
For both ANS and retinol fluorescence measurements, the peak position, A.max,
and the peak heights at