Spectrophotometry & UV detection

From The University of Reading, Department of Food Science and Technology

BSc Module FS460

Investigating Protein Structure and Function

Introduction

Proteins contain several chromophores that absorb light in the ultra-violet and infra red regions. Many also display fluorescence. The most important chromophores are the aromatic rings of Phe, Tyr and Trp.

UV absorbance and fluorescence are useful probes of structure and structural changes. This is due to the fact that chromophores display shifted spectra upon increasing or decreasing polarity of their environment, with changes in wavelength of maximum absorbance (lambda max) and molar extinction coefficient possible.

Spectral characteristics of chromophores in proteins

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Quantitation of tryptophan and tyrosine

It is often very difficult to accurately determine the number of Trp and Tyr residues in a protein. Conventional amino acid analysis frequently underestimates the content of these two important amino acids which are frequently involved in enzymic catalysis.

A useful and simple approach is the use of 4^th derivative spectroscopy of unfolded proteins. In this approach the fourth derivative of the uv-spectrum is calculated (usually automatically by the spectrophotometer). This results in a series of peaks and troughs rather than a broad absorbance peak.

The spectra were recorded for model compounds of tryptophan and tyrosine. The solid line is the spectrum for tryptophan, the dotted line is the spectrum for tyrosine.

One of the troughs on the trace is unique to tryptophan, the trough at 292 nm. The concentration of tryptophan in a sample of protein can then be determined with reference to a standard curve determined with the tryptophan model compound. If the concentration of protein is known then the number of tryptophans per molecule can be calculated.

The tyrosine content is a little more elusive as there is no absolutely unique peak or trough. The peak at 282 nm has a small contribution due to tryptophan – this can be taken into account by determining another standard curve for the tryptophan model compound at 282 nm. A correction factor can then be used to determine tyrosine concentration from the peak at 282. This is usually done by standard addition of tyrosine model compound to a solution of the protein.

The extrapolated x-axis intercept gives the number of tyrosines / molecule protein.

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Determination of amino acids exposed to solvent

UV spectroscopy can be used to determine the number of aromatic amino acids that are exposed to the solvent. This is a very useful probe of conformational changes occurring in proteins and can be used to detect the presence of aromatic amino acids in active site clefts of enzymes.

The usual approach is to perform “difference spectroscopy” in a dual beam spectrophotometer. One cell of the spectrophotometer contains the protein dissolved in buffer and the other has the protein in buffer with a perturbant such as glycerol.

The difference in absorption is quantified and compared to the absorbance of completely denatured protein. This allows the determination of the relative amount of solvent- exposed aromatic amino acids.

Difference spectroscopy of ribonuclease. This method determined that three of the six tyrosines present in the protein are exposed to solvent

Solvent perturbation method

Calculate the content of significant chromophores (usually tryptophan and tyrosine) in the protein using a spectrum determined in water and by comparison with spectra of model tryptophan or tyrosines, or by amino acid analysis.
Record the difference spectrum of denatured protein Vs native protein and identify extinction coefficient and lambda max.
Record difference spectrum in the presence of a perturbant such as 20% ethylene glycol and determine extinction.
Calculate ratio of extinction in 20% ethylene glycol to that of denatured protein (the fraction of the total that are exposed to solvent) and multiply by the number of chromophores known to be present to give the number of solvent exposed chromophores.

Surface exposed amino acids are frequently found buried in crevices on a proteins surface – particularly if they are involved in the active site of an enzyme. It is possible to gain information about the size of such a cleft by using a range of perturbants of differing molecular sizes and hence differing accessibilities.

This method determined that three of the six tyrosines present in the protein are exposed to solvent.

A useful property of tryptophans is that they can be oxidised which destroys their absorbance. The amino acid sequence can be compared before and after oxidation to identify particular tryptophans – this can be useful to place particular sequences at the protein surface.

pH titration of surface exposed tyrosines

The lambda max of tyrosine shifts from 274 to 295 nm as pH increases:

Curve A: all of the tyrosines are on the surface
Curve B: some of the tyrosines are exposed and some become exposed at high pH when the protein becomes denatured
Curve C: internal tyrosines are in a polar environment and can be titrated

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Circular dichroism (CD)

CD is a technique based upon the interactions of proteins with circularly-polarised light.
Circularly-polarised light consists of two components and components. These components interact differentially with amino acids resulting in an ellipse of polarisation. This is characterised as the molar ellipticity of a protein:

where [ ] is in degrees cm-2 Mole-1. The molar ellipticity varies with wavelength to describe the CD spectrum.

Distinct spectra are seen for polypeptides in random coil, alpha-helix and beta-sheet conformations. The CD signal at any given wavelength is given by the sum of the contributions of different secondary structures:

The spectra can be deconvoluted by computer to give accurate relative amounts of helix, coil and sheet. A simple estimation of alpha-helical content can be obtained at one wavelength. For instance at 208 nm the values of are -4,000 for beta sheet and random coil and -33,000 for alpha-helix:

Such estimates are fairly crude and most modern CD spectrophotometers have software to calculate secondary structure content.

