Molecular Fluorescence Analysis of Proteins

ANALYTICAL CHEMISTRY LAB 1 (CHEM 30107)

Introduction
When a molecule absorbs a photon of energy, a change in the electronic state of the molecule occurs. An electron from the singlet ground state is promoted to the singlet excited state. The measurement of this absorption of energy is the basis of molecular absorption spectroscopy. The excited molecule can return to the ground state in four different ways (see Figure). Collisional deactivation (external crossover) occurs when the excited molecule loses its excess energy by transferring it to other molecules with which it collides. Internal crossover occurs when the molecule looses its excess energy through a number of successive vibrational transitions. No photon emission results in these cases. Fluorescence and phosphorescence involve the emission of a photon of lower energy (longer wavelength) than the absorbed photon. Fluorescence occurs within about 10-8 sec after absorption and produces photon emission as the excited electron moves from the first singlet state to the ground state. Phosphorescence occurs when the excited electron enters the lowest triplet state from the excited singlet state by intersystem crossing. A photon is then emitted as the electron returns from the lowest triplet state to the singlet ground state. These triplet-singlet and singlet-triplet transitions require electron spin reversal, which has a low probability of occurrence; thus there is a longer period of time between absorption and phosphorescence than absorption and fluorescence. This time period is from 10-2 seconds to several minutes after absorption.

For any fluorescent molecule there are two characteristic spectra which can be obtained. The excitation spectrum plots the fluorescence intensity at a constant emission wavelength as a function of the excitation wavelength, whereas the emission spectrum plots the fluorescence intensity as a function of the emission wavelength during irradiation at a particular excitation wavelength.
The relationship between fluorescence and concentration can be derived from Beer’s Law (consult Skoog, Holler, and Nieman for the derivation).  For dilute solutions in which less than 2% of the excitation energy is absorbed, the fluorescence is proportional to the power of the excitation beam, Po, the molar absorptivity of the fluorescent molecules, ??, the fluorescence efficiency (quantum yield), ?, and the concentration of the sample, c.  There is also a factor, g, that depends on instrument geometry.

F = g?Po??c

Most of these factors can be kept constant so that fluorescence simply becomes proportional to the concentration of the sample.  Remember, this applies only to dilute, weakly absorbing samples.

F = kc

A schematic diagram of a fluorimeter is shown below.  White light from the source is passed through a filter or monochromator, which allows a specific wavelength to irradiate the sample.  If the sample molecules fluoresce at this excitation wavelength, the emission can be picked up at 90o to the excitation beam. The emission is analyzed by a second filter or monochromator, which admits a specific wavelength of the emitted light to the sample photomultiplier tube (PMT) detector.  The photomultiplier produces an electric current that is proportional to amount of light it receives at the specific wavelength, and a meter can read this current.  Thus, a plot of emission intensity versus emission wavelength can be created. However, the source often fluctuates in intensity, due to changes in the line voltage, and this leads to fluctuations in the fluorescence intensity and noise in the signal.  To compensate for the source fluctuations, a reference photomultiplier is used to constantly monitor the source intensity and subtract any effects it might have on the sample signal. A difference amplifier performs this subtraction.

Experimental Procedure

You will look at the fluorescence of several different proteins and amino acids in this lab. Part A is qualitative, part B is quantitative. In part A, you will look at serum albumin and two of its component amino acids, tryptophan and tyrosine, in order to discover which is responsible for the fluorescence of serum albumin. In part B, you will examine the vitamin riboflavin, which has a higher quantum yield, to investigate the sensitivity of fluorescence.

As soon as you come to lab, you should make sure that the fluorimeter has been turned on (so that the source has time to warm up) and the temperature control box has been turned on and set to room temperature. Then make up the stock solutions you will need. Since this is a trace analysis lab, you will need to be careful to use clean glassware, and use only the HPLC water provided, which the TA’s tell you to use. Fluorescence experiments are easily ruined by contaminants in water, on glassware, etc.  Lastly, obtain the quartz cuvette from the TA and sign it out.

A) Fluorescence of Serum Albumin

1)    Make 100 mL solutions of 10 µM tyrosine (MW 181.19) and tryptophan (MW 204.23) in phosphate buffer. In order to do this you should prepare 100 mL of 1mM solutions and dilute them to 10 µM (Serial Dilution)(Be ready with the calculation of this part and show to TA). Use ca. 10 ml of ethanol to dissolve tryptophan, if necessary. 10 ppm serum albumin in phosphate buffer will be provided by the TA’s.

