User:Dhea Patel/Notebook/Experimental Biological Chemistry Notebook/2012/11/13

Objective

• to run UV-vis on Au/HRP solutions
• to run AAS on Au/HRP solutions and Au/Lysozyme solutions
• to run ADA activity assay of purified ADA protein fractions using adenosine.

Description

• UV-vis was run on 3mL samples of the Au/HRP solutions, scanning 200-800nm. The data was collected and plotted, correcting for water.

• AAS was run on Au/HRP solutions and Au/Lysozyme solutions.
  • HCl was used as the blank for AAS
  • 8 standards were run to create the calibration chart.
  • The AAS tubing was placed in water in between each standard and each sample.

• In order to run the ADA activity assay, 50mL of 0.05M sodium phosphate buffer was prepared. The pH was adjusted to 7.4 by adding 12M HCl drop-wise until the pH-meter read the correct pH.
• 5mL of 0.1mM adenosine was prepared by dissolving 100mg of adenosine in 1mL of 0.05M sodium phosphate buffer to create a 0.00373M adenosine solution and then diluting 0.134mL of this new solution to 5ml with the 0.05M sodium phosphate buffer.
• Please refer to Melissa's entry for calculations.

Data

• The absorbance of AuNP/HRP solutions indicates that the optimum of Au/HRP reaction was around the AuHRP molar ratio 100. In this UV-vis graph, there are three peaks of interest: ~300nm (narrow), ~525nm, and ~616nm (broad). The 300nm and 616nm were unexpected, though not a complete shock, because the solutions were a transparent deep violet/blue color (see below for image of the solutions).
• Based on this data, solutions should be made in 20 molar ratio intervals between AuHRP molar ratios of 50 and 150 in order to see any aggregate formation.

AAS of Au-Lysozyme

• Concentration of Au vs. the Au/Lysozyme molar ratios.
• The AAS detected the presence of Au in the solutions ranging from 20 to 60 Au/Lysozyme molar ratios, with 60 Au/Lysozyme having the highest Au concentration.
• The AAS data also indicates that all the Au in the 70-130 Au/Lysozyme solutions were all in the protein fiber aggregates, as the Au concentrations according to the AAS was negative concentrations, indicating the lack of Au and AuNP. The negative value of Au could be due to the blank being HCl instead of deionized water.

AAS of Au-HRP

• Concentration of Au vs. the Au/HRP molar ratios.
• The AAS detected an increase of Au as the molar ratio of Au/HRP increased, indicating that no fibers were formed at any of the molar ratios prepared.

Notes

• Because the Au concentrations varied in each sample, the concentrations of the samples need to be converted to ppm (for both the AAS analysis of Au-lysozyme and Au-HRP) in order to accurately determine the loss of Au in each sample.