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Infra-red spectroscopy

This form of spectroscopy is based on the vibrational frequencies of atoms and bonds within a molecule. For proteins, seven major absorbance bands are seen, all derived from the peptide bond.

Amide bands I, II and III are most prominent and sensitive to secondary structure. Most studies use the amide band I derived from C=O stretching in the peptide bond. This depends on hydrogen bonding pattern, hence on secondary structure. For polylysine, a protein model compound:

alpha-helix frequency = 1650-1658
beta-sheet frequency = 1625-1640
random coil frequency = 1640-1648

In real proteins rather than polylysine, the absorbance maxima overlap

Second derivative spectroscopy is needed to deconvolute the spectrum

Infra-red spectroscopy is most sensitive to beta-sheet.

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Quick Guide

How does it work?

Monitors the absorbance of aromatic amino acids, tyrosine and tryptophan or if the wavelength is lowered, the absorbance of the peptide bond. Higher order structure in the proteins will influence the absorption

Detection Limitations

20 μg to 3 mg

Advantages

Quick
Sample can be recovered
Useful for estimation of protein before using a more accurate method
Well suited for identifying protein in column fractions

Disadvantages

Highly susceptible to contamination by buffers, biological materials and salts
Protein amino acid composition is extremely important, thus the choice of a standard is very difficult, especially for purified proteins
Absorbance is heavily influence by pH and ionic strength of the solution.

General Considerations

This is often used to estimate protein concentration prior to a more sensitive method so the protein can be diluted to the correct range

Procedure

Quantitative Procedure

Zero the spectrophotometer with a buffer blank
Make a standard curve using your standard of choice in the expected concentration range, using the same buffer that your unknown sample is in.
Take the absorbance values at 280 nm in a quartz cuvette
Place sample into quartz cuvette (make sure concentration is in the range of 20 μg to 3 mg
Take absorbance at 280 nm

Estimation Procedure

Zero spectrophotometer to water (or buffer)
Take the absorbance at 280 nm in a quartz cuvette
Change wavelength to 260 nm and zero with water (or buffer)
Take absorption at 260 nm in a quartz cuvette
Use the following equation to estimate the protein concentration
[Protein] (mg/mL) = 1.55*A₂₈₀ – 0.76*A₂₆₀

Discussion

Determination of protein concentration by ultraviolet absorption (260 to 280 nm) depends on the presence of aromatic amino acids in proteins. Tyrosine and tryptophan absorb at approximately 280 nm. Higher orders of protein structure also may absorb UV light or modify the molar absorptivities of tyrosine and tryptophan and thus the UV detection is highly sensitive to pH and ionic strength at which measurement is taken. Many other cellular components, and particularly nucleic acids, also absorb UV light. The ratio of A₂₈₀/A₂₆₀ is often used as a criterion of the purity of protein or nucleic acid samples during their purification. The real advantages of this method of determining protein concentration are that the sample is not destroyed and that it is very rapid. Although different proteins will have different amino acid compositions and thus different molar absorptivities, this method can be very accurate when comparing different solutions of the same protein.

To make an accurate determination of protein concentration, you will have to produce a standard curve (A₂₈₀) with known amounts of purified protein. You will also have to provide a blank that is appropriate for the sample and contains the same concentrations of buffer and salts as the sample. It is often convenient to dialyze the sample and measure the absorbance of the retentate (still in the dialysis sack) using the dialysate as the blank. Care must be taken to use quartz cuvettes, since glass absorbs UV light. A handy equation to estimate protein concentration that is often used is

[Protein] (mg/mL) = 1.55*A₂₈₀ – 0.76*A₂₆₀

However, it is also a good idea to always use a standard curve and suggested that you evaluate the agreement of the results using the above equation with results using a standard curve.

This method is the least sensitive of the methods discussed here. For increased sensitivity, the wavelength can be lowered to the range of 210 to 225 nm. This measures the amide bond in proteins. However it is much more subject to interference from many more biological components and compounds used to make buffer solutions.

If you don’t know what the protein concentration of an unknown sample is likely to be, the ultraviolet method might be a good starting point. Prepare a standard curve for the absorbance at 280 and 260 nm. After you have the data for the standard curve, rezero the spectrophotometer with water. Place your samples into a dry 1 mL quartz cuvette and read the absorbance. If the A₂₈₀ of your unknown sample is less than 2, you should probably not dilute your sample further. If the absorbance is <2, dilution will be required. When you are finished with the first measurement, the unknown can be returned to its original tube with minimal loss.

References

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Book MCB Equipment

Probing Protein Structure by Spectroscopy