User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/11/13

Experimental Biological Chemistry I

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Purpose Au/HRP solution were run under UV-vis spectrometer from 200nm to 800nm to obtain spectrum of supernatant Au/HRP and Au/ADA solutions were run using Atomic Absorption spectrometer to test the concentration of gold present in solution supernatant Adenosine stock solution was prepared to perform test to calculate ADA enzymatic activity Procedure on UV-vis spectrometry of Au/HRP Uv-vis spectrometer was set to scan Au/HRP samples made on 2012/11/07 from 200nm to 800nm 3mL of distilled water was loaded into quartz cuvette with 10.0mm pathlength and placed into base line standard 3mL of supernatant from following ratios of Au/HRP samples made from 2012/11/07 were loaded into quartz cuvette with 10.0mm pathlength separately to obtain spectrum. 10 - 50 - 100 - 150 - 200 - 250 - 300 - 350 - 400 - 450 The supernatant fro Au/HRP solutions were carefully pipetted out without including fibers in the samples to make sure more accurate measurement. Spectrum were run from 200nm and 800nm, and results were collected and plotted and shown in Data section below. Data on UV-vis spectrometry of Au/HRP Graph of Absorbance versus wavelength scanned were shown below. All ratios of Au/HRP solutions were tested and plotted on the following graph: According to the graph, the ratio of Au/HRP with the highest absorbance is 100 Au/HRP. There is a linear relationship between absorbance obtained and the concentration of Au/HRP gold nanoparticles present in samples. This means that Au/HRP has the highest amount of gold nanoparticles formed in the supernatant. Also according to the graph, 100Au/HRP yields the highest concentration of gold nanoparticles in solution, while the rest of ratios tested yields not as high. At ratios around 100Au/HRP: 50Au/HRP and 150Au/HRP, the concentrations of gold nanoparticles were decreasing. This result suggests a more in depth test with smaller increments between the range of ratios 50Au/HRP and 150Au/HRP to determine the exact ratio of Au/HRP that yields the maximum concentration gold nanoparticle in solution. With this information, one can predict the ratio of gold to HRP at which the gold nanoparticles in solution starts to form fibers. The absorbance at 525nm were looked at separately to show a better relationship between absorbance and different ratios of Au/HRP. The wavelength 525nm was choosen because this wavelength best measures the amount of gold nanoparticles in solution according to Bakshi, et al, a paper that investigates protein films made using gold nanoparticles formed between gold and BSA. A table of absorbance at different ratios of Au/HRP was listed below: Ratio of Au/HRP Absorbance at 525nm 0 -0.009 10 -0.003 50 0.0560 100 0.208 150 0.129 200 0.120 250 0.023 300 0.004 350 0.001 400 0.000 450 0.001 The data points were graphed on a separate graph to show the absorbance of solutions as ratios of Au/HRP change from 10 to 450. A graph of Absorbance at 525nm versus Ratios of Au/HRP ranging from 10 to 450 is shown below: From the graph above, it can be seen that the concentration of gold nanoparticles in supernatant increases drastically from 10Au/HRP and peaked at 100Au/HRP. Then the concentration of gold nanoparticles decreases about 20% around 100Au/HRP to 200Au/HRP, then decreases back to 0 absorbance starting at 300Au/HRP. Thus, it can be predicted that under 85°C, the gold nanoparticles forms in solution until 100Au/HRP, then fibers starts to form after 100Au/HRP. All gold nanoparticles aggregate and form fibers when the ratio of Au to HRP reaches 300. It was concluded to test ratios between 50 to 150 in smaller increments for more accurate conclusion on when protein aggregates form. Procedure on Atomic Absorption Spectrometry of Au/lysozyme and Au/HRP solutions 8 of HCl samples with the concentrations below were run to set up calibration curve: 5ppm - 8ppm - 10ppm - 15ppm - 20ppm - 25ppm - 30ppm - 40ppm The absorbance obtained were graphed with absorption versus concentration of HCl. The slope of the plot was used to calculate the concentration of gold in Au/HRP and Au/lysozyme solutions. Because the absorption data and graph were not saved during experimental process, the results cannot be displayed in Data section. Supernatant of Au/HRP samples made on 2012/11/07 with a ratio ranging from 10 to 450 were run using Atomic Absorption Spectrometry. Supernatant of Au/lysozyme samples made on 2012/10/31 with a ratio ranging from 20 to 130 were also run using Atomic Absorption Spectrometry. Distilled water was prepared in a 280mL flask to rinse off samples after Au/HRP and Au/lysozyme samples were run. Samples with different ratios of gold to either HRP or lysozyme was run by inserting sample tube into the supernatant of samples. The purple fibers in the samples were avoided to prevent clogging. After the absorbance of samples were taken, the sample tubes were inserted into distilled water for rinsing. This process was repeated until all samples were run. The absorbance for each Au/HRP and Au/lysozyme samples were collected by the atomic absorbance spectrometer. The absorbance for each sample were fitted into the slope of calibration curve to obtain the concentration of gold in unit of ppm. Because the raw data were lost, the numbers cannot be shown here. Data on Atomic Absorption Spectrometry of Au/ADA and Au/HRP solutions The concentration of gold in unit of ppm was calculated by converting absorbance into concentration using the HCl calibration curve. The concentration of gold for corresponding