Methods: Chapter

Heat treatment of protein solutions at 84 °C

Protein solution (250 g) was transferred into a glass beaker (400 ml) plus a magnetic stirring bar. The container was covered with transparent food wrap to prevent water evaporation during the heat treatment and placed into a waterbath above a magnetic stirrer plate. A thermometer was placed into the solution. The temperature in the solution reached 83 °C after 10 min. The maximal temperature of 84 ± 0.3 °C was reached after 15 min. Before heating, the solutions had room temperature and after heating they were put on ice immediately. After 30 min on ice they were stored at 5 °C.

Native PAGE (polyacryl amide gel electrophoresis) method

The method is suitable to qualify and quantify whey proteins and was adapted from Havea et al. (1998) and Patel (2007b). For all preparations and procedures extra pure demineralised water (Milli-Q water) was used. The term %C stands for % w/w of

crosslinking N,N'methylene-bis-acrylamide and %T stands for the

concentration/strength of acrylamide.

Native-PAGE resolving gel was prepared as follows: 2.0 ml native resolving gel buffer, 6.0 ml water and 8 ml acrylamide/bis 37.5:1 solution (30 %) were pipetted into a vacuum flask, mixed well and degassed while stirring with a magnetic bar under vacuum for 20 min. The native resolving gel buffer consisted of 3.0 M tris(hydroxymethyl)methylamine adjusted to pH 8.8 with hydrochloric acid. Immediately after the degassing procedure, 8 µl of TEMED and 80 µl ammonium persulphate (10 %) were added to the degassed solution and mixed gently to initiate the polymerisation reaction leading to polyacrylamide. Then 3.3 ml of the solution was

quickly pipetted between two glass plates, sealed on the bottom and at the sides (Mini- Protean® 3 Cell equipment). Water (0.5 to 1 ml) was pipetted on top of the solution between the glass plates, to give the gel a smooth and level surface upon setting. After 30 min at room temperature, the resolving gel (15 % T, 2.67 % C) had formed and the water was removed from the top layer using filter paper.

Native-PAGE stacking gel was prepared by mixing 2.0 ml stacking gel buffer, 5.0 ml water and 1.0 ml acrylamide/bis 37.5:1 solution (30 %) in a vacuum flask, followed by the degassing procedure described above. The native stacking gel buffer consisted of 0.5 M tris(hydroxymethyl)methylamine adjusted to pH 6.8 with hydrochloric acid. Immediately after the degassing procedure, 40 µl ammonium persulphate (10 %) and 8 µl TEMED were added to the degassed solution and mixed gently. The mixture was then pipetted between the glass plates on top of the pre-formed resolving gel. A plastic comb with ten slots was inserted, to form sample loading wells in the stacking gel. It was taken care that no air bubbles were embedded during the insertion. After 90 min at room temperature, the glass plates were removed from the apparatus, as the gel formation was completed.

Two gels, both consisting of a stacking and resolving gel part situated between two glass plates, had been prepared at a time. They were placed into an electrophoresis chamber filled with native-PAGE electrode buffer solution, pH 8.3 (which consisted of 0.125 M tris(hydroxymethyl)methylamine and 0.95 M glycine, diluted 1:4 with water).

Prepared sub-samples (10 µl) were charged into the sample loading wells on top of the stacking gel with a Hamilton syringe (Hamilton Company, Reno, Nevada, USA). The sub-samples consisted of a mixture of 50 µl protein solution (original sample) plus 950 µl native gel sample buffer (pH 6.8). Native gel sample buffer (pH 6.8) had been

prepared by combining 200 ml native stacking gel buffer (0.5 M

tris(hydroxymethyl)methylamine adjusted to pH 6.8 with hydrochloric acid), 20 ml Bromphenol blue solution (0.4 %), 600 ml water and 80 ml glycerol.

Then, electrophoresis was carried out at a maximum voltage of 200 V, with a final current of 70 mA, for a run time of approximately 70 min; power was turned off when

the bromphenol blue dye band reached the bottom of the resolving gel. The gels were then removed from the glass plates and transferred to polyethylene containers for the staining and destaining procedure.

