Fluorescence-Experiment, Fluorescence Spectroscopy to Study Unfolding of Apomyoglobin

Dr. Du Deguo, Instructor

Group# _________

DATE = _________/ 2013

1. Stock Solutions used in the experiment: Stock #1: Tryptophan (Trp) ____ M. Stock #2: Apomyoglobin (ApoMb) ____ M. Stock #3: 8 M urea solution. Stock #4: 500 mM phosphate buffer (pH 7.4). 2. Trp Fluorescence: Calculate the volume of each stock solution needed in order to prepare 1mL each solution below (Fill the table), and prepare these four sample solutions. Stock #1 (L)
Blank 1 (0uM Trp, 0M urea) Sample 1 (4uM Trp, 0M urea) Blank 2 (0uM Trp, 5M urea) Sample 2 (4uM Trp, 5M urea) 0 0

Stock #3(L)
0 0

Stock #4 (L)
40 40 40 40

H2O (L)

3. Take fluorescence spectrum of each solution sample. The excitation wavelength is set at 280 nm, and the emission spectrum is collected from 300500 nm. 4. ApoMb Fluorescence: Calculate the volumes of each stock solution needed to prepare 1mL each solution below (Fill the table), and prepare these sample solutions. Stock #2 (L)
Blank 3 (0 M ApoMb, 0M urea) Sample 3 (2 M ApoMb, 0M urea) Blank 4 (0 M ApoMb, 1M urea) Sample 4 (2 M ApoMb, 1M urea) Blank 5 (0 M ApoMb, 2M urea) Sample 5 (2 M ApoMb, 2M urea) Blank 6 (0 M ApoMb, 3M urea) Sample 6 (2 M ApoMb, 3M urea) Blank 7 (0 M ApoMb, 4M urea) Sample 7 (2 M ApoMb, 4M urea) Blank 8 (0 M ApoMb, 5M urea) Sample 8 (2 M ApoMb, 5M urea) 0 0 0 0 0 0

Stock #3(L)
0 0

Stock #4 (L)
40 40 40 40 40 40 40 40 40 40 40 40

H2O (L)

5. Take fluorescence spectrum of each solution sample. 6. Data analysis: Subtract the Blank spectrum from the Sample spectrum (e.g., Sample7 Blank7), and you will get the real" fluorescence spectrum of each biomolecule sample. 7. Describe the change of fluorescence spectra of ApoMb (e.g., wavelength shift) in presence of urea. Record the peak position of each spectrum. Describe the relation between the peak postion and urea concentration. Do Trp spectra show significant wavelength shift in presence of urea? Record the fluorescence intensity at 320nm (I320) and 380 nm (I380) of each ApoMb spectrum. Plot and describe the relation between the I320/I380 value and urea concentration. Determine the concentration of urea where the I320/I380 value shows the fastest change.