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Fluorescence methods in life sciences
Ctirad Hofr
Absorption spectroscopy
in the analysis of
biological molecules
Overview
• Determination of DNA concentration
• Determination of protein concentration
• Circular dichroism in determination of
biomolecular structure
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UV spectroscopy of DNA and proteins
• All atoms absorb in the UV region of the
spectrum, because this radiation has sufficient
energy to excite the outer electrons.
• UV spectroscopy is used to determine the
concentration of DNA and proteins and to define
the ratio of the DNA / protein in solution
• Beer-Lambert law is used to determine the
concentration of biological molecules
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Beer-Lambert law
• Light is absorbed in material
• For the absorption of monochromatic
light
• Beer-Lambert law:
The absorbance is directly proportional to the
concentration and thickness of the solution layer
lc
II ⋅⋅−
⋅= ε
100
I
I
lcA 0
10log=⋅⋅= ε
ε = molar extinction coefficient, c - concentration, l - optical path length
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Absorption spectrum of DNA
• DNA spectrum is composed of
contributions of individual bases
• Heterocyclic purines A, G
absorb the most, C, T less
• Absorbance of DNA is
measured at the maximum i.e.
260 nm
• The ratio A260/A280 is for pure
DNA 1.8
• The ratio less than 1.8 indicates
the presence of proteins or
contaminants. Abs
λ(nm)260 280
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Determination of the approximate
concentration of nucleic acids (NA)
• If the solution of NA has Abs 260=1 in 1 cm cuvette, then
concentration of:
• double-stranded dsDNA 50 µg/ml
• single-stranded ssDNA 30 µg/ml
• single-stranded RNA 40 µg/ml
• From this we can calculate the molar concentration using
the average Mr of nucleotides (320)
• Thus calculated concentration refers to 1 nucleotide!
• For the conversion of concentrations applies:
320 µg/ml ~ 1 mM
• It is the molar concentration of nucleotides in solution!
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Determination of the extinction
coefficient of oligonucleotides
• by measuring – Analytical determination
phosphor analysis
• by calculating – the most accurate is
based on the sequence considering the
influence of neighboring base
lcA ⋅⋅= ε
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Phosphorous analysis in the
determination of DNA concentration
• Precise analytical method
• Determines the concentration of phosphate groups
• Before the analysis it is necessary to cleave DNA
• It enables to determine ε also for DNA analogues
and in modification of DNA e.g. fluorescent labels,
which affect significantly ε
• I uses colorimetry – the degree of coloration of the
solution is directly proportional to the amount of
PO4 (i.e. the amount of DNA)
Murphy, J.H. and Trapane, T.L.,1996, Analytical Biochemistry, 240, 273-282.
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Calculation of extinction coefficient
of the DNA
• The extinction coefficients for the individual bases
contribute to the resulting ε of the entire DNA
according to rule of nearest neighbor
• Interaction of neighboring bases influence the level of
absorption
• Calculation of extinction coefficient of the
oligonucleotide with a length of n nucleotides
Warshaw, M.M. and Tinoco, I. (1966) Optical properties of sixteen dinucleoside phosphates. J. Mol., Biol., 20: 29-38.
Cantor, C.R. and Warshaw, M.M. (1970) Oligonucleotide interactions. Biopolymers, 9:1059-1103
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Example of calculation
• M13 sequencing primer 5'- gTA AAA CgA Cgg CCA gTg -3‚
= (368 000) – (185 400)
= 182 800 M-1cm-1
Individual bases nearest neighbor
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Calculators of the extinction coefficients
for DNA / RNA oligonucleotides
Calculation of ε
https://eu.idtdna.com/calc/analyzer
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Unit of optical density OD
OD is optical density unit
1 OD is the amount of DNA or protein which,
when is dissolved in one milliliter, has
absorbance 1 when is measured in a cuvette
with optical paths of 1 cm.