For the staining procedure, the gels where immersed in 50 ml amido black 10B dye (0.1 % (weight/volume) in 10 % acetic acid) for 60 min. Then the solution was removed and the gels drained. Afterwards, 100 ml acetic acid (10 % (volume/volume)) was added for destaining. The solution was replaced after 1 h and one more time after 10 h with 100 ml of acetic acid (10 % (volume/volume)). To support the staining and destaining processes they took place on a platform rocker. After 20 h destaining time, the gels were scanned and the colour intensity of the ß-lactoglobulin bands was quantified.

Four gels were prepared to run all together six replicates of each protein solution sample. On each gel two replicates of the protein solution sample without heat treatment (control) was run. The entire procedure was repeated once with a separate sub-sample preparation. The controls represented on each gel 0 % protein denaturation and showed the darkest stains. The colour intensity decreased, the higher the degree of denaturation was. The reduction in colour intensity of a stain in comparison to the average intensity of the two controls, represented the degree of protein denaturation.

Total sulfhydryl determination

Total sulfhydryls were determined as described in Section 3.6.4, Methods: Chapter 7.2. The same chemicals were used.

Free sulfhydryl determination

Protein solution (2000 µl) or RO water for controls, 200 µl phosphate buffer solution and 500 µl DTNB solution were added into test tubes, with vortex mixing after each addition (triplicates). The tubes then were put in the dark for 30 min at room temperature. The assay was other than that the same as for the determination of total sulfhydryls described inSection 3.6.4, Methods: Chapter 7.2. The same chemicals were used. For free sulfhydryl determination a standard curve was created using cysteine solutions (based on RO water) of different concentration levels instead of protein solution.

3.6

Materials and methods in Chapter 7

3.6.1 Materials: Chapter 7

The oxygen concentration was measured with an YSI model 57 Dissolved Oxygenmeter plus oxygen electrode from YSI Incorporated, Yellow Springs, OH, USA. For calibration the oxygen electrode was inserted into a 250 ml glass bottle, the bottom covered with 2-3 cm water. The bottle was immersed in a waterbath at 40 ˚C for approximately one hour until the temperature in the bottle had equilibrated to 37 ˚C (measured with the internal thermometer at the oxygen electrode). The needle of the oxygen meter was then adjusted to 6.73 mg/l, which is the solubility of oxygen in water exposed to water saturated air at 37 ˚C (YSI model 57 Operating Instructions).

The free radical agent 2,2`-azobis(2methylpropion-amidine) dihydrochloride (AAPH) (97 %), 5,5`-dithio-bis(2-nitrobenzoic acid) (DTNB) (99 %) and xylenol orange (no purity stated) were of the brand Aldrich from Sigma-Aldrich Co., St. Louis, Missouri,

USA. Sodium dihydrogen orthophosphate 1-hydrate (99.0 – 102.0 %),

tris(hydroxymethyl)methylamine (≥ 99 %), sodium dodecyl sulphate (SDS), ferrous sulphate 7-hydrate, hydrogen peroxide (30 %), sodium acetate-3-hydrate (99.5 %), phosphoric acid (~ 85 %) and trisodium phosphate-12-hydrate (99 %), all analytical reagents, were of the brand BDH from VWR International, West Chester, PA, USA. Disodium hydrogen orthophosphate 7-hydrate (≥ 99 %) and sodium hydroxide (NaOH, analytical reagent) were of the brand Ajax Chemicals from Ajax Finechem Pty. Ltd., Taren Point, Australia. Cysteine (no purity stated) was of the brand USB from Affymetrix Inc., Santa Clara, CA, USA. Oxygen free nitrogen, gas code 152, was purchased from BOC New Zealand Ltd., Auckland.

3.6.2 Methods: Chapter 7

Preparation of phosphate buffer (0.1 M), pH 6.0

Phosphate buffer solution (0.1 M) of pH 6.0 was prepared by combining 0.1 M solutions of sodium dihydrogen orthophosphate 1-hydrate and disodium hydrogen orthophosphate 7-hydrate in RO water.