User:Matt Hartings/Notebook/AU Biomaterials Design Lab/2016/09/02

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Objective

Measure lysozyme unfolding at pH 4 and 80 °C with no gold present. This is a complimentary measurement for here and here.

Description

measurement solution

  1. 12.5 μM lysozyme, pH 4
    1. 245 μL lysozyme stock solution from yesterday (153 μM)
    2. 500 μL 1 mM HCl
    3. 2255 μL water

(second sample)

  1. 1.25 μM lysozyme, pH 4
    1. 24.5 μL lysozyme stock solution from yesterday (153 μM)
    2. 500 μL 1 mM HCl
    3. 2475 μL water

(third sample)

  1. 0.51 μM lysozyme, pH 4
    1. 10 μL lysozyme stock solution from yesterday (153 μM)
    2. 500 μL 1 mM HCl
    3. 2490 μL water


Samples were run with the following conditions

  1. Excitation = 290 nm
  2. Emissionstart = 310 nm
  3. Emissionend = 540 nm
  4. Excitation slit width = 10.0 nm
  5. Emission slit width = 10.0 nm
  6. Scan Speed = 100 nm/min
  7. Data saved as: lysozyme_pH4_80C_time.sp

Data

The first two samples were too concentrated and saturated the detector.

I meant to take data every 5 minutes by took a sample at 3 minutes by mistake. It is labeled lysozyme_ph4_80C_05. The actual 5 minute sample is labeled as lysozyme_ph4_80C_05b.

From then, I keep the 1 sample every 5 min pace. So I have samples for: 0, 3, 5, 10, 15, 20, etc.


The three samples made at time 0 (Sample 3 was used for the rest of the measurements)

20160902 mrh LysozymeSamples.png


4 scans from the first 10 minutes of measurements. (Note: I highlight these individual spectrum because these are the spectra that correspond to the changes that are observed in the emission maximum versus time plot and the emission intensity versus time plot.)

20160902 mrh Lysozyme firstscans.png


Change in max wavelength as a function of time (signals the local environment of the tryptophan residues) Note that the wavelength of emission maximum changes from 352 nm to 362 nm. Any changes to much higher wavelengths (that you WILL see in your spectra) are likely due to energy transfer to nearby energy accepting chemicals.

20160902 mrh Lysozyme pH4 emissionmax.png


Change in integrated fluorescence intensity as a function of time

20160902 mrh Lysozyme pH4 IntegratedIntensity.png

Notes

Here's the rendered crystal structure of lysozyme (pdb code: 193L) highlighting the tryptophan residues.



Lysozyme tryptophan mrh 2016 09 05.png


Special notes about fluorescence and the measurements we're taking.



AU BDL Description Fluorescence1.png

AU BDL Description Fluorescence2.png

AU BDL Description Fluorescence3.png

AU BDL Description Fluorescence4.png

AU BDL Description Fluorescence5.png

AU BDL Description Fluorescence6.png