CHE 554 Spring 2012

J.P. Hoben & A.-F. Miller

Protein Fluorescence and Quenching Materials *Potassium Phosphate Buffer 10 mM potassium phosphate (K2HPO4), pH 7.4 *High Salt Buffer 2 M potassium chloride (KCl) in 10 mM potassium phosphate (K2HPO4), pH 7.4 *Guanidinium Buffer 6 M Guanidinium hydrochloride (GdHCl) in 10 mM potassium phosphate (K2HPO4), pH 7.4 *Quencher 5 M potassium iodide (KI) in 10 mM potassium phosphate (K2HPO4), pH 7.4 *BSA Stock Solution 5 mg/ml Bovine Serum Albumin in 10 mM potassium phosphate (K2HPO4), pH 7.4 *Lysozyme Stock Solution 10 mg/ml lysozyme in 10 mM potassium phosphate (K2HPO4), pH 7.4

Overview Fluorescence will be measured for increasing concentrations of a regent that is able to quench the excited state of the fluorophore. Each student will carry out the analysis for a single combination of one protein and one buffer (ie- one student, one sample type). To complete the post-lab, you will be asked to consider the data from whole class. The class data will be compiled within a helpful spreadsheet and emailed. Sample Preparation You will be assigned one of the three buffers and one of the two proteins. The stock solutions must be diluted with the assigned buffer. Use the following table to prepare 5 duplicate samples in 1.5 ml microfuge tubes. Protein Stock volume

Bufffer volume

BSA

75 uL

1.125 ml

Lysozyme

25 uL

1.175 ml

Samples prepared with guanidinium need to be incubated at 55oC for at least 20 min. Prior to fluorescence, all samples should be centrifuged for ~2 min at ~13,000 RPM.

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CHE 554 Spring 2012

J.P. Hoben & A.-F. Miller

Absorbance and fluorescence can be taken in any order and it does not matter which is data are collected first. Fluorescence To observe tryptophan emission in the range of 300-400 nm, the excitation wavelength will be set to 295 nm. Changes in fluorescence will be observed for the incremental addition of a quencher. •

Fluorescence will be performed in the instrument room (circular dichroism spectropolarimeter location). When you are ready to collect fluorescence data you will need to bring the following to the instrument room: a. 2 of the 5 samples you prepared b. USB jumpdrive/storage device (the instruments are NOT on the web) c. Lab notebook

•

While collecting fluorescence data you will have an opportunity to familiarize yourself with key parameters of the instrument. Please make an effort to record parameter settings in your notebook, ask questions, and think critically about data acquisition.

1. Transfer 2 ml of the prepared sample to the fluorescence cuvette Notebook observation: Notice anything different about the cuvette? 2. Record fluorescence 3. Add 2.5 ul 5 M KI, mix, and record fluorescence. 4. Repeat step 3 an additional 4 times. 5. Add 5 ul 5 M KI, mix, and record fluorescence. 6. Add 20 ul 5 M KI, mix, and record fluorescence. 7. Familiarize yourself with the spectral analysis software. 8. Use the software to calculate the area from 340-355 nm (The area will simultaneously be calculated for all spectra shown). 9. Export data File Export  Sheet Save As

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CHE 554 Spring 2012

J.P. Hoben & A.-F. Miller

Absorbance The absorbance will increase upon addition of the quencher (5M KI). Absorbance of the sample at the excitation (285 nm) and the emission (350 nm) wavelengths will be used to account for the loss of fluorescence intensity (inner filter effect):

Icorr=Iapp*

Eq. 1

Where Icorr is the corrected intensity, Iapp is the apparent or measured intensity, A295 is the absorbance at 295 nm, and A350 is the absorbance at 350 nm. 1. Transfer 3 ml of your assigned buffer to a quartz cuvette to use as a blank. 2. Load 3 ml of diluted protein solution into another cuvette (1 ml from each 3 of your prepared microfuge tubes). 3. Measure absorbance at 295 nm and 350 nm. Make sure to blank on the buffer when changing to a new wavelength. 4. Add 5 ul of quencher to diluted protein solution (5M KCl). To mix, use the 1000 ul pipette by pumping the liquid up and down 2-3 times. 5. Repeat step 3 (measure). 6. Repeat step 4 (add 5 ul) 7. Repeat step 3 (measure) 8. Repeat step 4 (add 5 ul) 9. Repeat step 3 (measure) 10. Repeat step 4 but using 20 ul of quencher. 11. Repeat step 3 (measure) 12. The series of measurements can be used to make a standard curve to relate quencher concentration to absorbance. The standard curve can be used to obtain the Icorr to account for the loss in fluorescence intensity. Analysis and questions 1. Use your observed fluorescence intensities at 350 nm in conjunction with your standard absorptions curves and calculate corrected fluorescence intensities for each concentration of quencher (including zero). 2. Also calculate the change in fluorescence ΔI = I0,corr - Icorr where Icorr is the corrected fluorescence intensity at a given concentration of quencher and I0,corr is the corrected fluorescence intensity in the absence of quencher.

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CHE 554 Spring 2012

J.P. Hoben & A.-F. Miller

I 0,corr vs. the concentration of quencher [Q]. Is your maximum quencher I corr concentration saturating ? (See figure 7.7.6 A in Pain).

3. Plot

€4. Plot I 0,corr vs. 1/[Q]. Do your data conform to the expectations of the modified ΔI Stern-Volmer model ? (In the simple case, the modified Stern-Volmer equation I 1 1 + predicts a straight line 0,corr = with intercept 1/fA where fA is the ΔI f A K D [Q] f A €

fraction of the tryptophan fluorescence susceptible to quenching (fraction of total fluorescence that is eventually quenched at infinite [Q]). The slope then yields KD, the Stern-Volmer quenching constant for the quencher and fluorophore in € question. (See figure 7.7.6 B in Pain (2004)). 5. From your own data, what fraction of the tryptophan fluorescence is susceptible to quenching ? 6. Measure the emission maximum (the wavelength at which emission is strongest) for each of your spectra and tabulate the values obtained in the absence of quencher and in saturating quencher. Compare your values with those reported by classmates using the same protein in different buffers. (This information will be distributed to all once all three groups have collected their data.) 6a. When there is no guanidinium HCl (a denaturant), which is more quenched: short-wavelength emission or longer-wavelength emission ? Does this make sense ? Why ? 6b. Comparing emission maxima in the absence of quencher, how does the emission maximum in the absence of denaturant compare with the maximum in the presence of denaturant. Does this make sense ? Why ? References Pain, R. H. 2005. Determining the Fluorescence Spectrum of a Protein. Current Protocols in Protein Science. 7.7.1–7.7.20.

Questions

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CHE 554 Spring 2012

J.P. Hoben & A.-F. Miller

Draw a Jablonski diagram including the processes of absorption, non-radiative decay, fluorescence. For each, write a sentence or two saying what information it provides in this lab exercise and explaining how (the logic as well as the physics). How many Trp residues are there in lysozyme ?

BSA ?

Based on your experiments, how many Trp appear to be exposed to quencher in lysozyme ? BSA ?

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