Denaturation of Proteins

Topics: Protein, Protein structure, Denaturation Pages: 8 (1919 words) Published: August 15, 2013
Almero, K. J. Date Due: 3 July 2013 Dante, R. A. Date Submitted: 3 July 2013 EXPERIMENT NO. 3 DENATURATION OF PROTEINS

Abstract

The experiment aimed to use the concept of viscosity to study the effects of different denaturants on 1% albumin extract. An Ostwald viscometer was used to measure the flow time of 5 mL of the blank and native protein. These were then denatured by adding 1 mL of denaturant and had their flow time measured. The flow time from the blank to denatured protein is increasing. The specific viscosity and reduced viscosity, ηred, were calculated from the data obtained. The best denaturant was determined based on the ηred of the denatured protein. NaCl had the highest percent change in viscosity from the native to denatured protein therefore it is the best denaturant for 1% albumin. The experiment can be deemed successful because of the high viscosity obtained in the results, indicating that the protein had been denatured as expected.

Results

Proteins are very sensitive to external conditions. To be able to preserve an extracted protein, one must know and maintain the favorable environmental conditions so that denaturation would be prevented. Denaturation is any alteration or modification in the native proteins’ secondary, tertiary or quaternary structure.1 Conditions that induced denaturation include pH changes, organic solvents, detergents, oxidation and reduction, salt concentration, metal ions, temperature changes and mechanical stress. For most researches and experiments, denaturation is avoided as much as possible because proteins have to be in their native/natural state. However, there are also circumstances wherein the proteins are needed to be denatured. An example of this is during the study of the folding/unfolding of proteins and rate of enzyme reactions.1

There are available methods in determining protein denaturation. The Circular Dichroism and Optical Rotatory Dispersion or CD-ORD detects changes in the secondary structures of proteins. Other methods include Nuclear Magnetic Resonance (NMR), fluorescence and viscometry which detect tertiary and quaternary structures’ alterations.2 In the experiment, only the viscometry was performed and it was done by measuring the viscosity of proteins and different denaturants in an Ostwald viscometer.

Two trials were done for the measurements of the flow time, t. Table 1 below shows the average flow time measured for the blank, natured protein, denaturant, and denatured protein in succession respectively. Different viscometers were used for each set of solutions. Generally, it can be seen that the flow time from the blank solution to the denatured protein solution is increasing.

Table 1. Average Flow Time Measurements
Denaturant| Average Time (s)|
| to| tp| td| tdp|
HCl| 111| 111| 116.5| 112.5|
NaOH| 209| 211.5| 228| 221|
NaCl| 207.5| 212| 227.5| 246.5|
Urea| 220.56| 218.49| 225.35| 222.06|
SDS| 64.5| 65.5| 68.5| 68.5|
β-mercaptoe-thanol| 103.94| 104.54| 106.96| 107.35|
Formalde-hyde| 190.85| 192.85| 232.7| 196.6|
Heat| 230.25| --| --| 223.75|

From the data obtained in table 1, the specific viscosity can be calculated. Table 2 below shows the specific viscosity of each natured protein, denaturant, solution, and denatured protein per set of runs. The specific viscosity of the solution was calculated to determine the ηsp of the denatured protein. The value of the reduced viscosity, ηred, is calculated by simply dividing the ηsp by the concentration.

Table 2. Calculated Specific Viscosity Measurements
Denatu-rant| ηsp (native)| ηsp(dena-turant)| ηsp(sol’n)| ηsp(dp)|...


References: [1] Switzer, R. and Garrity L. 1999. Experimental Biochemistry, 3rd ed.; United States of America: W. H. Freeman and Company. 89, 90
[2] Biochemistry Laboratory Manual, 2013 ed.; Quezon City: University of the Philippines – Diliman
[3] White, A. 1973. Principles of Biochemistry, 5th ed.; United States of America: McGraw Hill Book Company. 162
[4] Campbell, M
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