heating times and temperatures of ashing and atomization, as well as suitable diluents. Urine samples were diluted with an equal volume of 0.3 mol/liter. HNO3,.
C H A P T E R
F O U R
Absorption Spectroscopy Sanjay M. Nilapwar,* Maria Nardelli,† Hans V. Westerhoff,*,†,‡,§ and Malkhey Verma* Contents 1. Introduction 2. Theory of Absorption Spectroscopy 2.1. Origins of spectra 2.2. The Beer–Lambert law 3. Hardware 3.1. Instrumentation for absorption spectroscopy 3.2. Choice of materials for sample holders and buffer/solvents 4. Applications of UV–Visible Spectrometry 4.1. Estimation of protein concentration at 280 nm (A280) 4.2. Estimation of DNA melting temperature by absorption spectroscopy 4.3. Biomolecular interaction analysis 4.4. Enzyme kinetics 4.5. Measurement of intracellular metabolites 4.6. Measurement of fluxes 5. Perspective Acknowledgments References
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Abstract Absorption spectroscopy is one of the most widely used techniques employed for determining the concentrations of absorbing species (chromophores) in solutions. It is a nondestructive technique which biologists and biochemists and now systems biologists use to quantify the cellular components and characteristic parameters of functional molecules. This quantification is most relevant in the context of systems biology. For creating a quantitative depiction of a metabolic pathway, a number of parameters and variables are important * Manchester Interdisciplinary Biocentre, The University of Manchester, Manchester, United Kingdom Doctoral Training Centre for Integrative Systems Biology, The University of Manchester, Manchester, United Kingdom { Manchester Centre for Integrative Systems Biology, The University of Manchester, Manchester, United Kingdom } Netherlands Institute for Systems Biology, VU University Amsterdam, Amsterdam, The Netherlands {
Methods in Enzymology, Volume 500 ISSN 0076-6879, DOI: 10.1016/B978-0-12-385118-5.00004-9
#
2011 Elsevier Inc. All rights reserved.
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and these need to be determined experimentally. This chapter describes the UV–visible absorption spectroscopy used to produce experimental data for bottom-up modeling approaches of systems biology which uses concentrations and kinetic parameters (Km and Vmax) of enzymes of metabolic/signaling pathways, intracellular concentrations of metabolites and fluxes. It also briefly describes the application of this technique for quantification of biomolecules and investigating biomolecular interactions.
1. Introduction Absorption spectroscopy is widely used to obtain the absorbance spectra of specific molecules in solution and as solids. In the previous century, it has evolved as the preferred method for qualitative and quantitative determination of molecules present in solution. Absorption spectroscopy is also used as collective term for describing various spectroscopic techniques, such as UV–visible, fluorescence, circular dichroism (CD), and infrared spectroscopy. This chapter briefly covers the theoretical details and experimental aspects of UV-visible absorption spectroscopy for quantitative and qualitative measurement of biomolecules.
2. Theory of Absorption Spectroscopy 2.1. Origins of spectra Light visible to the human eye can be defined as electromagnetic radiation with wavelength between 400 and 780 nm (Threlfall, 1993). The apparent color of a material is complementary to the color of the incident light absorbed (see Table 4.1). So when white light is irradiated on a sample, the light will be partially reflected giving out a white color to the sample. If the light is fully absorbed, the substance will appear as black. Selective absorption of a color such as yellow results in the remaining light lacking the color yellow being reflected and the object in this example appearing blue (see Table 4.1). Blue is then referred to as the complementary color. Table 4.1 also describes the relationship between absorbed radiation (nm) and apparent color. The UV-visible spectral region (200–780) is normally expressed in terms of nanometers (nm) or angstroms (A˚) of the corresponding wavelengths. The units are interrelated as ˚ 1 nm ¼ 10 A It is customary to use nanometers rather than the pre-SI Angstrom unit.
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Table 4.1 Apparent color and the complementary absorbed color Wavelength range (nm)a
Apparent color
Absorbed color
400–465 465–482 482–487 487–493 493–498 498–530 530–559
Violet Blue Greenish-blue Blue–green Bluish-green Green Yellowish-green
559–571 571–576 576–580 580–587
Yellow–green Greenish-yellow Yellow Yellowishorange Orange Reddish-orange Red
Yellow–green Yellow Orange Red–orange Red Red–purple Reddishpurple Purple Violet Blue Blue
587–597 597–617 617–780 a
Greenish-blue Blue–green Blue–green
Wavelength range given is approximate (Burns, 1993).
