Biochemistry of Proteins. Isolation of Ovalbumin: Characterisation of Thiol Groups and Separation by Gel Filtration

With these absorbance numbers a concentration curve was constructed and the unknown solution was determined by finding the point of absorbance on the curve.

3ml of sample was taken first flask at 4 minutes and added to the appropriate tube of sodium hydroxide, from the second flask at 4.5 minute and so on, each flask was sampled at 30 second intervals. The sampling was then repeated starting at 8,12,16 minutes. The final sample from the last flask was taken at 18.5 minutes. Once the sampling was completed, measurements of absorbance were obtained for solution in each tube at 405 nm.

After this, the solution was poured into a volumetric flask just about to the 1dm3 line and then it was left there to cool to the same temperature as the room before filling precisely to the 1dm3 line with distilled water. The molar mass of CuSO4.5H20 was 249.5 so that means 249.5g of copper sulphate was needed to dissolve, in order to make a standard solution, into 1dm3of distilled water. Following this, a linear dilution of the CuSO4.5H2O was made in order to be used to make a calibration curve after using the colorimeter to write down the absorbance of each sample. A linear dilution is diluted with distilled water in order for it to make the concentration weaker and weaker. For this investigation, the dilutions made ranged from 0.01 to 0.1 M/l . It was essential to only make up 10cm3

10 microliters of the sample is then added and the assay absorption is measured at 340nm. If absorbance was above 1.5, samples were diluted.

Time) because it had a correlation closest to 1. All three orders were graphed and a linear regression was used to see which graphed order was closest to 1. The order was determined by comparing the concentration and time to the mathematical predictions made using the integrated rate laws. Analyzing each graph and finding each correlation helped determine which graph was closest to 1. The more concentrated a solution is, the higher the absorbance of that solution. This is due to Beer’s Law. The law measures the absorbance of a solution by determining how much light passes through a solution. As the concentration of a solution increases, fewer wavelengths of light are able to pass through the concentrated solution. The absorbance at 60 seconds was 0.573 (Figure 1: Table1). To calculate the concentration (molarity), the Beer’s Law equation was used, Abs = slope(m)+b. Plugging in what is known into the Beer’s Law equation resulted in 0.573 = 3.172e+004 + 0, where the concentration is determined by M = 0.573-0/ 3.172e+004. So, the concentration at 60 seconds using the equation (M = 0.573-0 / 3.172e+004) was 1.824e-5 M. The 1st order graph resulted in k=0.006152 (Figure 1: Graph 1). Other groups also resulted in their decolorization of CV to be the 1st rate

Scientists use an instrument called a spectrometer to quantitatively determine the amount of light absorbed by a solution. The primary inner parts of a typical spectrometer are described below. The spectrometer has a light source that emits white light containing a vast mixture of different wavelengths of electromagnetic radiation. The wavelength of interest is then selected using a monochromator (“mono” meaning one and “chromate” meaning color) and an additional exit slit. The separation of white light into different colors (wavelengths) is known as diffraction. The selected light then reaches the sample and depending on how the light interacts with the chemical compound of interest, some of the light is absorbed and some passes straight through. By comparing the amount of light entering the sample (P0) with the amount of light reaching the detector (P), the spectrometer is able to tell how much light is absorbed by the sample.

The Beers Law calibration experiment used many concentrations of crystal violet solutions. Each of these solutions were test and analyzed in order to determine the absorbance of each concentration The results were than graphed and produced a slope of 1.00E05 with an intercept of -2.21E-02.

The dark, navy blue colored graph represented the absorbance curve for the S1 sample. The red colored graph represented the absorbance curve for the S2 sample. The green colored graph represented the absorbance curve for the P1 sample. The purple colored graph represented the absorbance curve for the P2 sample. The gaps between the P2 curve was due to the oversaturation that led to the inconclusive spectrophotometer readings. The blue colored graph represented the absorbance curve for the P1 low salt sample. The orange colored graph represented the absorbance curve for the P2 low salt sample. The light blue colored graph represented the absorbance curve for the P1 medium salt sample. The light pink colored graph represented the absorbance curve for the P2 medium salt sample. The light green colored graph represented the absorbance curve for the P1 high salt sample. The light purple colored graph represented the absorbance curve for the P2 high salt

The values of color absorbance are effective because color absorbance has a linear relationship with concentration values, which in turn, allows us to easily find concentration values for many solutions. Beer’s law describes this phenomenon since the absorbance is directly proportional to concentration. We observed that as the color absorbance increased, the concentration of the FeSCN2+ complex ion increased. This is because as the FeSCN2+ concentration increases, the blood-red color becomes darker due to more presence of the blood-red FeSCN2+ ion. Therefore, the color absorbance increases because there is more blue color absorbed by the darker red color. We then graphed the absorbance and concentration values and created a line of best fit. Using the line of best fit, we were able to predict the equilibrium concentrations of the FeSCN2+ solutions and find the change required to reach equilibrium. Since we already knew the initial concentration of FeSCN2+ and since we already found the equilibrium concentration of FeSCN2+, we can calculate the change in equilibrium. Using this data, we were able to calculate the equilibrium concentration of all of the species in this lab, since we already knew the change from the initial concentration to the equilibrium change. Q is less than K because there was no initial concentration of FeSCN2+, but after the system reached

Having removed the detergent, the protein will refold. As shown by the Anfinsen experiment the polypeptide sequence determines the folding and therefore the three dimensional structure. As the polypeptide sequence is unaltered refolding can occur through the process of nucleation aggregation and compaction. In order to test that the protein was no longer denatured, the absorbency of the solution at 412nm could be measured and compared with the graph in figure 1 above, it should match the plot of standard ovalbumin in the absence of SDS.

1.2 1.0 0.8 0.6 0.4 0.2 0.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 Concentration (mM) y = 0.1897x R2 = 0.99

3. The spectrophotometer was set at 420nm. Distilled water was also used as the ‘blank’.

The absorbance is measured using a Plate reader and a Standard curve is generated. Also, the different types of pipetting techniques are assessed in this Assay.

Incorporation of assay controls included setting up a spectrophotomer and running the chart recorder with a full-scale deflection before the start of the assay. The set recorder had a corresponding value of 1 for the change in the absorbance. Therefore, prior testing was done to observe whether a change occurred in the readings. This helped to indicate that the results were valid, as they could have been affected by a fault during the setting up of the spectrophotometer. On the other hand this was considered as one of the controls for the experiment. Nevertheless, a new cuvette had to be used for each assay.

concentration, record the absorbance readings at a fixed wavelength, and plot the absorbance vs. concentration data. The wavelength of 520 nm was selected for experiment Part