Physical Methods in Biochemistry
Fall 2011
Optical Spectroscopy (I): Absorption
Peter Qin
LJS-251, UPC
Tel. (213) 821-2461
e-mail: pzq@usc.edu
Summary of previous lecture
Interaction between EM radiation and molecules
leads to:
• 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.
• Scattering: Redirection of light due to
its interaction with matter. Might or
might not occur with a transfer of
energy.
Summary of previous lecture
Description of an EM wave
wave number
angular frequency
amplitude
phase constant
E  E0  cos(k  z    t   )
phase
complex notation
E  E0  e
i ( k z t  )
Optical spectroscopy relates to electronic
excitation and molecular vibration.
Applications of Optical Spectroscopy in Biology
• Detection
DNA sequencing; Genetic analysis by fluorescence in situ
hybridization (FISH); Fluorescence microscopy for cellular
localization; Flow cytometry for cell identification & sorting ...
• Quantification
Absorption measurements for quantifying protein or nucleic
acids,..
• Monitoring interaction and conformational phenomena of
biological molecules.
Monitoring protein folding, protein/DNA binding, ...
Advantages:
• High sensitivity – Single Photon/Molecule Detection .
• Wide range of time resolutions.
• In vivo detection.
• Simple instrumentation.
Limitation:
• Spatial Resolution (being improved).
UV-Vis absorption spctroscopy
• The ultraviolet region falls between 190-380 nm
The visible region fall between 380-750 nm
• UV and visible radiation causes electronic transitions: promotion
of electrons from the ground state to a high energy state.
• only pi to pi* and n to pi*
transitions occur in the UV-vis
region are observed.
Possible electronic transitions
Observed electronic transitions in functional groups
Compound
l (nm)
Intensity
(e)
CH4
122
intense
s-s* (C-H)
CH3CH3
130
intense
s-s* (C-C)
CH2=CH2
165
16,000
s-s* (C-C)
187
950
p-p* (C=O)
273
14
n-p* (C=O)
CH3COCH3
model compound for
the peptide bond
transition with
lowest energy
UV-Vis Absorption Spectroscopy
• The wavelength (or frequency) of the absorption
is determined by DE = h n.
• The intensity of absorption: Beer-Lambert Law
Beer-Lambert Law (continued)
1. Transmittance vs. Absorbance
Transmittance: T = I/I0
Absorbance: A = log(I0/I)
and A = -log(T)
2. Determination of concentration
A = log(I0/I)
= -log(I/I0)
= (k/ln10) [C] l
= e [C] l
and
[C] = A / (e l)
A: absorbance; e : extinction coefficient, unit in L mol-1 cm-1
3. Linear addition for non-interacting species.
A = e1[C1] l + e2[C2] l + ...
Extinction coefficient e(l)
• Relates to the strength of the transition dipole.
• Wavelength dependent – characteristics of individual
molecules.
• Environmental sensitive.
UV-Vis absorption spectra of proteins
• Peptide group dominate far
UV absorption (<210 nm).
poly-lysin in aqueous solution
• Aromatic side-chains
dominate near UV absorption.
Absorption spectra of major nucleic acid bases
rATP
extinction coefficient (e)
rCTP
rGTP
rUTP
dTTP
200
220
240
260
280
300
wavelength (nm)
320
340
Environmental effects due to solvent
• Solution spectra are
broad due to molecular
collisions.
• Solvent polarity and
H-bonding ability might
affect the transition
dipole moment, and
therefore could affect
the intensity, band
shape, or wavelength of
absorption.
Absorption spectra of anisole in gas,
perfluorooctane (F), cyclohexane (C), dioxane (D)
and water (W)
Environmental effects due to chromophore
interaction: Hypochromicity
• When chromophores are close in space, they could interact electronically.
This perturbs their electronic states, and could affect the UV-Vis absorption
spectra.
• A parallel stack of dipoles are mutually repelling,
making it more difficult to create a transition dipole
(red) on the absorbing chromophore. The results in a
smaller dipole moment, which means less absorption - hypochromism.
• Stacking of DNA and RNA
gives large hypochromicity –
up to 30% less absorption is
observed in stacked DNA.
UV-Vis Absorption Spectrometer
Schematic of a single beam UV-Vis spectrometer
UV region (190 – 380 nm):
deuterium discharge lamp
visible and NIR (380 – 800 nm):
tungsten-halogen lamp
photodiode, phototube, or
photomultiplier tube (PMT)
Double beam Absorption Spectrometers
Diffraction grating
• A surface covered by a regular pattern of parallel lines
• Simplest form – a photographic slide with a fine pattern of black lines
• For practical applications, most gratings have grooves or rulings on
their surface rather than dark lines.
• Such gratings can be either transparent or reflective.
• The distance between the lines is comparable to the wavelength of light
• Light rays that pass through such a surface are bent as a result of
diffraction. This diffraction angle depends on the wavelength of the light.
Incident white light

dsin  nl
d = slit width

Incident white light
Detectors
Photo Multiplier Tube (PMT)
The initial detector in a PMT is a photo-emissive cathode surface, made
from alkali metal compounds, which ejects a single electron in response to
a photon absorption. A series of other "secondary electron emissive"
surfaces (the "dynode chain") amplifies this into a burst of ~106-7 electrons.
