UV and Fluorescence Spectral Studies of Proteins

Introduction and overview

The average protein contains3.5% phenylalanine, 3.5% tyrosine and 1.1% tryptophan: these three amino acids contribute the majority of the proteins absorbance and fluorescence properties in the 250-300nm wavelength range, although if the protein contains significant numbers of disulphide bonds these too will contribute to the absorption properties in the wavelength range 250-280nm. [the peptide bond and other amino acids contribute to absorbance properties in the 210-220nm region]. In theory the protein absorption properties in the range 250-300nm could be calculated from the absorption properties of the individual amino acids if the content of the particular protein was known. Alternatively, the amino acid composition of these three amino acids could be calculated from the protein absorption spectrum if the properties of the individual amino acids are known. This presumes that each of the three amino acids contributes to the absorption properties of the protein to the same extent as they do individually in solution. Is this true?

What aspects of the local environment may change the absorption properties of a given amino acid? Polarity is the major contributing factor. During the absorption transition an electron must go from a ground state to an excited state with a consequent charge separation and hence the local polarity will effect the energy difference [ie absorption wavelength] between the ground and excited states. Nàp* and pà p*

transitions will behave somewhat differently in this respect but both are subject to wavelength shifts as the polarity of the local environment is changed [see the appendix on uv absorption spectroscopy for more details]. Another consideration is the overall pH environment of the chromophore: are the absorption properties of the individual amino acids subject to pH induced changes in properties?

The local environment [and hence contribution to the overall absorption properties of the protein] of an individual amino acid in a protein is governed by the native conformation of the protein: what would happen to the absorption properties of the individual amino acids if the native conformation was destroyed by a chemical denaturant such as Guanidine Hydrochloride or Urea?

In the various experiments in this laboratory you will explore whether or not the absorption properties of the individual amino acids: phenylalanine, tyrosine and tryptophan can be used to reconstitute a spectrum of a protein if appropriate consideration of local environment of each amino acid residue contributing to the absorption properties of the protein is applied and you will establish that under certain conditions the absorption spectrum of the protein can give a reasonably accurate estimate of the number of phenylalanine, tyrosine and tryptophan residues per mole of protein. You will also establish the local environment effects on the absorption properties of these three amino acids. In a subsequent laboratory you will investigate the fluorescence properties of these amino acids and the effects of local environment in a protein. In this way you will learn how the spectral properties of a protein can be used to investigate the conformation of a protein and conformational changes in proteins. Depending upon the level of detail that can be obtained about the contribution of individual amino acids to the spectral properties of a protein [these can be investigated using site directed mutagenesis of individual side chains or chemical modification of individual side chains] spectral changes in the protein may be able to give quite detailed information about conformation or conformational changes.

Laboratory 1

Absorption Properties of Amino Acids

You are provided with the following solutions:

1mg/mL solutions of N-acetyl phenylalanine, N-acetyl tyrosine and N-acetyl tryptophan

0.1M Phosphate Buffers at pH 4.0, 7.0 and 10.0

0.2M Phosphate Buffer at pH 7.0

8M Guanidine Hydrochloride in 0.1M Phosphate Buffer, at 7.0

Isopropanol

And equipment:

Scanning uv-vis spectrophotometer

Quartz cuvettes
 
 

Experiment 1

Determination of the uv Spectrum of the Individual Amino Acids

Using the quartz cuvettes carefully record a blank spectrum [220-320nm] using 0.1M phosphate buffer at pH 7.0

Add 50mL of the stock phenylalanine solution and record the spectrum of the amino acid

Repeat this process for each amino acid that you are given.

Plot the molar absorption coefficient versus wavelength for each amino acid.
 
Amino Acid lmax Molar Absorptivity at lmax
     
     
     

Experiment 2

Examining the Effects of pH on the Absorption Properties of these Amino Acids

For each amino acid determine the absorption spectrum at pH 4.0, 7.0 and 10.0 using the approach in experiment 1.

For each amino acid construct a plot of the molar absorption coefficient versus wavelength showing the effects of pH on the absorbance properties of the amino acid.
 
Phenylalanine lmax Molar Absorptivity at lmax
pH 4.0    
pH 7.0    
pH 10.0    
Tyrosine lmax Molar Absorptivity at lmax
pH 4.0    
pH 7.0    
pH 10.0    
Tryptophan lmax Molar Absorptivity at lmax
pH 4.0    
pH 7.0    
pH 10.0    

Explain your observations.
 
