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8.1 8.1 Absorption Spectroscopy (UV and visible) Overview 1. Introduction 2. Theory 3. Methods 4. Transitions and chromophores 5. Applications 6. CD 1. Introduction One of the main goals of biochemical (and related) research is to understand the essence of biological life on a molecular level. Many experimental (physical) methods provide valuable information such as chromatography or centrifugation. The most detailed data, including structure information at atomic level, stems however from spectroscopic methods, i.e. from the interaction of electromagnetic radiation with biological molecules. Three types of spectroscopy can be distinguished: absorption (respectively transmission), emission and scattering. One of the most important methods of the latter type is x-ray crystallography. A typical example of emission spectroscopy is fluorescence. Here, we discuss absorption spectroscopy with a focus on radiation from the visible and UV spectrum. The principle is simple: radiation of a given frequency interacts with the sample (usually in solution) and the transmitted radiation is studied. The following figure summarizes frequency, wavelength and energy ranges of absorption spectroscopy. Electromagnetic spectrum with wavelengths from 1 nm to 1 m x-rays UV vis. IR micro radio nucl. electron molecular electron nuclear trns. transitions vibrations spins spins Möss- absorption IR/Raman EPR NMR bauer x-ray UV/vis. 1 2 3 4 5 6 7 8 9 wavelength (nm; powers of 10) 17 16 15 14 13 12 11 10 9 8 frequency (s-1; powers of 10) 7 6 5 4 3 2 1 0 -1 energy (J/mol; powers of 10) 8.2 2. Theory Electromagnetic radiation is characterized by its frequency and its wavelength , which are connected by = c with c = 2.998*108 m/s. Visible light with a wavelength of 300 nm has a frequency of 1015 s-1. The energy of a photon is E=h where h = 6.63*10-34 Js. A photon with = 300 nm, has an energy of 6.63*10-19 J. Various energy units are used in spectroscopy: - With 1 eV = 1.602*10-19 J the above energy corresponds to 4.139 eV. - In terms of "wave numbers" this energy is (for in nm) 107/ = 3.3*104 cm-1. - Finally, one may indicate the energy of one mol of photons: 6.02*1023*6.63*10-19 = 3.99*105 J/mol. For comparison, consider the following energies at room temperature T=300ºK: - kT: 0.026 eV ~ 4.1*10-21 J ~ 160 cm-1 (per molecule) k = 1.381 10-23 J/ºK - RT: 2.5 kJ/mol R = 8.314 J/(ºK mol) When energy from electromagnetic radiation is transferred to a molecule, the latter is brought to an excited state. Thus, the energy difference E between the two states of the molecule must correspond to the incoming radiation energy: E = h . Electronic transitions in a molecule occur with a certain probability. These depend on selection rules, which depend on symmetry considerations, spin conservation etc. Due to alterations in the system, induced for example by internal vibrations, symmetries are not perfect and as a consequence "forbidden" transitions become possible (although remain weak). Absorption also depends on the populations of the different states, which in turn assume in an equilibrium situation a Boltzmann distribution, i.e. they are proportional to exp(E/kT). In an absorption spectrum, peaks indicate the energy values for which radiation interacts with the sample. The line widths of these peaks provide an energy interval that arises, besides from instrument artifacts and alike, from Heisenberg's uncertainty rule: E h/ with E the energy uncertainty and the lifetime of a state. Consider light with a certain frequency and intensity I0 that enters a sample, i.e. a solution containing the molecule under investigation at a given concentration C (in mol/m3 or mM). The probability of absorption by the molecules is proportional to the number of molecules encountered. Passing a layer of the solution of thickness dx, the intensity change dI becomes dI = - const I0 C dx const is a proportionality constant. Integration over the thickness of the sample d yields I = I0 exp(const C d); introducing the absorbance or extinction coefficient = const / ln(10) one writes: I = I0 10 C d Lambert-Beer's law. In the case of d=1 cm the exponent C d is called optical density OD. It is of advantage to choose the concentration such that OD is about 0.5. 8.3 3. Methods A typical spectrometer consists of the following elements: - a light source, e.g. a conventional lamp for visible light or a deuterium lamp for UV; - a monochromator to obtain the desired frequency (or to sweep through an interval of frequencies); - a rotating mirror (or similar) to split the beam into two parts; - two cuvettes with solvent+solute and solvent only, each in the path of one beam; - detectors for each beam; - an electronic subtraction device for the two beam intensities and a plotter (or similar). Spectrometer source monochromator mirrors sample + detectors subtraction reference plotting 4. Chromophores In the context of biological molecules, chromophores (i.e. atomic groups that efficiently absorb electromagnetic radiation) are often related to ring systems. Examples are porphyrins in hemoglobins, cytochromes or in chlorophyll. High extinction coefficients are also observed for coenzymes such as NADH or FADH2. Another example is the retinal in rhodopsins. On a more general ground one can observe the aromatic amino acids Phe, Tyr and in particular Trp as well as the bases of nucleotides. Finally, absorption measurements of the peptide bonds between amino acids in proteins are sensitive tools of the local conformation. The following table summarizes some chromophores, their wavelength of maximal absorption and the extinction coefficient at this wavelength. 