The purpose of today's lab work was to use the Ocean Optics spectrophotometer in order to measure how the absorbance of a sample of AuNP fiber sample changed over time as it was digested by alpha-chymotrypsin. From that data, we will be able to determine the rate at which alpha-chymotrypsin degrades the AuNP fibers.
- First, we spun down 4 samples of the AuNP fibers that Dr. Hartings synthesized on September 2 and pipetted off the supernatant
- We then suspended the samples in 1 mL of Tris Buffer
- The buffer was 50mM Tris and 20mM CaCl2 with pH=8
- Next, we brought up one of our stock volumes of alpha-chymotrypsin to 1mL in the Tris buffer. The final concentration of the alpha-chymotrypsin was 53.517µM.
- We calculated the volume of each component that we needed to add to our cuvette:
- We needed the final volume in the cuvette to be 3mL (including the alpha-chymotrypsin), and we wanted the final concentration of alpha-chymotrypsin in the cuvette to be 1µM. Thus, we did the following calculation to determine how much alpha-chymotrypsin (designated as a) we would need to add to the cuvette (designated as c):
[(1µmol a)/(1L c)] * [(1L)/(1000mL)] * [3mL] * [(1L a)/(53.517µmol a)]
=56µL of the alpha-chymotrypsin would be needed
- We needed to add 1mL of the AuNP fiber sample. Thus, the volume of buffer that we would need to add to the cuvette in order to bring the final volume of the cuvette up to 3mL was:
3000µL total -1000µL AuNP fiber sample - 56µL alpha-chymotrypsin
=1944µL of Tris buffer
- We then added the AuNP fibers and the Tris buffer to the cuvette and began taking measurements
- We saved the graph and set the parameters to be 2min between scans and to stop taking absorbance measurements after 2.5 hours
- We clicked save, then play
- After the second file was created, we added the alpha-chymotrypsin to the sample.
Data and Analysis
The Ocean Optics software measured the absorbance of our sample every two minutes using wavelengths of incident light from 190.96 to 891.81nm. We added the alpha-chymotrypsin 2 minutes into the experiment.
In order to correct the data for the "blank", which was at 2 minutes (just before we added the alpha-chymotrypsin), I subtracted the absorbance value of the blank at each wavelength from the corresponding absorbance value of each "sample" (i.e. each 2 minute measurement). I then corrected for noise by subtracting the average absorbance values for the last 60nm of light from each "sample".
Because the AuNP fibers absorb most strongly a wavelength of light of 530nm, I then plotted the absorbance value at an incident wavelength of 530nm as a function of time. The graph is shown below.
Figure 1: Absorbance of AuNP Fibers Degraded with alpha-Chymotrypsin as a Function of Time (min) at a Wavelength of 530nm
From the figure above, it's clear that the AuNP fibers absorb much more greatly from about 0-5min, which was just before and after the alpha-chymotrypsin was added. After that, the absorbance decreases sharply and levels off at a low absorbance value of about 0.8. Because the absorbance decreases so sharply, and because the AuNP fibers are really the only substance that is absorbing light at this wavelength, the data indicates that alpha-chymotrypsin degrades AuNP fibers very rapidly and thoroughly.