 
 
 

Experiment 3

Examining the Effects of Polarity on the Absorption Properties of the Aromatic Amino Acids

Using pH 7.0 buffer examine the effects of varied concentrations [by %] of isopropanol on the absorption properties of tryptophan, tyrosine and phenylalanine. You should use isopropanol concentrations upto 50%

These are easily achieved by using varied volumes of isopropanol added to a total volume of 1mL. To maintain the 0.1M phosphate buffer used in the earlier experiments each cuvette should contain 0.5mL of the 0.2M phosphate buffer, some volume of isopropanol to give the desired % and H2O to bring the volume to 1mL total [prior to addition of the amino acid solution]. Use the set up sheet below to guide the set up of the individual cuvettes:

Set Up Sheet: Experiment 3
 
Cuvette # mL 0.2M Phosphate % Isopropanol mL Isopropanol mL H2O
1 0.5 0 0 0.5
2   10    
3   20    
4   30    
5   40    
6   50    

Record each spectrum as in experiment 1 and construct a plot of molar absorptivity versus wavelength showing the effects of varied Isopropanol on the absorption properties of each amino acid.

Record your conclusions from this data using the table below:

Effects of Isopropanol on the Absorbance Properties of Amino Acids
 
Amino Acid Effect on lmax Effect on Molar Absorptivity
Phenylalanine    
Tyrosine    
Tryptophan    

Experiment 4

The Effects of Guanidine Hydrochloride at pH 7.0

For each of the amino acids determine the absorption spectrum in 6M Guanidine Hydrochloride at pH 7.0

You will need to calculate how much guanidine hydrochloride solution to add per 1mL cuvette, making up the remaining volume with the 0.1M phosphate buffer at pH 7.0.

As before, record a blank spectrum for each amino acid using 6M Guanidine Hydrochloride and then add 50mL of the amino acid and record the spectrum.

Construct a plot of the effects of Guanidine Hydrochloride on the Absorption properties of the individual amino acids using the data you obtained in experiment 1 for reference.
 
Amino Acid lmax Buffer Molar Absorptivity Buffer lmax GudHCl Molar Absorptivity GudHCl
Phenylalanine        
Tyrosine        
Tryptophan        

Experiment 5

uv Spectrum of a Protein and the Effects of Guanidine Hydrochloride

Using approaches similar to those described in experiments 1-4 determine the absorption spectrum of a protein [a concentration of approximately 0.2-0.5mg/mL in the cuvette will work well for most proteins] in 0.1M Phosphate Buffer and in 6M Guanidine Hydrochloride [in 0.1M Phosphate Buffer]
 
 

Laboratory 2

Fluorescence Properties of Amino Acids and Proteins

You are provided with the following solutions:

1mg/mL solutions of N-acetyl tyrosine and N-acetyl tryptophan

0.1M Phosphate Buffers at pH 4.0, 7.0 and 10.0

0.2M Phosphate Buffer at pH 7.0

8M Guanidine Hydrochloride in 0.1M Phosphate Buffer, at 7.0
 
 

And equipment:

Scanning Fluorometer

Quartz fluorescence cuvettes [4 Optical sides]
 
 

Experiment 1

Determination of the Fluorescence Spectra of Tyrosine and Tryptophan

Introduction to Fluorescence Spectra

When an electron is excited from the ground state to the singlet excited state by the absorption of a photon, the absorbed photon can be released as an emitted photon [fluorescence] or can return to the ground state via a number of other pathways[ for a complete discussion see the appendix on fluorescence spectroscopy]. Thus for a fluorophore, two separate spectra can be collected: an absorption spectrum- usually referred to as an excitation spectrum- where the fluorescence emission is observed at a fixed wavelength and the excitation wavelength is scanned; and a fluorescence emission spectrum, where the excitation wavelength is fixed and the emission wavelength scanned. Both spectra are useful and will be explored in these laboratories.

When scanning excitation and emission spectra how do you decide what the fixed wavelength will be? With emission spectra it is usual to initially fix the exciting wavelength at the absorption maximum for the molecule and scan through longer wavelengths with the emission monochrometer to establish the emission maximum. Once the emission maximum has been established, the emission monochrometer is set at that wavelength and the excitation monochrometer scanned to define the excitation spectrum. Finally, if the excitation maximum is different from the absorption maximum, the emission spectrum would be re-scanned using the new excitation wavelength. When examining the various spectra, three parameters are important to consider: the intensity of the fluorescence, the wavelength of the maximum, and the shape of the spectrum: for a pure fluorophore for example, the shape and wavelength of maximum emission should not change if the excitation wavelength is changed.
 
 

Emision Spectra:

Using an absorption maximum for tyrosine of 275nm and for tryptophan of 283nm determine the emission spectrum of each fluorophore.

Using the quartz cuvettes carefully record a blank emission spectrum [290-400nm] using 0.1M phosphate buffer at pH 7.0

Add 50mL of the stock tyrosine solution and record the emission spectrum of the amino acid

Repeat this process for each amino acid that you are given.

Plot the fluorescence emission intensity versus wavelength for each amino acid.
 
Amino Acid lmaxemission Emission Intensity at lmax
     
     
     

Excitation Spectra:

Using the emission wavelengths determined above for each amino acid, determine the excitation spectrum of each, scanning the excitation wavelength from 240 to 5nm below the fixed emission wavelength.

