Cooking Egg White : Monitoring Protein Unfolding

Introduction : Egg White is predominantly a mixture of water and 20-40% protein (albumin). When it is clear, the protein molecules form small balls floating in liquid. The protein molecules are approximately 20 Angstroms or so in size.

When egg white is heated, the protein molecules gain energy. The protein chain begins to flop and wiggle more and more until eventually it comes undone. As the protein unfolds it is likely it will get tangled with neighbouring protein molecules. This means the mean particle size increases for two reasons – each molecule is unfolded and molecules start to clump together. The tangles of protein eventually become so large they scatter light as well as X-rays. That’s when the egg turns white.

When you cool egg white the molecules don’t have time to untangle themselves leaving them stuck in the unfolded state.

Protein folding and unfolding is of great interest in biology, medicine and biotechnology. By studying what conditions make proteins fold and unfold, and how fast they do it we hope to work out the rules to making particular proteins.

Experiment :

1. Take a fresh (or refrigerated) egg, break it open and separate the white from the yolk.

2. Transfer about 10 microLitres of egg white to a diameter=1mm glass X-ray capillary and flame seal it.

3. With the sample at 20C, measure the Small Angle X-ray Scattering at least over the range (0.5 < Scattering Angle < 4 Degrees for λ=1.54 Angstroms radiation). On our beamline a good image takes about 200 seconds.

4. Heat the sample to 90C. Remeasure the SAXS pattern.

Results :

Figure One : X-ray Scattering from Uncooked Egg White (1mm capillary, 200 second Exposure, Image is 200 pixels square taken a distance of 3485 pixels from the sample, X-ray Intensity = 10 000 000 X-rays per sec, lambda=1.54 Angstroms, Peak Intensity = 60 Detector Units). Note how the scattering is fairly dim and spreads over a wide angle.

Figure Two : X-ray Scattering from Cooked Egg White (1mm capillary, 200 second Exposure, Image is 200 pixels square taken a distance of 3485 pixels from the sample, X-ray Intensity = 10 000 000 X-rays per sec, lambda=1.54 Angstroms, Peak Intensity = 130 Detector Units). Note how the scattering is bright and confined to a very small angle.

Figure Three – Comparison of Scattering from Cooked and Uncooked Egg White. The plot is of Scattering Intensity per Pixel (ie. proportional to X-rays scattered at that angle) as a function of Scattering Angle. The uncooked egg white scatters brightly out to 1 degree or more. The cooked egg white scatters to less than 0.6 degrees. Clearly the cooked egg white has much larger particles in it than the uncooked egg White

Figure Four : Fitting Scattering from Uncooked Egg White. The Fitted curve is for a molecule with a Radius of Gyration (mean size) of 23 Angstroms.

Figure Five : Fitting Cooked Egg White Scattering Curve. The fitted curve is for a mean protein size of 40 Angstroms. The fitting curve is not particularly good. This either suggests there are a range of particle sizes (some cooked protein and some uncooked protein) or that our model of individual particles is not accurate – instead, the egg would be in a gel-like state.