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.

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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 nTrp [3] 1 1490 nTyr 1 125 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

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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. Eftink MR (1991) In: Suelter CH (ed.) Methods of Biochemical Analysis, vol. 35, p. 127. New York: Wiley. The difference spectrum shows several bands between bAbsorption coefficients at 280 nm; average values, as found for 280 nm and 300 nm, which originate from the nine folded proteins; data are from Pace CN and Schmid FX (1997) tyrosines and the single tryptophan residue. Generally, How to determine the molar absorption coefficient of a protein. In: the absorbance differences between the native and the Creighton TE (ed.) Protein Structure: A Practical Approach.p. unfolded forms of a protein are small, but extremely useful 253. Oxford: IRL Press. for monitoring conformational changes of a protein. ‘aromatic’ region. Disulfide bonds that form between two cysteine residues also show an absorbance band near Protein unfolding 260 nm. Many cofactors of proteins absorb light in the UV- vis region. Reduced nicotinamide–adenine dinucleotide Native proteins can be unfolded by heat or by denaturants (NADH) and reduced flavin–adenine dinucleotide such as urea or guanidinium chloride (GdmCl). Protein unfolding transitions can thus be measured by following (FADH2) show spectra in the near-UV; haem groups and copper-containing cofactors absorb in the visible region. the absorbance changes at 287–292 nm as a function of Therefore haemoglobin is red and plastocyanin is blue. temperature or denaturant concentration. The absor- When the peptide groups and the aromatic residues are bances of native and of unfolded protein molecules can part of an asymmetric structure, or when they are also depend on temperature. The refractive index decreases immobilized within an asymmetric environment (as in slightly with temperature, as well as the protein concentra- folded proteins), left-handed and right-handed circularly tion (due to the thermal expansion of the solution), and the polarized light are absorbed to different extents. This ionization of dissociable groups can change. Together, phenomenon is called circular dichroism. these effects influence protein absorbance only to a minor The absorbance properties of the aromatic amino acids extent and therefore the dependence on temperature is are shown in Table 1. In the near-UV the molar absorbance usually small in the absence of structural transitions. When of phenylalanine is much smaller than that of tyrosine and denaturants are added, the absorbances of tyrosine and tryptophan, and the spectrum of a protein (such as tryptophan at 287 nm and at 291 nm, respectively, increase ribonuclease T1, Figure 1) between 240 and 300 nm is slightly, even in the absence of structural transitions. This therefore dominated by the contributions from the Tyr and originates from the change in refractive index (i.e. the Trp side-chains. Phe residues contribute fine structure polarity) of the solvent with the concentration of GdmCl or (‘wiggles’) to the spectrum between 250 and 260 nm. The urea. Similar effects are also observed when other aromatic amino acids do not absorb above 310 nm, and denaturants are employed. therefore protein absorbance should be zero at wave- lengths greater than 310 nm. Proteins without Trp residues do not absorb above 300 nm. Absorbance of Nucleic Acids Dependence on environment Molecular origin of nucleic acid absorbance The absorption spectra of the aromatic amino acids are sensitive to changes in their environment. In general, shifts Nucleic acids show a strong absorbance in the region of in the wavelength of maximal absorption predominate: a 240–275nm. It originates from the p!p* transitions of the blue-shift of the spectra is observed when the polarity of the pyrimidine and purine ring systems of the nucleobases. The solvent increases. For example, the maximum of the bases can be protonated and therefore the spectra of DNA absorption of tyrosine is shifted by about 3 nm, from and RNA are sensitive to pH. At neutral pH the absorption 277 nm to 274 nm, when the solvent is changed from maxima range from 253 nm (for guanosine) to 271 nm (for carbon tetrachloride to water. This spectral shift, com- cytidine), and, as a consequence, polymeric DNA and bined with minor changes in the strength of absorbance RNA show a broad and strong absorbance near 260 nm. and in the fine structure of the spectrum, leads to maxima in Spectra for the individual bases and for nucleic acids are