ratios of Au/HRP and Au/Lysozyme were displayed in table below: Au/HRP ratio Concentration of Au[ppm] Au/Lysozyme Ratio Concentration of Au[ppm] 10 -1.5443 20 0.0057 50 1.8191 30 1.9388 100 11.7714 40 2.7586 150 13.1419 50 3.806 200 23.6748 60 4.5002 250 24.4588 70 -2.4599 300 27.876 80 -2.4839 350 25.4761 100 -2.5856 400 35.1173 120 -2.6394 450 25.6198 130 -2.6454 A graph was made with concentration of gold in ppm in supernatant versus mole ratio of Au to HRP and displayed below: From the graph above, increase in mole ratio of Au/HRP leads to an linear trend of increase in gold concentration in supernatant. This indicates no protein aggregation would form as mole ratio of Au/HRP increases. The result provide a contrast with results from 2012/10/17 with the maximum concentration of gold in supernatant at low mole ratios of Au/BSA and minimum concentration of gold in supernatant once the mole ratios of Au/BSA reaches over 134. The result indicate that protein aggregation is less likely to form when HRP is used to replace BSA. This might be caused by the nature of HRP compare to BSA and different behaviors when proteins unfold and refold. Gold concentrations in Au/Lysozyme samples were also determined and plotted onto the graph "Concentration of Au in Solution of Au-Lysozyme" below: In the graph above, the concentration of gold in Au/Lysozyme supernatant increased from mole ratio of 20 to 60, and dropped immediately from mole ratio of 60 and on. From mole ratio of 60 and on, there are no gold present in supernatant. The samples with mole ratio from 60 and on appear transparent as oppose to purple. This result indicates that protein started to form aggregation from mole ratio of 60 and on. Compare to the result with Au/HRP from above and Au/BSA from 2012/10/17, the behavior of Au/Lysozyme resemble more like Au/BSA samples in terms of protein aggregation formation. This result also indicates that BSA and lysozyme behavior similarly when forming gold nano particles. It can also indicate that they have similar protein folding and unfolding kinetic mechanism. Graph and data plotting the percent concentration change before and after in unit of ppm for both Au/HRP and Au/Lysozyme is shown below: Au/ADA ratio ADA added [uL] HAuCl4 [uL] Water [uL] Mol Au [mol] Mass Au [mg] Initial [Au] [ppm] Final [Au] [ppm] Change in [Au] [ppm] Percent Change [Au] [%] 60 137.1 45.71 7817.2 4.79955E-07 0.094535296 11.81691206 1.3572 10.45971206 88.51476601 70 137.1 53.3 7809.6 5.5965E-07 0.110232582 13.77907269 -0.2608 14.03987269 101.8927253 80 137.1 60.9 7802 6.3945E-07 0.125950548 15.74381852 3.6762 12.06761852 76.64988328 90 137.1 68.6 7794.4 7.203E-07 0.14187533 17.73441626 -1.0337 18.76811626 105.8287794 100 137.1 76.2 7786.8 8.001E-07 0.157593297 19.69916209 -1.331 21.03016209 106.7566326 110 137.1 83.8 7779.1 8.799E-07 0.173311263 21.66390791 -1.2499 22.91380791 105.7695038 120 137.1 91.4 7771.5 9.597E-07 0.18902923 23.62865374 -1.3351 24.96375374 105.6503431 130 137.1 99 7763.9 1.0395E-06 0.204747197 25.59339956 -1.4923 27.08569956 105.8308002 140 137.1 106.7 7756.3 1.12035E-06 0.220671978 27.58399731 -1.2172 28.80119731 104.4127034 150 137.1 114.3 7748.7 1.20015E-06 0.236389945 29.54874313 -1.4464 30.99514313 104.8949629 The table above listed the calculated initial gold concentration in unit of ppm and final measured concentration of gold in ppm in Au/ADA dialyzed samples. The change in gold concentration indicates how much gold nanoparticles were formed. If the percent change of gold is creater than 1, then it means that most of gold were incorporated into gold nanoparticles. When percent change goes below 1, data indicates that there were still some percentage of gold left in solution that were not nucleated within proteins to for gold nanoparticles. The graph plotting the percent change in concentration of gold in solution versus the mole ratios of Au/ADA were shown in graph below: It can be seen that th percent change in gold concentration were relatively the same. The lowest percent change in gold concentration appeared to be 80Au/ADA. However, this might be due to accidental suspension of nanoparticle fibers when Atomic absorption spectra were taken. Aside from the 80Au/ADA data point, the second lowest change in percent change in gold concentration happened to be 60 Au/ADA. This result might indicate that the concentration of gold in solution in 60 folds encouraged less nanoparticle formation compared to Au/ADA samples with greater mole ratios. Lastly, the percent chage in gold concentration were roughly the same in remaining Au/ADA samples as the mole ratios goes above 90. This indicates that as the mole ratio increase, more gold were incorporated within gold nanoparticles. This indicates that high mole ratio in solution help facilitate gold nanoparticle nucleation. Procedure on making adenosine Adenosine free base was obtained in powder form from aMR-ESCO®. Sodium phosphate dibasic Heptahydrate was obtained from Fisher Scientific®. Diluted adenosine in liquid form was made to prepare for ADA activity assay. 0.05M sodium phosphate buffer was made in 50mL of distilled water. The pH of sodium phosphate buffer was adjusted to 7.4 using HCl. 0.1mM of adenosine was made in 5mL of sodium phosphate buffer. This procedure was not done by writer, thus was not described in details. For details of the calculations, see Melissa's Notebook, which includes calculations for making sodium phosphate buffer, pH adjustment of sodium phosphate buffer, calculations to weight out the adenosine, and calculations on the amount of sodium phosphate buffer to add to adenosine in order to reach 0.1mM adenosine.