Plot fluorescence intensity at lmaxemission versus excitation wavelength.
 
 
 
Amino Acid lmaxexcitation Emission Intensity at lmax
     
     
     

If you feel it necessary, repeat the emission spectra you previously determined using lmaxexcitation
 
 

Experiment 2

Examining the Effects of pH on the Fluorescence Properties of these Amino Acids

For each amino acid determine the excitation and emission spectra at pH 4.0, 7.0 and 10.0 using the approach in experiment 1.

For each amino acid construct the appropriate plots of the fluorescence versus wavelength showing the effects of pH on the fluorescence properties of the amino acid.
 
 
 
Tyrosine lmaxexcitation Fluorescence Intensity at lmax
pH 4.0    
pH 7.0    
pH 10.0    
Tryptophan lmaxexcitation Fluorescence Intensity at lmax
pH 4.0    
pH 7.0    
pH 10.0    
Tyrosine lmaxemission Fluorescence Intensity at lmax
pH 4.0    
pH 7.0    
pH 10.0    
Tryptophan lmaxemission Fluorescence Intensity at lmax
pH 4.0    
pH 7.0    
pH 10.0    

Explain your observations.
 
 
 
 

Experiment 3

Examining the Effects of Polarity on the Fluorescence Properties of Tyrosine and Tryptophan

In experiment 1 you determined the fluorescence properties of tyrosine and tryptophan in aqueous solution. In this experiment you will repeat those determinations but using the non-polar solvent hexane in place of the phosphate buffer that you used in experiment 1.
 
 

Emision Spectra:

Using an absorption maximum for tyrosine of 275nm and for tryptophan of 283nm determine the emission spectrum of each fluorophore.

Using the quartz cuvettes carefully record a blank emission spectrum [290-400nm] using isopropanol as the solvent

Add 50mL of the stock tyrosine solution and record the emission spectrum of the amino acid

Repeat this process for each amino acid that you are given.

Plot the fluorescence emission intensity versus wavelength for each amino acid.
 
Amino Acid lmaxemission Emission Intensity at lmax
     
     
     

Excitation Spectra:

Using the emission wavelengths determined above for each amino acid, determine the excitation spectrum of each, scanning the excitation wavelength from 240 to 5nm below the fixed emission wavelength.

Plot fluorescence intensity at lmaxemission versus excitation wavelength.
 
 
 
Amino Acid lmaxexcitation Emission Intensity at lmax
     
     
     

If you feel it necessary, repeat the emission spectra you previously determined using lmaxexcitation

Carefully compare the results you obtained in this experiment with the results that you obtained in experiment 1. Discuss the differences that you find.
 
 

Experiment 4

The Effects of Guanidine Hydrochloride at pH 7.0

For tyrosine and tryptophan determine the excitation and emission spectra in 6M Guanidine Hydrochloride at pH 7.0

You will need to calculate how much guanidine hydrochloride solution to add per 3mL cuvette, making up the remaining volume with the 0.1M phosphate buffer at pH 7.0.

As before, record a blank spectrum for each amino acid using 6M Guanidine Hydrochloride and then add 50mL of the amino acid and record the spectrum.

Construct a plot of the effects of Guanidine Hydrochloride on the fluorescence properties of the individual amino acids using the data you obtained in experiment 1 for reference.

Emision Spectra:

Using an absorption maximum for tyrosine of 275nm and for tryptophan of 283nm determine the emission spectrum of each fluorophore.

Using the quartz cuvettes carefully record a blank emission spectrum [290-400nm] using hexane as the solvent

Add 50mL of the stock tyrosine solution and record the emission spectrum of the amino acid

Repeat this process for each amino acid that you are given.

Plot the fluorescence emission intensity versus wavelength for each amino acid.
 
Amino Acid lmaxemission Emission Intensity at lmax
     
     
     

Excitation Spectra:

Using the emission wavelengths determined above for each amino acid, determine the excitation spectrum of each, scanning the excitation wavelength from 240 to 5nm below the fixed emission wavelength.

Plot fluorescence intensity at lmaxemission versus excitation wavelength.
 
 
 
Amino Acid lmaxexcitation Emission Intensity at lmax
     
     
     

Compare and contrast the results you obtained with 6M Guandine Hydrochloride with those obtained in phosphate buffer or hexane.

Experiment 5

Fluorescence Spectra of a Protein and the Effects of Guanidine Hydrochloride

Using approaches similar to those described in experiments 1&4 determine the emission and excitation spectra of a protein [a concentration of approximately 0.2-0.5mg/mL in the cuvette will work well for most proteins] in 0.1M Phosphate Buffer and in 6M Guanidine Hydrochloride [in 0.1M Phosphate Buffer]. For both solvents repeat the emission spectrum using a different excitation wavelength [10nm from the original lmaxexcitation

Discuss your results.