Array-Detector Spectrophotometer
These spectrometers use
photodiode arrays (PDAs) or
charge-coupled devices
(CCDs) as the detector. All
wavelengths pass through
the sample. The light is
dispersed by a diffraction
grating after the sample and
the separated wavelengths
fall on different pixels of the
array detector.
UV-Vis absorption for proteins and nucleic acids
UV and visible radiation causes electronic transitions: Promotion of
electrons from the ground state to a high energy state.
p -> p * and n -> p *
transitions occur in the
UV-Vis region
The occupied and unoccupied orbitals of the amide chromophore showing
possible transitions from the filled orbitals to unoccupied orbitals.
Three classes of chromophores in protein
1. Peptide bond
2. Amino acid side chains
3. Prosthetic groups
1. Peptide bond (amide group)
The peptide n -> p* absorption band is
typically observed at 210-220 nm
with a very weak intensity (emax 100)
Much stronger p -> p* transition (emax
7000) is observed at far UV
region around 190 nm
Absorption is influenced by secondary structures
•  helix,  sheet, or
random coil give
sufficiently distinctive
absorption spectra
• This can be used for
diagnostic purposes for
the native protein
(pH 10.8, 52oC)
(pH 6.0, 25oC)
(pH 10.8, 25oC)
UV absorption spectrum of poly-L- lysine
in aqueous solution (K. R.and P.D.
PNAS 47:1775 (1961))
Amino Acid Side Chains
• A number of amino acid side chains have electronic transitions
overlapping with in the strong amide transition (190-220 nm). Absorption
of these side chains are difficult to detect
Asparagine (Asn); Aspartic acid (Asp); Glutamine (Gln); Glutamic acid
(Glu); Arginine (Arg); and Histidine (His)
• The following aromatic amino acids have p -> p* absorption bands
between 230 - 300 nm.
e
Tryptophan
(Trp)
Tyrosine
(Tyr)
Phenylalanine Cysteine
(Phe)
(Cys)
Distinctive absorption maximum is observed
around 280 nm in the aromatic amino acids
Extinction coefficients at 280 nm
Residue
e280 L Moles-1 cm-1
Trp
5,690
Tyr
1,280
Cystine (s-s bond)
300
If a protein contained 3 Tyrosines and one Tryptophan
its extinction coefficient would be:
e =3 x 1,280 + 1 x 5,690
at 280 nm
Protein concentration estimated from UV absorbance
Monitors the absorbance of aromatic amino acids,
tyrosine and tryptophan
Advantages
•Quick
•Sample can be recovered
•Useful for estimation of protein before using a more accurate
method
•Well suited for identifying protein in column fractions
Disadvantages
• Highly susceptible to contamination by buffers, biological materials
and salts
• Some extinction coefficients are influenced by pH and ionic
strength of the solution.
• Light scattering due to aggregates can cause overestimation of
material
Example
Annexin B12
0.45
Absorbance
0.40
A280 = 0.21
0.35
0.30
e = 12287.8
0.25
0.20
l = 0.1 cm
0.15
0.10
0.05
0.00
-0.05
240
260
280
300
320
340
360
Wavelength (nm)
More accurate colorimetric assays are available:
• Lowry
• Biuret
• Bradford assay
• Bicinchoninic Acid (BCA) assay.
Quantitation of protein in HPLC/FPLC
Prosthetic Groups (co-factors)
• A number of prosthetic groups have intense absorption
bands in the UV-Vis region.
• The absorption is generally sensitive to to the local
environment and the state of oxidation and/or coordination.
heme
heme-CO free and
w/ peptide
heme-CO bind to full
length & truncated
PrP
from: Pato et.al., “Heme as an optical probe of a conformational transition of
ovine recPrP”. Protein Sci, 2004, 13:1100-1107
UV absorption by nucleic acids
• All nucleic acids (DNA and
RNA) have strong near-UV
absorption (260 nm) due to
the aromatic nitrogenous
bases.
• The sugar phosphate
backbone has an
insignificant contribution to
absorption at l > 200 nm
Estimation of DNA purity
• The major absorption band for purified DNA occurs at
about 260 nm
• Protein material, the primary contaminant of DNA, has a
maximum absorption at 280 nm
• The ratio A260/A280 is often used as a relative measure of
the nucleic acid/protein content of a DNA sample
• The typical A260/A280 for pure isolated DNA is 1.8
• A smaller ratio indicates increased contamination with
protein.
Extinction coefficients at 260 nm for nucleic acids
(*) Individual base
(*) In an oligonucleotide, e is determined by both base
composition and base order (sequence). This is due to
stacking interaction between bases (Hypochromicity).
(*) For large DNA and RNA, an average of e = 10,000
M-1cm-1 per nucleotide is used.
Thermal denaturation of DNA and RNA
The transition from one conformation (double strand) to
another (single strand) leads to reduce stacking. This
reduces hypochromicity and goes along with an increase in
absorption. The Tm-value (blue line) corresponds to the
temperature at which half of the molecules is single and the
other half double stranded.