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The purpose of the experiment was to test the free sulphydryl groups that may be important for the enzyme activities as well as the roles that the disulphide bonds plays n the stabilizing of the structure that is 3 dimensional of the enzymes and proteins. The latter purpose is vital in the study of proteins. The results for tube 2 showed that N= 6.588 and that tube 3 showed that N= 0.153 sulphide groups in the samples. Tube 2 contained Urea and sodium borohydrate. When the proteins were denatured partially, it ...view middle of the document...
One drop of Octyl alcohol was added to each tube, it was mixed well and shook to dissolve the urea. Tubes were placed in a water bath at 37°C and allowed the reduction reaction to proceed for 30 min. Then 0.5ml of 1M KH2PO4 which contained 0.2M Hydrochloric Acid was added to each tube. Wetted the sides of the tubes of destroy the Borohydride. 2 ml of acetone was added to each tube after 5 min mixed well. Then bubble nitrogen was passed through each tube for 2 min. 0.5ml of 10mM DTNB in 0.05M phosphate buffer pH 8 was added immediately after purging with nitrogen. Finally, bubble nitrogen was passed through each tube for 2 min to fill the gas space. Stopper the tubes and allowed to stand for 15 min at 37°C. 3ml glass cuvettes and Varian spectrophotometer were used for reading the absorbance at 412 nm. Then the instrument was zeroed on water. The tubes were taken straight from the water bath to the cuvette keeping the contents warm to prevent phase separation which will cause cloudiness and prevent a true absorbance reading.
Table 1a of Lysozyme.
|Absorbance and Concentrations: Solutions |Absorbance (AU) |
|1 |0.065 |
|2 |0.928 |
|3 |0.116 |
|4 |0.095 |
Our data was faulty and thus could not be used for this calculation; this was due to errors in concentration of our protein samples. Hence we used other results that were more accurate.
Table 1b: Rectified, more accurate results
|Solutions |Absorbance (AU) |
|1 |0.185 |
|2 |0.193 |
|3 |0.104 |
|4 |0.089 |
Graph 1: Standard curve of BSA .
From the table the absorbencies at 412nm of Tube 1, 2, 3 and with no proteins were 0.270, 0.655, 0.193 and 0.182 respectively. Absorbance values of the tubes of 1, 2 and 3 were subtracted from the tube 4 which had no protein. After subtraction, the absorbance values were...