Some insight into quantum theory is useful for appreciating practical UV-visible absorption spectroscopy. First, light of a specific color is not just a collection of waves with various wavelengths, it is also a collection of particles with a precise kinetic energies (nonrest masses in relativity theory), called photons. The wavelength and energy are related by E¼h
c l
where, h is Planck’s constant and c the velocity of light in vacuo. The energies of photons in the region of 200-780 nm allow excitation of outer valence electrons and inner shell d–d transitions with associated vibrational levels (Burns, 1993). When molecules are closely but irregularly packed together, as they are in spectroscopic measurements of solutions, they influence each other’s energy levels which become broadened blurring the sharp spectral lines present in the vapor states into wide spectral bands. These effects can be seen as a difference between the spectrums of benzene as a vapor and in the liquid state (Burns, 1993). The color of a substance (Table 4.1) is related to its electronic structure. The absorption of ultraviolet or visible light by the substance will be accompanied by changes in the electronic state of the molecules in the sample, but only if such change is possible; light absorption by substances is quantal.
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s* n ® s*
s ® s*
e
p*
p ® p* e
n Nonbonding p
e e
s
Bonding
Energy
n ® p*
Antibonding
Only certain energy changes are possible, because the structure of the molecule in its ground state only allows for certain changes to take place. The energy provided should enable electrons in an orbital in the molecule to transit to an orbital of higher energy. Because photon absorption is too fast for heat to escape from the absorbing molecule, the difference in energy between the ground state and the excited state of the molecule should be (almost, see above) precisely equal to the energy of the photon. In most stable molecules, all bonding orbitals are fully populated by electrons already and transition needs to be very fast; they do not allow the electrons to change the position and must therefore between overlapping orbitals. Consequently, the bonding electrons jump mostly to the corresponding antibonding orbitals. The ground state orbitals involved are s, p, and n (nonbonding) orbitals, while antibonding orbitals are s*(sigma star) and p* (pi star). Transition of an electron from a bonding to the corresponding antibonding s orbital is referred as s–s* transition, and the transition of one electron from a p orbital to an antibonding p* orbital is referred as p–p* transition (see Fig. 4.1). Other electronic transitions also occur due to the absorption of UV–visible light and include n to s* and n to p*. Both s to s* and n to s* transitions require high energy and occur in the far ultraviolet region of 150–250 nm. These transitions are relatively weak absorbers. Most of the UV–visible spectra involve n to p* and the more intense p to p* transitions (Table 4.2); it is these orbitals that overlap most. A spectrum is the absorbance of the substance as a function of the wavelength of the light. The spectrum exhibits peaks at the wavelengths that correspond to the energy differences between n and p* and between p and p* orbitals. Every single covalent bond in a molecule will have a s and a corresponding s* orbital. Every double bond will have a p and a corresponding p* orbital. Every lone electron pair in a molecule has an n orbital. Two lone pairs typically occur on oxygen atoms, because these have six electrons and at most two covalent bonds and one lone pair occurs on nitrogen atoms, because these have five electrons and mostly three covalent bonds.
Figure 4.1 Electronic transition of p, s, and n electrons.
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Table 4.2 Orbital transitions and lmax of common organic molecules e
Transition
lmax (nm)
s to s*
135
s to s* n to s*
150 183
Acetone
n to p*
290
Benzene
p to p*
254
200
p to p*
180–200
1000
n to p* p to p*
275 200
17 5000
Molecule name
Chemical structure
Ethane
H H H
H
H O
Methanol
H
H
O
H
Ethylene
H
H H
H
N O
Nitromethane H
H
Here e is molar absorptivity. (Thermo Spectronic, 2011)
Clearly, already a small molecule will exhibit an absorption spectrum in the UV region with many peaks, which because of their broadening (see above) tends to become a number of connecting broad absorbance bands.
