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UV-Visible Spectrophotometry

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UV-Visible Spectrophotometry Biological Macromolecules: Introductory article UV-visible Article Contents . Introduction Spectrophotometry . Protein and Nucleic Acids Concentrations . Absorbance of Proteins Franz-Xaver Schmid, University of Bayreuth, Germany . Absorbance of Nucleic Acids . Applications in Enzyme Kinetics Biological macromolecules such as proteins and nucleic acids absorb light in the UV-visible region of the spectrum. Absorbance measurements are used for measuring concentrations, for the detection of conformational changes and of ligand binding, and for following enzyme reactions. Introduction A spectrum is obtained when the wavelength of the Spectroscopy is a technique that measures the interaction incident light is changed continuously. In diode-array of molecules with electromagnetic radiation. Light in the spectrophotometers the sample is illuminated by the full near-ultraviolet (UV) and visible (vis) range of the lamp light. After passage through the cuvette the electromagnetic spectrum has an energy of about 150– transmitted light is spectrally decomposed by a prism into 400 kJ mol 2 1. The energy of the light is used to promote the individual components and quantitated by an array of electrons from the ground state to an excited state. A diodes, often in intervals of 2 nm. In diode-array spectro- spectrum is obtained when the absorption of light is photometers the entire spectrum is recorded at the same measured as a function of its frequency or wavelength. time and not by a time-dependent scan as in conventional Molecules with electrons in delocalized aromatic systems instruments. Thus spectral changes can be followed often absorb light in the near-UV (150–400 nm) or the simultaneously in a wide range of wavelengths. visible (400–800 nm) region. The buffers used for absorbance measurements should Absorption spectroscopy is usually performed with not absorb light in the wavelength range of the experiment. molecules dissolved in a transparent solvent, such as in For work in the near-UV, buffer absorbance should be aqueous buffers. The absorbance of a solute depends small above 220 nm, and indeed most of the solvents linearly on its concentration and therefore absorption commonly used in biochemical experiments do not absorb spectroscopy is ideally suited for quantitative measure- in this spectral region. Buffers that contain carboxyl and/or ments. The wavelength of absorption and the strength of amino groups absorb light below 220 nm, and therefore absorbance of a molecule depend not only on the chemical should not be used when working in this wavelength range. nature but also on the molecular environment of its Buffers with very low absorbance in the far-UV include chromophores. Absorption spectroscopy is therefore an phosphate, cacodylate and borate. excellent technique for following ligand-binding reactions, enzyme catalysis and conformational transitions in pro- teins and nucleic acids. Spectroscopic measurements are Protein and Nucleic Acids very sensitive and nondestructive, and require only small amounts of material for analysis. Concentrations Lambert–Beer law Spectrophotometers The concentrations of proteins or nucleic acids in solution Spectrophotometers are standard laboratory equipment. can be easily and accurately determined by absorbance They usually contain two light sources: a deuterium lamp, measurements. The absorbance (A) is related to the which emits light in the UV region and a tungsten–halogen intensity of the light before (I0) and after (I) passage lamp for the visible region. After passing through a through the protein solution by eqn [1], and the absorbance monochromator (or through optical filters) the light is depends linearly on concentration, according to the focused into the cuvette and the amount of light that passes Lambert–Beer law (eqn [2]). through the sample is detected by a photomultiplier or a A 52log (I/I ) [1] photodiode. In double-beam instruments a cuvette with 10 0 buffer is placed in the reference beam, and its absorbance is subtracted from the absorbance measured for the sample. ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net 1 Biological Macromolecules: UV-visible Spectrophotometry A 5 ecl [2] differently to the absorption coefficient. These differences are small, however, typically smaller than 5%. In eqn [2], c is the molar concentration, l is the pathlength in Accordingly, the absorption coefficient e of a protein can cm, and e (L mol 2 1 cm 2 1) is the molar absorption be calculated in a simple fashion. First the numbers of its coefficient. The concentration of a substance in solution Trp, Tyr and Cys disulfide bonds (nTrp, nTyr and nSS, can thus be determined directly from its absorbance using respectively) are counted, and then e is calculated by use of eqn [2]. The measurement of absorbances higher than 2 eqn [3] as the linear combination of the individual should be avoided, because only 1% of the incident light is contributions of these amino acid residues. transmitted through a solution with an absorbance of 2 2 1 2 1 (and is quantitated by the photomultiplier). e280 (L mol cm ) 5 5500 Â nTrp [3] 1 1490 Â nTyr 1 125 Â nSS Absorption coefficients of proteins The molar absorbances of Trp, Tyr and Cys disulfide bonds (5500, 1490, and 125 L mol 2 1 cm 2 1 respectively) Proteins usually show absorption maxima between 275 represent average values for the chromophores in folded and 280 nm (Figure 1), which are caused by the absorbance proteins. e280 values calculated by this simple procedure of the two aromatic amino acids tryptophan (Trp) and show an accuracy of about + 5%. A paper by Pace et al. tyrosine (Tyr) and, to a small extent, by the absorbance of (see Further Reading) compares calculated and experi- cystine (i.e. of disulfide bonds). The absorbances of Trp mental e280 values for many proteins. and Tyr depend on the microenvironment of their If more accurate e280 values are required, two solutions chromophores, and they are slightly red-shifted when with identical protein concentrations must be analysed: transferred from a polar to a nonpolar environment, such one containing just buffer and one containing buffer plus as in the interior of a globular protein (see below). As a 6 mol L 2 1 guanidinium chloride. The absorbance of the consequence, in native proteins, the residues that are unfolded protein with solvent-exposed chromophores can exposed to solvent and those that are buried will contribute then be modelled by using reference e280 values for Trp, Tyr and the disulfide chromophore determined in 6 mol L 2 1 guanidinium chloride. 20000 ) Concentrations of nucleic acids –1 15000 The concentrations of nucleic acids in solution are cm –1 routinely determined from their strong absorbance at 10000 260 nm. In fact, amounts of nucleic acid are often given as (L mol Molar absorbance ‘A260 units’. For double-stranded deoxyribonucleic acid 5000 (DNA) one A260 unit is equivalent to 50 mg DNA; for (a) single-stranded DNA it is equivalent to 33 mg DNA; and 0 for single-stranded ribonucleic acid (RNA) it is equivalent 4000 to 40 mg RNA. All these amounts would cause an A of 1 ) 260 –1 3000 when dissolved in 1 ml and measured in a 1-cm cuvette. cm 2000 Proteins absorb much more weakly than nucleic acids. –1 Contaminating proteins therefore hardly affect the con- 1000 (b) centrations of nucleic acids, as measured by A260. In a 1:1 (L mol 0 mixture of nucleic acids and proteins, the proteins Absorbance difference –1000 contribute only about 2% to the total absorbance at 240 260 280 300 320 340 260 nm. Wavelength (nm) Figure 1 (a) Ultraviolet absorption spectra of the protein ribonuclease T1. The spectrum of the native protein (in 0.1 mol L 2 1 sodium acetate, pH 5.0) is shown by the continous line, the spectrum of the unfolded protein (in 6.0 Absorbance of Proteins mol L 2 1 guanidinium chloride in the same buffer) is shown by the broken line. Ribonuclease T1 contains nine Tyr residues, which give rise to the Molecular origin of protein absorbance maximum at 278 nm, the single Trp residue leads to the shoulders between 280 and 300 nm. The small contributions of the four Phe residues near 260 The peptide groups of the protein main chain absorb light nm are barely detectable. (b) Difference spectra between the native and in the ‘far-UV’ range (180–230 nm). The aromatic side- the unfolded protein. The major difference at 287 nm arises from the exposure of the Tyr residues in the unfolded protein, the shoulders between chains of Tyr, Trp and Phe also absorb light in this region 290 and 300 nm originate from exposure of the Trp residue. Spectra of 15 and, in addition, they absorb in the 240–300 nm region mmol L 2 1 protein were measured at 258C in 1-cm cuvettes. (Table 1). This region is called the ‘near-UV’ or the 2 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net Biological Macromolecules: UV-visible Spectrophotometry Table 1 Absorbance of the aromatic amino acids the difference spectra in the descending slope of the original spectrum, that is in the 285–288 nm region for tyrosine and ea e b max 280 around 290–300 nm for tryptophan. 2 1 2 1 (L mol (L mol In folded native proteins, the aromatic residues that are Compound l 2 1 2 1 max (nm) cm ) cm ) buried in the hydrophobic core of the molecule also show a Tryptophan 280 5600 5500 small red shift in their absorbance, which is reversed when Tyrosine 2751400 1490 they become exposed to the aqueous solvent upon Phenylalanine 258 200 unfolding. This is illustrated by the spectra obtained for a the protein ribonuclease T1 (Figure 1). The maximal Absorption coefficient at lmax in water at neutral pH; data are from differences in absorbance occur in the 285–295nm region.
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