Absorption Spectroscopy of Biopolymers Overview

Electromagnetic Wave

Units in different Spectroscopy Visible & near-UV region wavelength (nm) Microwave & radiowave region frequency (Hz) Infared region wavenumber (cm-1) Far-UV, x-ray, g-ray energy (DE=hn)

Absorption & Emission Rapid process(10-15s)

Absorption & Emission

Radiation-Induced Transition Absorption Stimulated emission Spontaneous emission

Stimulated emission Stimulated emission is the process by which an electron, perturbed by a photon having the correct energy, may drop to a lower energy level resulting in the creation of another photon. The perturbing photon is seemingly unchanged in the process (cf. absorption), and the second photon is created with the same phase, frequency, polarization, and direction of travel as the original.

Stimulated emission

Spontaneous emission Spontaneous emission is the process by which a light source such as an atom, molecule, nanocrystal or nucleus in an excited state undergoes a transition to the ground state and emits a photon. The phase of the photon in spontaneous emission is random as is the direction the photon propagates in.

UV-Visible Spectroscopy Ultraviolet-visible spectroscopy involves the absorption of ultraviolet/visible light by a molecule causing the promotion of an electron from a ground electronic state to an excited electronic state. Ultraviolet/Visible light: wavelengths (l) between 190 and 800 nm

UV-visible spectrum The two main properties of an absorbance peak are: Absorption wavelength lmax Absorption intensity Amax Housecroft and Sharpe, p. 466

Beer-Lambert Law e = A/cb A = ebc A = ec (when b is 1 cm) log(I0/I) = ebc e = A/cb A = ebc A = ec (when b is 1 cm) I0 = intensity of incident light I = intensity of transmitted light = molar absoptivity coefficient in cm2 mol-1 c = concentration in mol L-1 b = pathlength of absorbing solution in cm-1 A = absorbance = log(Io/I) ℓ 0.1 cm http://www.hellma-worldwide.de/en/default.asp

Beer-Lambert Law A Absorbance or optical density (OD) e absorptivity; M-1 cm-1 c concentration; M T transmittance

Transmittance, Absorbance, and Cell Pathlength http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/beers1.htm

Deviations from the Beer-Lambert Law Low c High c The Beer-Lambert law assumes that all molecules contribute to the absorption and that no absorbing molecule is in the shadow of another http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/beers1.htm

Absorbance Measurements

Sample Concentrations Solution too concentrated Diluted five-fold

UV-visible spectrum of 4-nitroanaline Molecular mass = 138 Solvent: Ethanol Concentration: 15.4 mg L-1 Pathlength: 1 cm Harwood and Claridge, p. 18

UV-visible spectrum of 4-nitroanaline Determine the absorption maxima (lmax) and absorption intensities (A) from the spectrum: lmax = 227 nm, A227 = 1.55 lmax = 375 nm, A375 = 1.75 2. Calculate the concentration of the compound: (1.54 x 10-2 g L-1)/(138 g/mol) = 1.12 x 10-4 mol L-1 Determine the molar absorptivity coefficients (e) from the Beer-Lambert Law: e = A/cℓ e227 = 1.55/(1.0 cm x 1.12 x 10-4 mol L-1) = 13,900 mol-1 L cm-1 e375 = 1.75/(1.0 cm x 1.12 x 10-4 mol L-1) = 15,700 mol-1 L cm-1

Molar absorptivities (e) The molar absorption coefficient, e (l), expresses the ability of a molecule to absorb light in a given solvent. In the classical theory, molecular absorption of light can be described by considering the molecule as an oscillating dipole, which allows us to introduce a quantity called the oscillator strength, which is directly related to the integral of the absorption band as follows:

Molar absorptivities (e) Molar absoptivities are very large for strongly absorbing chromophores (e >10,000) and very small if the absorption is weak (e = 10 to 100). The magnitude of e reflects both the size of the chromophore and the probability that light of a given wavelength will be absorbed when it strikes the chromophore. A general equation stating this relationship may be written as follows: = 0.87 x 1020P x a where P is the transition probability (0 to 1) a is the chromophore area in cm2 The transition probability depends on a number of factors including where the transition is an “allowed” transition or a “forbidden” transition http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/uvspec.htm#uv2

Examples of molar absorption coefficients, e

UV-visible spectroscopy definitions chromophore Any group of atoms that absorbs light whether or not a color is thereby produced. auxochrome A group which extends the conjugation of a chromophore by sharing of nonbonding electrons. bathochromic shift The shift of absorption to a longer wavelength. hypsochromic shift The shift of absorption to a shorter wavelength. hyperchromic effect An increase in absorption intensity. hypochromic effect A decrease in absorption intensity.

Absorption and Emission of Photons http://micro.magnet.fsu.edu/optics/lightandcolor/frequency.html

Absorption and Emission Absorption: A transition from a lower level to a higher level with transfer of energy from the radiation field to an absorber, atom, molecule, or solid. Emission: A transition from a higher level to a lower level with transfer of energy from the emitter to the radiation field. If no radiation is emitted, the transition from higher to lower energy levels is called nonradiative decay. http://www.chemistry.vt.edu/chem-ed/spec/spectros.html

Singlet and Triplet Excited States http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/lumin1.htm

Absorption and emission pathways McGarvey and Gaillard, Basic Photochemistry at http://classes.kumc.edu/grants/dpc/instruct/index2.htm

Selection Rules In electronic spectroscopy there are three selection rules which determine whether or not transitions are formally allowed: Spin selection rule: DS = 0 allowed transitions: singlet  singlet or triplet  triplet forbidden transitions: singlet  triplet or triplet  singlet Changes in spin multiplicity are forbidden http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/lumin1.htm

