User:Sara K. Mattson/Notebook/Chemistry 671/2016/09/14

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Fluorescence

Purpose

Use fluorescence to examine the rate at which myoglobin unfolds at pH5

Procedure

  1. Sample was made Myoglobin: 0.25mM Au, 12.5uM Myoglobin, 0.1mM HCl 3mL total
    1. Au 4.57mM
      1. (0.25mM)(3.0mL)=(XmL)(4.57mM)
      2. XmL= .164mL= 164uL Au
    2. Myoglobin 184uM
      1. (12.5uM)(3.0mL)=(XmL)(184uM)
      2. X=0.315mL= 315uL Myoglobin
    3. HCl 0.1mM
      1. (3.0mL)(0.01mM)=(1.0mM)(XmL)
      2. XmL=0.03mL= 30uL HCl
    4. Water
      1. Volume of water= 3000uL- 164uL-274uL-30uL= 2532uL
  2. Absorbance measurements were taken and saved every 5 minutes for 3 hours
  3. The instrument was set at 80 degrees Celsius
  4. The scan range parameters were start at 310nm and ending at 540nm
  5. The emission and exit slit were set at 10nm

Solutions for UV-VIS preparation

Stock Concentrations:

  1. Au 4.57mM
  2. Myoglobin 198mM
  3. Fructose 13.0mM
0 mM Fructose
pH μL of 1 mM HClstock μL of 1 mM NaOHstock μL of 1 M NaOHstock μL of AuCl3stock μL of Fructosestock μL of Myoglobinstock μL of Water
4 500 0 0 274 0 315 3917
5 50 0 0 274 0 315 4360
6 5 0 0 274 0 315 4405
7 0 0 0 274 0 315 4410
8 0 5 0 274 0 315 4405
9 0 50 0 274 0 315 4360
10 0 500 0 274 0 315 3910
11 0 0 5 274 0 315 4405
12 0 0 50 274 0 315 4630
0.0625 mM Fructose
pH μL of 1 mM HClstock μL of 1 mM NaOHstock μL of 1 M NaOHstock μL of AuCl3stock μL of Fructosestock μL of Myoglobinstock μL of Water
4 500 0 0 274 24 315 3910
5 50 0 0 274 24 315 4360
6 5 0 0 274 24 315 4405
7 0 0 0 274 24 315 4410
8 0 5 0 274 24 315 4405
9 0 50 0 274 24 315 4360
10 0 500 0 274 24 315 3910
11 0 0 5 274 24 315 4405
12 0 0 50 274 24 315 4360


0.125 mM Fructose
pH μL of 1 mM HClstock μL of 1 mM NaOHstock μL of 1 M NaOHstock μL of AuCl3stock μL of Fructosestock μL of Myoglobinstock μL of Water
4 500 0 0 274 48 315 3910
5 50 0 0 274 48 315 4360
6 5 0 0 274 48 315 4405
7 0 0 0 274 48 315 4410
8 0 5 0 274 48 315 4405
9 0 50 0 274 48 315 4360
10 0 500 0 274 48 315 3910
11 0 0 5 274 48 315 4405
12 0 0 50 274 48 315 4360

Results

  • Wavelength

WavelengthflouresceneCH092016AU.png

  • Integrated Intensity

IntegratedintensityflourescenceCH092016AU.png

  • First 15 Minutes

First15flourescenceCH092016AU.png

  • 35-55 Minutes

35-55flourescenceCH092016AU.png

Notes

The actual concentration of the myoglobin used in fluorescence was 184mM, but the calculations were done for 119mM.