2.2. The Beer–Lambert law In absorption spectroscopy, the Beer–Lambert law, also known as Beer’s law or Beer–Lambert–Bouguer law, relates the absorption of light to the properties of the material through which the light passes. The Beer’s law states that the absorbance of a beam of collimated monochromatic radiation
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Table 4.3 Standard parameters used in UV–visible spectrophotometry
Parameter
Symbol Synonym
Absorbance
A
Extinction value Molar extinction coefficient Path length Transmittance
A1% 1 cm e l T
Mathematical representation
OD (optical density), log I0/I D, E E1% 10e/M 1 cm Molar absorptivity A/lM M ¼ mol/dm Cell length b or d Transmission I0/I
in a homogenous isotropic medium is proportional to the absorption path length l, and to the concentration c, or, in the gas phase, to the pressure of the absorbing species (McNaught and Wilkinson, 2006). Beer’s law can be expressed as A ¼ log10
I0 ¼ elc I
where I0 and I are intensities of incident and transmitted light, respectively. The portionality constant e is called molar absorption coefficient. For l in cm and c in mol/dm3 or M in mol/l, e will result in dm3/mol/cm or M/cm is the commonly used unit. Although the SI unit of e is m2/mol. The Beer– Lambert law holds only if the spectral bandwidth of the light is narrow compared to spectral line widths, otherwise the effective e decreases with increasing bandwidth of the radiation. Some of the standard parameters used in UV–visible spectroscopy are listed in Table 4.3.
3. Hardware 3.1. Instrumentation for absorption spectroscopy An absorption spectrophotometer is a device used to measure absorbed light intensity as a function of wavelength. In UV–visible spectrophotometers, a beam of light from a suitable UV and/or visible light source is passed through a prism or diffraction grating monochromator. The light then passes through the sample to be analyzed before reaching the detector (Fig. 4.2). UV–visible spectrophotometers have five main components: the light source, monochromator, sample holder, detector, and interpreter. The standard light source consists of a deuterium arc (190–330 nm) and a
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A
Monochromator Sample
Diffraction Grating
Detector
Light source
B
Mirror
Monochromator Diffraction Grating
Reference
Detector
Light source Mirror Sample
Detector
Figure 4.2 Typical optical layout of an absorption spectrophotometer. (A) Single beam spectrophotometer and (B) double beam spectrophotometer.
tungsten filament lamp (330–800 nm), which together generates a light beam across the 190–800 nm spectral range. The monochromator produces a compact optical path and reduces optical aberrations. Modern instruments use grating monochromators in reflection mode as the dispersing element. There are two classes of spectrophotometers: single and double beam. In single beam configuration, the components are in single beam sequence; this instrument is cheaper to build and easy to use and maintain. A single beam spectrometric instrument requires a reference sample to be measured separately from the test sample. In a double beam spectrophotometer, the light from the source is split into two separate beams after passing through the monochromator (Fig. 4.2B). One beam is used for the sample, while the other one is used for reference determination. This configuration is advantageous because sample and reference reading can be conducted simultaneously so that the measurement becomes independent from variations in the intensity and spectral composition of the light source; a single beam spectrophotometer should always be switched on some time before use to allow the lamp to reach a constant temperature. Some double beam spectrophotometers employ a beam chopper, in which by blocking one beam at a time, measurement of sample and reference beams by a single photomultiplier at essentially the same time becomes possible.
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3.2. Choice of materials for sample holders and buffer/solvents Sample cells are available in a wide variety of materials and pathlengths, with most routine measurements being made using 10 mm rectangular cell. These are conventionally being fabricated using materials such as plastic, glass, fused quartz, and synthetic silica. Quartz and silica have an advantage over the other materials in that they can be used to measure absorbance down to 190 nm, compared to 300 nm for glass and plastic cuvettes. For specialized experiments, numerous designs and sizes of sample (0.2–4.0 ml) cuvettes/cells are available. The solvent or medium used for estimation of UV–visible absorbance needs to be selected carefully as every solvent has a specific UV absorbance wavelength cutoff. The solvent cutoff can be defined as the wavelength below which the solvent itself will absorb all the light. If the solvent cutoff wavelength is close to the solute detection wavelength, a different solvent should be used. Table 4.4 provides some of the examples of commonly used solvents.
4. Applications of UV–Visible Spectrometry Spectrometry has been extensively used in wide variety of applications. UV-visible spectroscopy can provide qualitative and quantitative information of compounds, examples of which are discussed below.
