The purpose of today's lab work was to use a Fluorescence Assay in order to determine the rate at which 1µM alpha-chymotrypsin digests AuNP fiber samples.
Protocol
First, we made a 1mL stock solution of our protease, alpha-chymotrypsin, by adding 1mL of phosphate buffer to one of the stock masses of alpha-chymotrypsin that we weight out earlier in the semester. The final molarity of this sample was 47.266µM.
Next, we prepared AuNP fiber samples for use in our experiment.
Dr Hartings had synthesized the AuNP fiber samples on October 2nd.
We had seven AuNP samples total, one for each incubation time that we were interested in:
10 min
15 min
30 min
45 min
1 hour
1.5 hours
2 hours
We spun all of our samples down at 300 RPM for 10 minutes. Our goal was to get the fibers to collect in the bottom of the tube without crushing the fibers together into an unreactive mass.
We pipetted off as much supernatant as we could.
After prepping the AuNP fiber samples, we calculated how much alpha-chymotrypsin stock and phosphate buffer we would need to add to each sample and blank. (Our goal was to make one AuNP fiber sample and one alpha-chymotrypsin blank for each of the incubation times listed in step 2.2. All of these solutions would have a concentration of alpha-chymotrypsin of 1µM. All solutions would be brought up to a total volume of 1mL using phosphate buffer).
To determine how much of our stock of alpha-chymotrypsin we needed: Let M1=concentration of alpha-chymotrypsin stock=47.266µM V1=volume of alpha-chymotrypsin stock we need to add to the AuNP fiber sample M2=final concentration of alpha-chymotrypsin in the AuNP fiber sample=1µM V2=final volume of alpha-chymotrypsin sample=1mL M1V1=M2V2 V1=(M2V2)/M1 V1=[(1µM)(1mL)]/(47.266µM) V1=0.212mL=21.2µL
We then subtracted the volume of alpha-chymotrypsin stock we needed to use (21.2µL) from the total volume of the solution (1mL, or 1000µL) in order to determine the volume of phosphate buffer to add: 1000µL-21.2µL=978.8µL of phosphate buffer
We added the calculated volumes to the samples and blanks. We vortexed all of the AuNP fiber samples (except for the 10min sample) and put all of the samples and blanks into the 37 degree Celsius water bath, except for the 10 minute sample and blank, for their respective incubation times.
As each sample and blank finished incubating, we:
Spun down the AuNP fiber sample (but not the blank) at 12,000 RPM for 1 minute
Combined, in a 600µL Eppindorf tube, the following:
20uL of the blank or sample
140uL of Assay Buffer
40uL of Assay Reagent
Measured the fluorescence with an excitation wavelength of 390nm and an emission spectrum from 400 to 600nm
We then repeated steps 4-5 for the 10 min sample and blank.
Data
Figure 1: Raw Data: Fluorescence of alpha-Chymotrypsin Blanks as a Function of the Wavelength of Incident Light (nm)
The figure above shows the raw data for the fluorescence of the alpha-chymotrypsin blanks as a function of the wavelength of incident light. The different curves on the graph represent different incubation times, as indicated by the legend.
Figure 2: Raw Data: Fluorescence of AuNP Fiber Samples as a Function of the Wavelength of Incident Light (nm)
The figure above shows the raw data for the fluorescence of the AuNP fiber samples, which incubated with alpha-chymotrypsin, as a function of the wavelength of incident light. The different curves on the graph represent different incubation times, as indicated by the legend.
Figure 3: Fluorescence Intensity of the alpha-Chymotrypsin Blanks and the AuNP Fiber Samples as a Function of the Incubation Time (min)
The above figure shows the fluorescence intensity of both the alpha-chymotrypsin blanks (in blue) and the AuNP fiber samples (in red) as a function of the amount of time that the solutions were incubated. The fluorescence intensity was determined by doing the following:
I corrected the alpha-chymotrypsin blanks for instrumental noise by subtracting the fluorescence for the last 0.5nm from all of the other fluorescence measurements for each blank
I did the same correction for instrumental noise for the AuNP fiber samples
I integrated the area under the fluorescence curve for each blank starting at 420nm. This gave the fluorescence intensity of each blank.
I integrated the area under the fluorescence curve for each sample starting at 420nm. This gave the fluorescence intensity of each sample.
I graphed both integrations
Interestingly, both the blanks and the samples seemed to reach a maximum fluorescence intensity at an incubation time between 30-45 minutes. The fluorescence intensity then decreased sharply for the blanks and then began to level off. The same pattern was observed in the AuNP fiber samples, though the drop in intensity was not as steep.
Figure 4: Fluorescence Intensity of the AuNP Fibers+Peptides as a Function of the Incubation Time (min)
The above figure shows the fluorescence intensity of the AuNP fibers+peptides as a function of the amount of time that the solutions were incubated. The fluorescence intensity was determined by subtracting the fluorescence intensity of the alpha-chymotrypsin blanks from that of the AuNP fiber samples. This effectively subtracted out any fluorescence intensity in the fiber samples that would have been from the alpha-chymotrypsin, giving only the fluorescence intensity of the intact AuNP fibers and the chymotrypsin-digested AuNP fibers (peptides).
Figure 5: Concentration of AuNP Fibers+Peptide (mg/mL) as a Function of Incubation Time in alpha-Chymotrypsin (min)
The above figure shows the concentration of the AuNP fibers+peptides as a function of the amount of time that the solutions were incubated. The concentration of the AuNP fibers+peptides was determined from the data in Figure 4: these data points were divided by the slope of the calibration curve from September 30 (figure 4), which was 475965. This converted the fluorescence intensity to the concentration of AuNP fibers+peptides.
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