2)     Load the applications software for the fluorescence instrument.  Ask the TA to show you how to save your spectra!

3)    Set up the following condition parameters in the setup menu: Excitation wavelength: 280 nm; Emission scan range: 290-380 nm; Slits: 5 nm.  Make sure the emission tab is clicked and then acquire an emission spectrum of the buffer solution.

You should see two peaks, one at 290 (may not be visible) and a second peak around 312 nm. The peak at 290 is the tail of the elastic scattering peak. Light from the source is elastically scattered (no energy loss), resulting in a very intense peak at the excitation wavelength of 280 nm. The second peak is due to Raman scattering of the water in the buffer. In general, Raman scattering of water is a weak interference in fluorescence. However, in this case, since we are looking at dilute solutions of proteins, it will be the most intense peak in some of the spectra that you will acquire.

4)    Using the same conditions as in (3), acquire a spectrum of tryptophan.  Locate the peaks and save the spectrum.

5)     Now acquire an excitation spectrum of tryptophan. Choose the optimal emission wavelength based on the emission spectrum.

6)  Now, using the same conditions as in (3), acquire a spectrum of tyrosine. Make sure to rinse the cuvette several times between the tryptophan and tyrosine spectra. (Use a little of your tyrosine solution to rinse the cuvette out to avoid contamination)

The fluorescence from tryptophan should have been easily resolved from the water Raman band. The tyrosine peak, however, appears as a shoulder on the left side of the Raman band.  To better locate the tyrosine band, it is necessary to use spectral subtraction. If you subtract the spectrum of the buffer acquired earlier from the tyrosine spectrum in part 6, the spectral components due to tyrosine should remain.  To subtract your spectrum, do the following:
1.    Figure out what spectra you need to subtract.  Ex: spectra A – spectra B.
2.    Open (display) the first spectra (spectra A) in your window.
3.    From the toolbar, select GRAPH then MATHS.  A spectral calculator should appear.
4.    Select your trace (spectra A) from the pull down menu.  The name of your spectra should appear in the calculation window.
5.     Open (display) the second spectra (spectra B) in your window.
6.    Select the trace (spectra B).  Now your calculator window should read “spectra A – spectra B”.
7.    Click the equal sign (as if you are using a calculator and making a subtraction).
8.    Your subtracted spectra should appear in a separate window.  Don’t forget to save this!
Show the spectral subtraction result to your TA, and save the spectrum. What is the location of the tyrosine peak?
7)    Without changing the conditions, acquire a spectrum of serum albumin. As with the other compounds, locate the peaks and save.

B) Quantifying Riboflavin (day 2)

1)    A stock solution of 10 ppm riboflavin will be available from the TA. Since riboflavin is heat and light sensitive, get several mL of the solution and immediately return it to the TA, so it can be returned to the refrigerator. Just pour a few milliliters into a clean beaker, DO NOT put pipettes in the bottle of stock solution.
Put 1.00 mL (accurately measured) into a 100 mL volumetric flask and dilute with buffer solution. This is the 100 ppb stock solution. Use this solution to prepare the standard solutions listed below. Be sure to use clean, volumetric pipets and flasks which have been rinsed with HPLC water. Before coming to lab, complete the table with the final riboflavin concentrations, and be sure to include it in your final report.

Solution Number    mL of stock solution    Dilute to (mL)    Final Concentration
1    1     (100 ppb)    50     (2 ppb)
2    2     (200 ppb)    50    (4 ppb)
3    10   (1000 ppb)    50    (20 ppb)
4    20   (2000 ppb)    50    (40 ppb)
5    25    (2500 ppb)    50   (50 ppb)
6    1 (Unknown soln)    50
NOTE: 1 mM = 1×10-3 M
Tyrosine= 1×10-3 * (181.19) / 10 = 0.01811 g
Tryptophan= 1×10-3 *(204.23) / 10= 0.02404 g
2)    For this part of the experiment, you will pick the correct excitation and emission wavelengths to make the quantitative measurements. Use the 50 ppb riboflavin solution for these measurements.

Set the emission wavelength to 540 nm, the slits to 5 nm, and scan the excitation wavelengths from 500 to 280 nm. (Use the SCAN command.) Pick the excitation wavelength for analysis and print the spectrum.
Use a fresh 50 ppb riboflavin sample to determine the emission wavelength. Set the excitation wavelength based on your results, and scan the emission wavelength from 10 nm above the excitation wavelength to 680 nm.
Pick the emission wavelength for analysis and save the spectrum.

