Ammonium Sulphate Was Required To Give Rise To Precipitation Essay

Analysis:

1. What volume of ammonium sulphate was required to give rise to precipitation?

Tube 1:
5.5mls of (NH₄)₂SO₄ was required to give rise to precipitation.
5.5ml/20ml *100% = 27.5%

Tube 2:
6.7mls of (NH₄)₂SO₄ was required to give rise to precipitation.
6.7ml/20ml *100% = 33.5%

Tube 3:
5.6mls of (NH₄)₂SO₄ was required to give rise to precipitation.
5.6ml/20ml *100% = 28.0%

Tube 4:
6.2mls of (NH₄)₂SO₄ was required to give rise to precipitation.
6.2ml/20ml *100% = 30.5%
1. From the absorbance readings before and after the precipitation, calculate the % recovery of the amylase?

Absorbance reading at start = 0.472 abs  33.7% T

Absorbance reading after precipitation: Calculate Transmittance of each sample:

Transmittance calculations,
% Recovery of Amylase
Tube 1: = 0.080abs  83.2% T Tube 1: 33.7/83.2 = 41.0%

Tube 2: = 0.160abs  69.2% T Tube 2: 33.7/69.2 = 48.7%

Tube 3: = 0.199abs  63.2% T Tube 3: 33.7/63.2 = 53.3%

Tube 4: = 0.220abs  60.3% T Tube 4: 33.7/60.3 = 55.9%

Table 1: Transmittance and Recovery calculations

1. Enzyme denaturation can occur during salting out; what does this mean? Water-soluble proteins have concentric "shells" of semi-ordered water molecules arranged around them, in much the same way that dissolved salts have associated water molecules making them soluble. If you add too much salt, the waters in the protein solvation shells are stripped out to dissolve the salt, precipitating the protein out of solution. High salt on the outside relative to the inside will lead to water diffusing out of the cytosol. If the water concentration in your cell gets low enough, it will destabilize your enzymes and they will denature.

2. Research and describe another method of protein/enzyme
Discussion

Salting out is an effect based on the electrolyte-nonelectrolyte interaction, in which the non-electrolyte could be less soluble at high salt concentrations. It is used as method of separating proteins. This experiment uses ammonium sulphate to force the precipitation of α-amylase using the salting out process. The solubility of a protein is dependent on the salt concentration in the solution. Ideally “the greater protein concentration, the better recovery you will have”, (Anfinsen, 1968).

Tube no. 4 showed the greatest recovery however this tube contained the second highest precipitation count, that honour going to tube two. By comparing tube two with four, we can see that time has a significant effect on the recovery %. This can also be seen by comparing tube threes’ recovery with tube one.

Ideally we would like to repeat this experiment, finding the precipitation point with tube one and then repeating that volume for the last three tubes, leaving us a standard to compare centrifuge times/speed with. I feel that a low power spin (rpm) with a longer time frame would achieve the best results and minimise risk, such as cell lyses etc. “For a typical cell homogenate, a 10 min. spin at low speed (400-500 x g) yields a pellet consisting of unbroken tissue, whole cells, cell nuclei, and large debris.” (Caprette,