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net 3 Biological Macromolecules: UV-visible Spectrophotometry found in the text book by Cantor and Schimmel (see determined by measuring either the decrease in substrate Further Reading). concentration or the increase in product concentration as In native DNA the bases are stacked in the hydrophobic a function of the reaction time. When the substrate (S) core of the double helix and accordingly their absorbance is and the product (P) differ in absorbance, the progress considerably decreased relative to the absorbance of single- of an enzymatic reaction can be followed directly by stranded DNA and even more so relative to short monitoring the change in absorbance as a function of time. oligonucleotides in which the aromatic bases are exposed The absorbance changes are linearly related with the to the aqueous solvent. The decrease in absorbance upon changes in concentration (via the Lambert–Beer relation, base stacking in the interior of DNA or RNA double see eqn [2]) and therefore the reaction rates (d[P]/dt or helices is called hypochromism. It provides a very sensitive 2 d[S]/dt) can be calculated directly from the absorbance and convenient probe for monitoring strand dissociation data when the absorption coefficients of the reacting and unfolding (‘melting’) of DNA double helices. species are known. NADH-linked enzyme reactions, such as those catalysed by the lactate, malate or alcohol Nucleic acid denaturation dehydrogenases provide excellent examples for absor- bance-based enzyme assays. The reduced nicotinamide The two strands in a double-helical DNA or RNA (a ring in NADH shows an absorbance maximum near 2 1 2 1 duplex) are held together by noncovalent forces, primarily 340 nm (e340 5 6220 L mol cm ), which is lost upon by hydrogen bonds between the bases and by hydrophobic oxidation to NAD 1 . Thus the activities of these dehy- and van der Waals interactions between the aromatic rings drogenases can be measured directly by following the (the so-called stacking interactions) of the bases. These decrease in A340 as a function of time. weak interactions can be broken by heating. The con- When no absorbance changes occur during the reaction comitant denaturation is usually referred to as ‘melting’ of of an enzyme with its natural substrate, often coloured the duplex structure. It can be followed conveniently by the derivatives can be synthesized with chromophoric reporter increase in absorbance near 260 nm. A plot of the groups. A commonly used reporter is 4-nitrophenolate, absorbance as a function of temperature is called a melting which absorbs near 400 nm. This group can be esterified curve, and the temperature at which the increase in with acetic acid in 4-nitrophenyl acetate (to serve as a absorbance is 50% complete is defined as the melting substrate for proteases), with phosphate (as a substrate for temperature of the double-helical nucleic acid that is phosphatases) or with sugars (to probe amylases or studied. glycosidases). The melting temperature of a double-helical DNA In favourable cases a ‘silent’ enzyme reaction with a molecule depends on the nature of the solvent and on its colourless product can be coupled with another enzyme relative content of G 1 C versus A 1 T. In double-helical reaction that uses the product of the first enzymatic DNA the negatively charged phosphate groups of the reaction for a conversion that leads to a change in backbone repel each other and thus lower the stability of absorbance. Often such silent reactions are coupled to an the duplex. This unfavourable electrostatic repulsion is NADH-consuming or NADH-producing step. As out- progressively screened when counter-ions (typically in the lined above, changes in NADH concentration are easily form of NaCl) are added in increasing concentrations. followed by the strong change in absorbance at 340 nm. It Thus, duplex stability increases strongly with the concen- is, of course, essential that in such coupled enzyme assays tration of salt in the solvent. GC base pairs form three the rate of the indicator reaction is much higher than the hydrogen bonds and are thus more stable than AT base rate of the primary reaction. This is usually achieved by pairs, which form only two hydrogen bonds. Therefore, the using the indicator enzyme in a high concentration. melting temperature of DNA duplexes increases with G 1 C content; in fact, the G 1 C content of a DNA can be determined from its melting temperature. Long DNA Further Reading duplexes contain regions with different G 1 C contents, Brown SB (ed.) (1980) An Introduction to Spectroscopy for Biochemists. which can melt independently of one another. Their London: Academic Press. melting curves are complex and encompass several melting Cantor CR and Schimmel PR (1980) Biophysical Chemistry, part II. San points; they can be identified as maxima in the first Francisco: W. H. Freeman. derivative of the melting curve. Harris DA and Bashford CL (1987) Spectrophotometry and Spectro- fluorimetry: A Practical Approach. Oxford: IRL Press. Pace CN, Vajdos F, Fee L, Grimsley G and Gray T (1995) How to measure and predict the molar absorption coefficient of a protein? Applications in Enzyme Kinetics Protein Science 4: 2411–2423. Schmid FX (1997) Optical spectroscopy to characterize protein An enzyme catalyses the conversion of one or several conformation and conformational changes. In: Creighton TE (ed.) substrates to one or several products. The rate of the Protein Structure: A Practical Approach, pp. 261–297. Oxford: IRL catalysed reaction or the activity of the enzyme can be Press.

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