Measurement of optical density OD is frequently
used in biology as a simple method for
determining the concentration, because in the
range of 0..1 there is an approximately linear
relationship between the concentration of the
biological material and the value of absorbance.
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Example of calculating DNA concentration
ε = 182 800 M-1cm-1
Total after synthesis 8,5 OD260
Add 500 ul H2O
What is the molar concentration of DNA?
c = 93 µM
The molar concentration of the whole strands!
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Hypochromic effect in the formation of DNA
• Double-stranded DNA absorbs less DNA than
single-stranded, and that less than individual
nucleotides
• Use: monitor melting-dissociation of
complementary strands
Reduction of absorbance :
Abs (nucleotides) >Abs (ssDNA) >Abs (dsDNA)
Abs
λ(nm)
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Monitoring of DNA melting
• DNA denaturation curve - dependence of
absorbance (260nm) on temperature
• Melting temperature Tm - the temperature
at which half of the molecules is denatured
Abs(260)
Temperature (°C)
Tm
+
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Calculator of DNA thermal stability
Calculation of melting temperature and its
dependence on the concentration of
oligonucleotide and salts
http://www.basic.northwestern.edu/biotool
s/oligocalc.html
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Absorption spectrum of proteins
• Spectrum of proteins is composed of contributions
of individual amino acids
• Tryptophan, tyrosine, and cysteine absorb the most
• Absorbance of proteins is measured at 280 nm
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Spectroscopic determination of
protein concentration
Analytical determination
Bradford method - change of the absorption
maximum in the presence of protein
Calculation of extinction coefficient
based on the amino acid sequence
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Bradford method to determine
the protein concentration
• The method is based on the shift of the absorption
maximum of acidic solution Coomassie Brilliant Blue
G-250 from 465 nm to 595 nm upon binding to the
protein. The change is caused by ionic and
hydrophobic interactions with the protein, that stabilize
the negatively charged form of dyes resulting in a color
change of the solution.
• Range of application of this method is 0.1 – 1.25 mg/ml
• In this range there is no significant change in the
extinction coefficient of the protein/Coomassie complex
Bradford, MM. Analytical Biochemistry 72: 248-254. 1976.
Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990).
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Coomassie
Other names:
Coomassie Blue, Brilliant Blue, Brilliant
Blue G, Acid Blue 90, C.I. 42655,
Brilliant Blue G 250, or Kunasty Blue
Named after the African
city of Kumasi in Ghana
Originally, this substance was used in
the textile industry for dyeing wool.
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Practical example of the determination
of protein concentration
• Standards of BSA (bovine serum
albumin or immunoglobulin G) were
prepared in the range of
0.125…1.5 mg/ml
• After addition of Coomassie solution
(e.g. 980 µl + 20 µl of protein
solution) and a short incubation
(5 min) Abs at 595 nm is measured
• Based on the calibration curve, the
concentration of the sample is
defined
• The most accurate determination is
in the range of 0.2 – 0.7 mg/ml 0
0,2
0,4
0,6
0,8
1
0 0,5 1 1,5 2
protein (mg/ml)
Abs(595nm)
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Absorption densitometry of gels
• In the analysis of molecules in the gel, the information
about the quality and quantity can be obtained
simultaneously
• Amount of absorbed light depending on 2D position is
measured
• Determination of DNA and protein concentration after
staining the gel with Coomassie or silver
Light source
Detector
Optical density
(Abs)
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Calculation of protein extinction
coefficient
εTotal = number of(Tyr).εTyr + number of(Trp).εTrp + number of(Cys).εCys
• The extinction coefficients for protein
measured in water at 280 nm:
εTyr = 1490, εTrp = 5500, εCys = 125
Gill, S.C. and von Hippel, P.H. (1989). Anal. Biochem. 182:319-326(1989).
http://www.expasy.org/tools/protparam.html
Circular polarization
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Circular dichroism in structural
analysis of molecules
• Circular dichroism is caused by asymmetry of molecular
structures
• Optically active molecules of sugars and amino acids are
part of biological molecules
• In the case of arrangement of monomeric units into a
helix, it leads to a significant enhancement of optical
activity of the whole macromolecule
• Optical activity of solutions of biological molecules is used
to describe their structure and especially their structural
changes
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Principle of CD
• The material is an optically active if it rotates the plane of polarized light
• Plane-polarized light can be decomposed into a left-handed and righthanded
component of circularly polarized light.