8.4 Chromophor max at max comment [nm] [mol-1 cm-1] Peptide * ~190 ~7000 Peptide n * ~220 ~100 "forbidden" Phe 257 200 206 9300 188 60000 Tyr 274 1400 222 8000 193 48000 Trp 280 5600 219 47000 Adenosine 259 14900 Guanosine 276 9000 Cytidine 271 9100 Thymidine 267 9700 Uridine 261 10100 FAD 438 60000 Chlorophyll a 435 60000 from plants 675 85000 Chlorophyll: a simple estimation Porphyrin groups occur in many biological systems, examples are hemoglobin (oxygen transport) and chlorophyll (photosynthesis). The following figures show the chemical structure of chlorophyll a and its absorption spectrum. 8.5 In an approximate calculation of chlorophyll we first note that porphyrines have 11 double bounds and thus 22 -electrons. Two of these bonds can be broken without major change of the absorption spectrum. Thus, 18 -electrons form a ring of de- localized electrons. Assuming a "circular potential box" with radius r where r is an average radius of the ring (~4Å), we can write a Hamiltonian for these electrons: H = h2/(2m)·2 = h2/(2m)·1/r2·/2 With the boundary condition r = |n| we get the solutions () = 1/(2)½·exp(in) n = …-2,-1,0,1,2… with energies En = n2h2/(2mr2). The 18 electrons fill orbitals with n=0,±1,±2,±3,±4 (two electrons in each). Thus the lowest energy transition is E = h2/(2mr2) * (52-42) yielding a wavelength of 571 nm, which lies between the observed two absorption bands (see table and figure above). The introduction of a periodic potential with the nuclei as wells and of electron-electron interactions splits the spectral line by 0.9 eV, which corresponds to a splitting of about 240 nm, and thus approximates the chlorophyll spectrum quite well. 5. Applications The following are three examples where absorption spectroscopy was applied to biological systems. (A) Poly-lysine Poly-lysine adopts different conformations under different conditions. At high pH it forms regular secondary structures: an -helix at room temperature and a -sheet at high temperature. At neural pH the molecules have no defined conformation (random coil). The following figure shows the extinction coefficient of poly-lysine at pH 10.8 and 25º C (thick line), at pH 10.8 and 52 ºC (dashed line) and at pH 6 and 25 ºC (thin line). Thus, absorption spectroscopy may serve to characterize the secondary structure of a molecule. 8.6 (B) Environment-dependent absorption The following figure shows the change in extinction of Trp when transferring the bee venom melittin (27 amino acids) from water to lipid membranes. It proves the tight interaction of melittin with membranes, and thus its toxicity. (C) Extinction of adenosine as a function of ordering When different forms of adenosine are subjected to absorption spectroscopy one observes a dependence with the degree of ordering. The following figure (left) displays the spectra for AMP (adenosine-mono-phosphate), single-stranded polyA with limited ordering, and a double helix with high ordering. The increase of absorption with increasing ordering is called hyperchromism. It allows observation of thermal denaturing of DNA, e.g. with different contents of GC (see figure on the right). 8.7 6. A short introduction to CD Light is considered (in classic theory) to be a wave with an electric and a magnetic field component, E and H, respectively. In linear polarized light the E-vector oscillates in one direction perpendicular to the propagation direction (and so is the H- vector). As the figure below shows, one can describe a linear polarized wave E as the sum of two circular polarized waves EL and ER, with the field vectors rotating around the propagation axis. (Similarly, circular polarized light can be described as a superposition of two linear polarized waves with different phases.) E EL ER Interaction with molecules can be different for the two circular polarized waves. For example, chiral molecules can absorb the two waves to different extents. The result is then a wave with |EL| |ER|, i.e. an elliptic wave. In Circular Dichroism (CD) experiments one measures the difference in extinction coefficients L - R for the two circular polarized waves. One reports the ellipticity = k··c·d with sample thickness d (cm), concentration c (g/cm-3) and in cm2/g; k = ln(10)·180/(2·)=33. Note the somewhat strange units due to historical reasons. In CD, optically active molecules must fulfill two conditions. A chromophore is needed to get absorption and a chiral center is required for 0. The two centers may be identical, overlap or be located very close two each other. Chirality may be due to an asymmetric atom such as tetrahedral carbon atoms with four different substituents. Chirality may also be caused by asymmetry in the structure, e.g. the spiral-like structure of composite rings or helical structures in proteins or DNA. Important in all cases is an asymmetry of the electron distribution. CD is a sensitive and easy-to-use tool to follow conformational changes in proteins and DNA, e.g. as a consequence of temperature or pH changes. The following figure shows its use to quantitatively determine the extent of regular secondary structure in the (unknown) 3D structure of a protein.
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