4.1. Estimation of protein concentration at 280 nm (A280) UV–visible absorbance is used widely for determining protein concentrations in sample solutions and can be carried out by a simple spectrophotometer. Proteins in solution absorb light, resulting in absorbance maxima at Table 4.4 Cutoff wavelengths for various solvents
Solvent
Acetone Benzene Dimethylformamide (DMF) Ethanol Toluene Water (Thermo Spectronic, 2011)
UV absorbance cutoff (nm)
329 278 267 205 285 180
Absorption Spectroscopy
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280 and 205 nm. Absorption of light in the near-UV range is largely dependent on amino acids, tryptophan (Trp), and tyrosine (Tyr), while phenylalanine (Phe) and the disulfide bonds present in protein solutions may also have minor effect. A280 for various proteins ranges from 0 to 4 for 1 mg/ml solution of protein, although most of the proteins give 0.5–1.5 (Kirschenbaum, 1975). The protein solution can also be measured in a wide range of buffers. The advantages of absorbance assays include fast and convenient operation and no additional reagents or incubations being required. Some of the disadvantages of this assay result from the fact that it is not strictly quantitative as it is based on strong absorbance of Phe, Tyr, and Trp residues. Different proteins may therefore have varying extinction coefficients, resulting in proteins containing neither Phe nor Tyr or Trp remaining undetected by UV–visible spectroscopy. Secondary, tertiary, and quarternary structure can also affect absorbance, along with factors such as pH and ionic strength. Other disadvantages include strong interference by nucleic acids and other chromophores. The extinction coefficient of nucleic acid in the near-UV (280 nm) region is as much as 10 times higher than that of proteins, resulting in a small amount of nucleic acid greatly influencing the total absorption (Aitken and Learmonth, 2002).
4.2. Estimation of DNA melting temperature by absorption spectroscopy DNA consists of two long polymers of simple units called nucleotides, with backbones made of sugars and phosphate groups joined by ester bonds. The nucleotides within DNA are bonded together such that the sugar of one nucleotide is always attached to the phosphate group of the next nucleotide. These two polymeric chains of DNA are bonded by two hydrogen bonds between adenine and thymine and three hydrogen bonds between guanine and cytosine from opposite chains. These hydrogen bonds are responsible for providing thermodynamic stability to the double-stranded DNA in solution. When DNA in solution is exposed to extremes of heat, pH, or solutes, such as urea or amides, the double-stranded helical structure of DNA shifts into a randomly unfolded single-stranded form called denatured DNA. During denaturation, the interactions between complementary base pairs are disrupted and this results in significant changes in a number of physical properties, such as an increase in UV absorption at 260 nm, an increase in buoyant density, and a decrease in viscosity. The effect of increase in UV absorption provides an appropriate method for monitoring the denaturation of DNA. In this method, the temperature of the DNA solution is increased in stepwise manner so that the DNA gradually denatures by strand separation. A temperature control accessory strapped to a UV–visible spectrophotometer is perfectly suited for these studies, as temperature can readily be controlled and monitored and the denaturation can simultaneously be
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recorded by observing changes at 260 nm. The absorbance versus temperature (thermal denaturation) curve obtained, after fitting with nonlinear curve fitting algorithm, gives the melting temperature (Tm) of DNA. UV absorption of nucleic acids related to the formation of double-stranded and/or single-stranded conformations has been used to examine the interaction with PNA or oligonucleotide probes with DNA. The determination of the thermodynamic stability and the kinetics of duplex formation are important. Analyses include the measurement of UV-absorption changes when nucleic acid targets form duplexes with antigene probes (Kushon et al., 2001).
4.3. Biomolecular interaction analysis The ability of a biomolecule (protein or nucleic acid) to absorb UV–visible light may allow the monitoring and characterization of complexes formed with ligands with concomitant alteration in the structure and function. One example is the protein serum albumin, which is involved in maintaining the colloid osmotic pressure and pH of blood and transport of hydrophobic substances by blood. Serum albumin has a characteristic absorbance spectrum with two peaks at 225 and 275 nm. An alteration of the spectrum after interaction with drugs has given useful information on the nature of the interaction and on the possible variation of protein activity as a consequence (Chi et al., 2010; Hu et al., 2006). DNA constitutes another example. Drugs targeting DNA molecules tend to alter the DNA absorbance at 260 nm, an event that can be easily observed and monitored (Ahmadi et al., 2011; Devi and Singh, 2011).