• If left-handed component of circularly polarized light goes through the
environment at different speeds (has a different refractive index n) than the
right-handed component of circularly polarized light => it causes twisting of
plane polarized light
• Left-handed component of circularly polarized light is absorbed differently
than right-handed component of circularly polarized light => it causes a
change from plane-polarized light to elliptically polarized
• Circular dichroism is defined as the difference between the extinction
coefficient of the left- and right-handed component of circularly polarized
light CD = ∆ε =εL- εP
• ellipticity θ − in degrees is often measured
• Twisting of plane polarized light and characterization
of elliptically polarized light gives information about
structure of molecules in solution
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Decomposition of plane-polarized
light to circularly polarized component
http://www.enzim.hu/~szia/cddemo/edemo0.htm
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Change of polarization in
asymmetrical environment
• In the environment where the left-handed circularly pol. light moves
differently than the right-handed, there is a change in mutual shift of
circularly polarized components, which causes the twist of the
polarization plane.
• In the case of different absorption of left- and right- circul. pol. light
also, the elliptically polarized light was formed
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Ellipticity
Ellipticity is angle that characterizes the rate of change
of plane-polarized light to elliptically polarized.
If the light is plane polarized θ =0
If the light is circularly polarized θ =45 °
)(298.3
][
180
)(
4
303.2
tan
PL
PL
LR
LR
EE
EE
εεθ
π
εεθ
θ
−⋅=
°⋅−=
+
−
=
ER vector of electric.
intensity of right-handed
pol. component
EL vector of electric.
intensity of left-handed
pol. component
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Principle of CD
• The material is an optically active if it rotates the plane of polarized light
• Plane-polarized light can be decomposed into a left-handed and righthanded
component of circularly polarized light.
• If left-handed component of circularly polarized light goes through the
environment at different speeds (has a different refractive index n) than the
right-handed component of circularly polarized light => it causes twisting of
plane polarized light
• Left-handed component of circularly polarized light is absorbed differently
than right-handed component of circularly polarized light => it causes a
change from plane-polarized light to elliptically polarized
• Circular dichroism is defined as the difference between the extinction
coefficient of the left- and right-handed component of circularly polarized
light CD = ∆ε =εL- εP
• ellipticity θ − in degrees is often measured
• Twisting of plane polarized light and characterization
of elliptically polarized light gives information about
structure of molecules in solution
1. photomultiplier
2. space for the cuvette
3. modulator
liquid crystal which is
piezoelectrically with
alternating current
compressed and stretched
and thus modulates
alternately the circularly
polarized beam to the right
and to the left
5. monochromator
10. lamp
Scheme of instrument for measuring circular dichroism
Kindly provided by prof. Vorlíčkovou
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Utilization of CD spectroscopy
• Determination of the secondary structure of
biomolecules
• Determination of the relative amounts of individual
conformations (α helix, β sheet for proteins)
• Observation of slight structural changes
• Structural transitions of DNA (A,B,Z)
and proteins
• Determination of thermal stability
• Observation of the protein-protein
and protein – DNA interactions
• Observation of protein tertiary structure
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CD spectroscopy of DNA
http://www.ibp.cz/cs/oddeleni/cd-spektroskopie-
nukleovych-kyselin/informace-o-oddeleni
Vorlíčková, M., Kypr, J. and Sklenář, V.: NUCLEIC ACIDS: (c) SPECTROSCOPIC
METHODS, Encyklopedia of Analytical Science, vol. 6, sec. ed., Elsevier, Oxford,
(2005) 391-399
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at a distance
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