User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/10/09

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Purpose

  • Calculate the activity rate of Horseradish peroxidase with fixed concentration of AAP and varying concentration of H2O2, as well as fixed concentration H2O2 and varying concentration of AAP
  • Run liquid portion of Au/BSA solution made on 2012/10/03 and calculate gold concentration in analyze

Procedure

  • Activity rate of Horseradish peroxidase measured with UV-vis spectrometer on 2012/09/18 was calculated using Excel.
    • Activity rate of HRP for fixed concentration of H2O2 and varying concentration of AAP was calculated by analyzing the corresponding sets of data measured with varying concentration of AAP. On the absorbance versus time data obtained from UV-vis output, the region with the steep slope was taken, representing the kinetic rate of the enzyme at the giving condition. The changes in values were averaged, yielding one data point of average value of absorbance versus time per concentration of AAP. The data points were graphed with "change in absorbance versus change in time" on the y-axis and different concentration of AAP on the x-axis. For the graph, please refer to Melissa's Notebook to see the actual graph: HRP Activity Rate with Varying AAP Concentration. The slope, which calculated to be -6 x 10-8 uM-1s-1 is the rate of horseradish peroxidase with concentration of AAP ranging from 0μM to 2700μM.
    • Activity rate of HRP for fixed concentration of AAP with varying concentration of H2O2 was also calculated. This time, the data obtained from UV-vis sample with corresponding sets of data were graphed with Absorbance versus Time. However, none of the solution yielded a platoed trend. Therefore, all of the obtained value were included. The slope of each sets of data were taken and was averaged to create one data point at each concentration of H2O2. The resulting values, representing 1/average rate in the unit of "change in seconds over change in absorbance" were graphed versus 1/[H2O2] in the unit of μM-1. The graph 1/average rate versus 1/[H2O2] was graphed as a line. Please refer to Melissa's Notebook for the actual graph: Enzymatic Activity of HRP at Fixed [AAP] (2.5mM AAP). The slope of the graph concluded to be 16779 change in second/change in absorbance/μM-1.
  • Au/BSA solutions made on 2012/09/11 were used for testing gold concentration in solution using atomic absorption spectroscopy.
    • Au/BSA solutions include small fiber segments all over the solution. Attempt to pipet out the solution from fibers was unsuccessful. Thus, x uM pore-size nylon filter paper was obtained to filter out the fibers from solutions. After filtration, the originally purple homologous mixture turned clear mixture. All Au/BSA samples were filtered through the nylon filter paper, and the same filter paper was used throughout whole filtration process. The filtered solution was poured from a 250mL Erlenmeyer flask into 15mL falcon tubes.
    • Atomic Absorption spectrometer was calibrated with gold standard solutions with the following concentrations:
   5ppm-8ppm-10ppm-15ppm-20ppm-25ppm-30ppm-40ppm
    • After standard calibration, the filtered Au/BSA solutions were fed through. The clear solution after filtration yielded no absorption, indicating no gold present in solution. Due to the lack of gold present, no relationship between Au/BSA ratios and gold concentrations in solution can be formed. Due to this error in procedure, data was discarded.

Results

  • From observation, filtration of Au/BSA solutions with uM pore-size nylon filter paper proved to be unsuccessful. The filtration yielded clear solutions as oppose to purple solutions. This could indicate a number of things:
    • Particles made with Au/BSA protocol produced not nanoparticles, but macroparticles that cannot go through the uM pore-size filter paper.
    • The gold nanoparticles present in solution might interact with the nylon filter paper and cause aggregation to prevent gold nanoparticles from filtering through. In this case, centrifugation method could be used instead of filtration to avoid gold nanoparticle aggregation.
    • Filters should be cleaned each time Au/BSA samples were filtered through to prevent contamination of one solution to another.
  • From analyzing the horseradish peroxide activity with H2O2 and AAP, it can be concluded that the horseradish activity has low enzymatic rate when reacting H2O2 with AAP at concentration in the uM range. Two suggests can be made for improvement:
    • One possible suggestions for increasing enzymatic rate is to increase the concentration of both substrate as well as the HRP enzyme itself.
    • Another suggestion for increasing the enzymatic rate is to mix all solutions in buffer as oppose to water. Buffer might yield a more stable and reactivation-friend environment for both substrate and enzyme. Buffer might also help protein folding that will further engage reaction of substrates.


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