Yezi Cho
American Community School of Abu Dhabi
Proteins are complex macromolecules composed of one or more chains of amino acids, or polypeptides. They play essential roles in many biological processes, including structural support, catalysis, and signaling pathways. Amino acids join together through a condensation reaction. A peptide bond is formed when the carboxyl group (–COOH) of one amino acid reacts with the amino group (–NH2) of another amino acid to form a dipeptide. A molecule of water (H2O) is released as a byproduct. Denaturation is a process in which the structure of a protein is altered causing it to lose function, usually permanently. Extreme changes in pH can alter the protein’s charge, changing protein structure. High temperatures can break the weak hydrogen bonds holding the protein structure together, causing the protein to unfold and lose its function.
A colorimeter is an instrument that measures the amount of light that passes through a sample in the form of absorbance at a particular wavelength. The absorbance is correlated to the concentration of a substance using the Beer-Lambert law. The more turbid the albumin solution, the greater the absorbance indicating higher levels of denaturation. Turbidity is a measure of cloudiness in a liquid. As albumin denatures, the protein molecules clump, becoming more turbid and absorbing more light.
First, the albumin solution was prepared by diluting albumin solution with an equal volume of distilled water. The diluted albumin solution was added to test tubes, which were each placed in water baths prepared in temperatures 0-5 °C, 40°C, 60°C, 80°C, and room temperature. The test tubes were submerged in their respective water baths for at least 10 minutes to equilibrate to the desired temperature.
To measure the turbidity, the colorimeter was calibrated using a cuvette with distilled water and was set to measure absorbance at 430 nm. Press the calibrate button. The control sample at room temperature was inserted to record the baseline absorbance. Each of the test tubes containing albumin at different temperatures were inserted and their absorbances were recorded.
Figure 2. Absorbance of albumin solution at 0-5, 20, 40, 60, 80°C (au)
Absorbance (au) (± 0.001) | |||||
Temperature (°C) | Trial 1 | Trial 2 | Trial 3 | Mean | SD |
0-5 | 0.157 | 0.235 | 0.248 | 0.213 | 0.049 |
20 | 0.121 | 0.155 | 0.148 | 0.141 | 0.018 |
40 | 0.071 | 0.079 | 0.05 | 0.067 | 0.015 |
60 | 0.162 | 0.136 | 0.98 | 0.426 | 0.480 |
80 | 1.18 | 1.12 | 1.357 | 1.219 | 0.123 |
Figure 2. Relationship between temperature (°C) of albumin solution and absorbance (au)
According to Graph 1, the mean absorbance(au) of the albumin increased for 0-5°C, 60°C, and 80°C, while it decreased for 40°C as compared to the standard temperature of 20°C. At 0-5°C, the diluted albumin solution started to freeze, causing the liquid to become white and turbid. At 40°C, the water molecules are interacting with the albumin, resulting in low absorbance due to dilution. The denaturation process begins at a temperature between 40°C and 60°C, as the 60°C solution with a high absorbance rate indicates that the albumin has denatured. The 80°C solution has a significantly higher denaturation rate than the 60°C solution, as its absorbance is much greater due to high turbidity.
Denaturation is the process in which the structure of a protein is altered. High temperatures can break the hydrogen bonds holding the protein structure together, resulting in the unfolding of the protein and loss of function. Albumin is a protein that is affected by high temperatures, becoming more turbid as denaturation occurs thus resulting in measurement of high absorbance by the colorimeter.
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