3)    Use the “GOTO” command to set the excitation and emission wavelengths for the rest of the experiment. Measure the fluorescence for each of the standard solutions, the unknown, and a blank (phosphate buffer). You do not need to acquire spectra in this case. Just note the number that appears in the top left corner of the window in your notebook. Be sure to rinse the cuvette well between each sample.
Plot the data as the fluorescence intensity versus concentration. Be sure to correct the sample and unknown values using the blank fluorescence intensity. Indicate where the fluorescence intensity for the unknown sample falls on the curve. If the curve is nonlinear, explain why. Calculate the slope, y-intercept and r2 (square of residuals) for the calibration curve using Excel. (For a refresher on linear regression please refer to Skoog, Holler and Nieman).

4) Use the 10 ppm and 100 ppb samples of Riboflavin to obtain a UV-VIS absorption spectrum at some point during the lab. Use the buffer solution for a blank. If the UV-VIS spectrometer is not already on, it should warm up for 5 minutes before it is used.   If you have not used the spectrometer before, the TA will help you. Acquire and print out (or save) an absorption spectrum over the wavelength range 250 -600 nm.

Lab Report
•    Introduction (5pts)
•    Procedure (5 pts)
•    Deviations from procedure: list any variance from the procedure outlined here (i.e. incorrect dilutions, lab partner forgot how to pipette, etc.) Also list any problems which developed. (5pts)
•    Results
Serum Albumin fluorescence
a)    Labelled spectrum of buffer (5pts)
b)    Uncorrected emission spectra of Serum albumin, tryptophan and tyrosine (in one plot) (5pts)
c)    Corrected emission spectra of Serum albumin, tryptophan and tyrosine (in one plot) (5pts)
d)    Excitation spectra of Serum albumin, tryptophan and tyrosine (in one plot) (5pts)
Quantifying Riboflavin
a) Table of riboflavin dilutions and final concentrations; three recorded intensities and average intensity (5pts)
b) Riboflavin fluorescence emission spectra (all concentration with unknown sample in one plot) (5pts)
c) Riboflavin excitation spectra (5pts)
d) Riboflavin UV/VIS spectra (both concentration) (5pts)
e) Calibration curve for riboflavin. The slope, y-intercept and r2 values should be calculated using Excel. (5pts)
f) Report the concentration of riboflavin in your unknown in ppb. (5pts)

•    Questions:

a)    Discuss the source of fluorescence in serum albumin. Compare the tyrosine and tryptophan spectra to the serum albumin spectrum. Look up the relative number of tyrosines and tryptophans in serum albumin. Explain your conclusions. (10pts)

Note: Serum albumin contains both tryptophan and tyrosine. Which one is responsible for the fluorescence in serum albumin?  Is this what you would predict based on the relative concentrations of the 2 amino acids in serum albumin? Explain and give your reasoning. (Consider the emission wavelength of tyrosine relative to the emission wavelength of tryptophan.)

b)    Compare the riboflavin and serum albumin spectra. Does the fluorescence come from the same components in the two proteins? (5pts)

c)    Compare the UV/VIS and fluorescence excitation spectra for riboflavin using the UV/VIS spectrum for the 10 ppm sample. How do the peak shapes and intensities compare? Do you see a UV/VIS spectrum for the 100 ppb sample? (10pts)

d)    Compare molecular absorption spectrophotometry and molecular fluorescence in terms of sensitivity and types of samples that can be analyzed. Explain both in general terms and using results from this lab. (10pts)

e)    Discuss possible sources of error in a quantitative fluorescence analysis at the ppb level. (5pts)

•    Conclusion (5pts)

Fluorimeter Instructions

1.    Make sure the computer is turned on
2.    Turn on Spectrometer (lower right switch on front) and make sure the light is green.
3.    Double click on the Cary Eclipse folder
4.    Double click on “SCAN” icon
5.    Click on “SETUP” located on the left of the window
6.    Set the parameters that are instructed in your lab report
•    Excitation wavelength = 280 nm
•    Emission scan ranges = 290-380 nm
•    Slits = 5 nm
7.    Click “OK”
8.    Open the blue green panel on top of spectrometer to access the sample compartment
9.    Insert the QUARTZ (FOUR-CLEAR-SIDED) CUVETTE with sample into the central sample holder
10.    Close the sample compartment door
11.    Click on the “ZERO” button on the left
12.    Click on “START” button at the top
13.    After the spectrum is complete, click the            icon to auto-scale the plot

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