Selection rules Laporte selection rule: there must be a change in the parity (symmetry) of the complex Laporte-allowed transitions: g  u Laporte-forbidden transitions: g  g or u  u g stands for gerade – compound with a center of symmetry u stands for ungerade – compound without a center of symmetry Selection rule of Dℓ = ± 1 (ℓ is the azimuthal or orbital quantum number, where ℓ = 0 (s orbital), 1 (p orbital), 2 (d orbital), etc.) allowed transitions: s  p, p  d, d  f, etc. forbidden transitions: s  s, d  d, p  f, etc.

s and s* orbitals http://www.cem.msu.edu/~reusch/VirtualText/intro3.htm#strc8a

p and p* orbitals http://www.cem.msu.edu/~reusch/VirtualText/intro3.htm#strc8a

Electronic Transitions: p  p* http://www.cem.msu.edu/~reusch/VirtualText /Spectrpy/UV-Vis/uvspec.htm#uv2 The p  p* transition involves orbitals that have significant overlap, and the probability is near 1.0 as they are “symmetry allowed”. McGarvey and Gaillard, Basic Photochemistry at http://classes.kumc.edu/grants/dpc/instruct/index2.htm

p  p* transitions - Triple bonds Organic compounds with -C≡C- or -C≡N groups, or transition metals complexed by C≡N- or C≡O ligands, usually have “low-lying” p* orbitals http://www.cem.msu.edu/~reusch/VirtualText/intro3.htm#strc8a

Electronic Transitions: n  p* http://www.cem.msu.edu/~reusch/VirtualText /Spectrpy/UV-Vis/uvspec.htm#uv2 The n-orbitals do not overlap at all well with the p* orbital, so the probability of this excitation is small. The e of the np* transition is about 103 times smaller than e for the pp* transition as it is “symmetry forbidden”. McGarvey and Gaillard, Basic Photochemistry at http://classes.kumc.edu/grants/dpc/instruct/index2.htm

Lycopene from Tomatoes http://www.purdue.edu/UNS/html4ever/020617.Handa.lycopene.html

b-carotene b-carotene                                   b-carotene http://www.ch.ic.ac.uk/wiki/index.php/It:Beta_Carotene

Chlorophyll

Lycopene

Chlorophyll B-carotene hemoglobin

E photon = hn = DE molecule = E upper state – E lower state

Conjugated System and Absorption UV-VIS 365 nm 466 nm 500 nm

Particle in a One-Dimension Box 波函數 y 出現機率

Particle in a One-Dimension Box Wave function Energy of each level

Example Dye A

Diphenyl polyene dyes

Quantitative Analysis A plot of absorption versus wavelength is the absorption spectrum

Solutions containing the amino acids tryptophan and tyrosine can be analyzed under alkaline conditions (0.1 M KOH) from their different uv spectra. The extinction coefficients under these conditions at 240 nm and 280 nm are A 10-mg smaple of the protein glucagon is hydrolyzed to its constituent amino acids and diluter to 100 mL in 0.1 M KOH. The absorbance of this solution (1 cm path) was 0.717 at 240 nm and 0.239 at 280 nm. Estimate the content of tryptophan and tyrosine in mol (g protein)-1

Isosbestic points Isosbestic wavelength the wavelength at which two or more components have the same extinction coefficient The occurrence of two or more isosbestics in the spectra of a series of solutions of the same total concentration demonstrates the presence of two and only two components absorbing in that spectra region.

Isosbestic points

UV spectrum of DNA from E. coli UV of Protein UV spectrum of BSA UV spectrum of DNA from E. coli

UV Absorption of amino acid

Effect of Secondary structure

UV of Nucleotide

Origin of Spectroscopic Changes Change in local charge distribution Change in dielectric constant Change in bonding interaction Change in dynamic coupling between different parts of the molecule

Absorption Spectroscopy APPLICATION

Light sensitive protein Human Eye http://www2.mrc-lmb.cam.ac.uk/groups/GS/eye.html Retina Light sensitive protein Retina Outer segment

Rhodopsin is a protein in the membrane of the photoreceptor cell in the retina of the eye. It catalyses the only light sensitive step in vision. The 11-cis-retinal chromophore lies in a pocket of the protein and is isomerised to all-trans retinal when light is absorbed. The isomerisation of retinal leads to a change of the shape of rhodopsin which triggers a cascade of reactions which lead to a nerve impulse which is transmitted to the brain by the optical nerve 1BRD http://www2.mrc-lmb.cam.ac.uk/groups/GS/rmovie.html

1BRD 1BM1

Heme Protein a near-planar coordination complex obtained from iron and the dianionic form of porphyrin. Derivatives are known with substituents at various positions on the ring named a, b, c, d etc. Heme b, derived from protoporphyrin IX, is the most frequently occurring heme.

                                                   Merck Index Summary: C34H32FeN4O4 mol. wt. 616.48; It is the color furnishing portion of hemoglobin. Absorption max in phosphate buffer at ph 7 is ~550, 575 nm. Sparingly soluble in glacial acetic acid; frreely soluble in prsence of xygen. Very unstable.

UV-Vis spectroscopy of H-NOX protein His102 Tyr140 Trp9 Phe78 Asn74 blue: FeII unligated complex red : FeII-O2 complex

These data clearly demonstrate that a tyrosine in the distal heme pocketof the H-NOX heme fold is necessary and sufficient for stabilization of an FeII–O2 complex and provide strong indication that one of the keys to ligand discrimination in sGC is the lack of a hydrogen bond donor in the distal pocket.

soret band : a very strong absorption band in the blue region of the optical absorption spectrum of a heme protein.