4.4. Enzyme kinetics The kinetic parameters of enzymes can be determined by measuring absorption of chromophore substrates or products of enzymatic reactions as discussed elsewhere in this volume. Low throughput assays are often carried out using single or double beam UV–visible spectrophotometers and a 1.0-ml cuvette. Medium throughput kinetic assays can be conducted using UV–visible plate readers using 96- and 384-well plates and manual pipetting. High-throughput assays can also be performed in microtitre plates containing up to 3456 wells using modern robotic liquid handling instruments (Bonowski et al., 2010; King et al., 2009). Measurement of kinetic parameters of hexokinase (HK), an enzyme which phosphorylates glucose (Glc) in the glycolytic pathway, is achieved using absorption spectroscopy. The Vmax and Km of HK were measured through coupled enzyme assay using glucose-6-phosphate dehydrogenase (G6PDH) as a coupling enzyme (Sheel and Neet, 1975). To measure the kinetic parameters for glucose, the assay was conducted in 100 mM Tris– HCl at pH 7.0, 5 mM MgCl2, 5 mM NAD, 50 U/ml G6PDH, 2 mM ATP
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at various glucose concentrations (0–20 mM). The kinetic parameters with respect to ATP can be measured under similar reaction conditions keeping the glucose concentration fixed (20 mM) and varying the ATP concentrations (0–2.5 mM). The activity was monitored at 30 C in a BMG POLARStar microplate reader by measuring change in the absorbance of cofactor NADH at 340 nm. The spectrograms for the HK assay at various concentrations of glucose and determination of the kinetic parameters by fitting the initial rates to the Michaelis–Menten equation are shown in the Fig. 4.3A. The spectrogram and progressive curve fitting for determination of kinetic parameters are shown in Fig. 4.3B. 3 1.22 mM Glc
2.5
0.31mM Glc
Rate (mM/min)
A
NADH (OD340)
0.15 mM Glc 0.08 mM Glc
2
0.04 mM Glc 0.02 mM Glc 0.01 mM Glc
1.5
0.005 mM Glc
0.06 0.05 0.04 0.03 0.02 0.01 0
Vmax = 62 mM/min
kcat = 6.3 min-1 Km = 0.145 mM
0
0.2
0.4 0.6 0.8 Glucose (mM)
1
1.2
1 0.5 0 0
B
500
1000
1500 2000 Time (s)
2500
3000
3500
1.4 1.2
NADH (mM)
1 Vmax = 63 mM/min kcat = 6.35 min-1 Km = 0.096 mM
0.8 0.6 0.4 0.2 0 0
500
1000
1500
2000 2500 Time (s)
3000
3500
4000
Figure 4.3 Absorption spectrograms of a hexokinase assay and determination of kinetic parameters by fitting the data using (A) Michaelis–Menten equation and (B) progressive curve fitting based on that equation.
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The activities of glycolytic enzymes and enzymes in other pathways can be measured in freshly prepared cell-free extract under defined assay conditions. The standard protocol for the preparation of cell-free extract for Saccharomyces cerevisiae is described below and see also the other Chapters on enzyme assays in this volume. 4.4.1. Cell-free extract preparation Fifty milliliter of cells at an OD600 nm of 1.0 were harvested by centrifugation in a precooled centrifuge at 4000g for 5 min at 4 C. The cell pellet was washed three times with 10 ml of ice cold (4 C) freeze buffer (10 mM potassium phosphate buffer, 2 mM Na2H2–EDTA at pH 7.5) to remove the growth medium completely. The final cell pellet was resuspended in 2.5 ml of sonication buffer (100 mM potassium phosphate buffer, 2 mM MgCl2 at pH 7.5) to obtain a final OD600 nm of about 20, which will give enough intracellular proteins to measure the activity of all the glycolytic enzymes using a cell extract volume of 5-10 ml. One milliliter of this cell suspension was transferred into a precooled screw top tube containing 0.7-0.8 g of glass beads (e.g., 425–600mm, Sigma). The cells were homogenized in the Mini bead-beater (8–10 bursts of 10 s each at a shaking speed of 3450 oscillations/ min each burst followed by an incubation of 1 min on ice). Homogenization can also be performed by vortexing the cells at maximum speed for 30 s 10 times and cooling the samples between two rounds of vortexing for 1 min on ice. The samples were then centrifuged at 4 C and 13000 rpm for 10 min. The supernatant (cell-free extract) was transferred to a new 1.5-ml eppendorf tube and stored on ice. The protein concentration in this cellfree extract was then measured, and 5–10 ml of cell extract was used for each enzyme assay under uniform/defined conditions.
4.5. Measurement of intracellular metabolites Intracellular metabolites with chromophore characteristics can be measured directly by UV–visible spectroscopy in tandem with HPLC (Teusink et al., 2000). Another method to measure the intracellular metabolite concentrations is through enzymatic assay using UV-visible absorption spectroscopy (Teusink et al., 2000). 4.5.1. Quenching and extraction of intracellular metabolites Intracellular metabolism is dynamic in nature and the flux through a pathway changes quickly when cells are disconnected from their extracellular environment. This affects the intracellular metabolites concentrations at a timescale equal to the ratio of their concentration to the flux through them. For major pathways such as glycolysis in yeast, these times are on the order of a second (Teusink et al., 2000). When cells are separated from their external environment, a very rapid (
