Background
BIOCHEMISTRY LAB
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CHE-554
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Experiment #1
Spectrophotometry
In day 1 we will use spectrophotometry as an analytical
technique using a known extinction coefficient to assess
the precision and accuracy of common operations in a
biological chemistry lab: pipetting.
In day 2 we will undertake an experiment wherein we will
determine the extinction coefficient of a protein and then
use it to learn the concentration in a solution.
Relevant material is provided in the text in experiment 1,
beginning page 15. However, we will use the Bradford
Professor Testa
reagent instead of Folin-Ciocalteau, we will omit studies of
riboflavin and adenine, we will instead measure A280 of
lysozyme protein when native and when denatured.
Thus this experiment will have two parts: 1-Bradford
reagent chromogenic assay and -2- A280 of lysozyme.
(Introductory material beginning on page 3 of the text may
also prove useful.)
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Photometry relates to the study of light.
An experimental tool for producing and
measuring a spectrum of light, visible or
ultraviolet, is the UV-VIS spectrophotometer.
The UV-VIS spectrophotometer produces
incident light and measures the light that passes
through the sample (is not absorbed). The
machine calculates how much light was
absorbed, and presents that to the user.
Solutions absorb at specific wavelengths (energy
levels) of light, and this is a function of the
material in the solution. Particular materials have
a characteristic absorption spectra through a
range of wavelengths. Therefore, one can obtain
information about a solution by measuring its
absorbance.
The absorption of a solution at a specific
wavelength also depends on the concentration of
sample. Therefore, one can measure the
concentration of known material via UV-VIS
spectroscopy.
In the visible range, wavelengths of light not
absorbed by the sample make up the color of the
sample that you see.
Theory of absorbance -2
Theory of absorbance -1
# photons absorbed = # photons entering dye x γNAπr2 C l
= # photons entering dye x ζ C l
d (# photons) = - # photons x ζ C dl
Each photon has a probability γ of being absorbed if it
encounters a molecule of dye (absorbing substance).
X photons incident
Upon passage through a small amount of solution, the path
length is very short: dl (a small change in position ‘l’)
(1-γ)X photons
transmitted
dP= -ζ C dl x P
dP/P = - ζ C dl
γX photons absorbed (γ = 0.333 here)
ln(P)-ln(Po) = -ζ Cl - -ζ C 0
ln(P/Po) = -ζ Cl
P/Po = e-ζCl
r
If a photon’s path passes through a solution with C x NA
molecules of dye per L , we consider that a photon affects
molecules within a cross-section of area πr2 and the length of
the path through the dye is l (letter ‘l’), then the photon is
expected to encounter C x NA x πr2 x l molecules and have a
probability γ C NA πr2 x l of being absorbed.
r is assumed to depend only on the molecule’s identity and
the wavelength of light. C is the concentration (moles L-1).
# photons absorbed = # photons entering dye x (γNAπr2) C l
ln (P/Po) = 2.303 log(P/Po)
log(P/Po) = -ζ Cl /2.303
= - εCl, ε=ζ /2.303
l
3
The number of photons changes
by a small amount: dP.
Incident light
transmitted light
Po (power at zero
thickness of absorber)
4
Pl (power at ‘l’
thickness of absorber)
l (letter l)
http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/beers1.htm
Theory of absorbance -3
log(Io/I) = A = εCl,
Eq. 1-7
C is concentration, l is path length,
ε is molar extinction coefficient.
ε (and therefore A) is a function of the wavelength of the
light.
If the dye is too concentrated, some molecules may be in
the shade of others and not have their expected probability
of absorbing a photon.
Non-linear regime, Beer-Lambert
law no longer holds for high C or
long path lengths.
The Beer-Lambert Law
A=εcl
This equation relates the concentration of the lightabsorbing compound and the path-length of
incident light to the absorbance of a solution.
A
ε is the extinction coefficient, which is a constant
that depends on the structure of the material, the
wavelength of incident light, and the solvent
C
5
Units of ε:
is the calculated concentration of the sample
l is the length of the path that the incident light
travels through the sample
Plot A/ Cl = ε
A = εCl, slope = εl
is the measured absorbance of the sample
M-1
cm-1
http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/beers1.htm
In these experiments, measure the absorbance
using a spectrophotometer and calculate the
concentration of sample in solution.
The electromagnetic
spectrum
Absorbance vs.
wavelength
λ (nm)
266.50 nm
349.00!
445.25!
ε (M-1 cm-1)
33000
11138
11051
in ethanol
band II band I
7
http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/spectrum.htm
8
http://omlc.ogi.edu/spectra/PhotochemCAD/html/riboflavin.html
Significance of
wavelength
λ = c/ν,
ν is the frequency,
λ is the wavelength and
c is the speed of light.
ΔE = h ν,
= hc/λ
h is Planck’s constant, = 6.6 x 10-34J/s
Transitions between
electronic states
λmax is the wavelength with the maximal ε for a given band.
It corresponds to the energy of the transition associated
with that band.
Long wavelength photons carry less energy, shortwavelength photons carry more energy.
Longer wavelengths ≈ n-π* transitions,
mid-wavelenths ≈ π-π* transitions.
Usually 260, 180 nm, respectively.
Eg. N-containing bases of DNA: 260 nm absorbance.
Hence the danger of UV light to DNA.
9
10
http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/spectrum.htm
The visible portion of the
EM spectrum
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•
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Violet: 400 - 420 nm
Indigo: 420 - 440 nm
Blue: 440 - 490 nm
Green: 490 - 570 nm
Yellow: 570 - 585 nm
Orange: 585 - 620 nm
Red: 620 - 780 nm
http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/spectrum.htm
Making a measurement
Spectrophotometer
Sample standard curve
Sample data set
Interpolation or the use of the
equation of the line allows
determination of the unknown
concentration.
Selecting a wavelength
Spectrophotometer
A
Slit
Sample
14
C (M)
A non-zero intercept may be real, for example due to a
reaction with the buffer.
In this case the unknown falls out
of range and requires
extrapolation, which is much more
dangerous than interpolation.
In this case the unknown falls out
of range and requires
extrapolation, which is much more
dangerous than interpolation.
15
A
Sample standard curve
A
Sample standard curve
C (M)
16
C (M)
Why use absorbance ?
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Validation of techniques and
refresher on uncertainties.
Bromophenol blue
It is often a MUCH more accurate
way to know concentrations than
the weights and volumes used to
produce them.
The advantage provided depends
on the magnitude of the extinction
coefficient (why ?)
Accuracy is different from precision
(how ?)
Concentration ?
c = mass/mw•vol
Make an ‘illegal’
measurement,
break BeerLambert’s law
and evaluate
error.
Dilute to A < 1
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17
We will compare the actual
concentration of a solution
prepared by weighing, dissolving
and diluting with the concentration
predicted based on the execution
plan.
Concentration ?
c = A/εl
Statistics based on independent repetitions of the dilutions
and absorbance measurements.
Validation based on comparison with authentic standard
18solution.
First experiment:
A chromophorogenic assay
Non-absorbing compounds can be
detected via a reaction that generates
a chromophore in proportion to the
compound’s concentration.
! Either a known ε or a standard curve
are used to relate the A to the starting
compound’s concentration. (The
standard curve in-essence yields ε).
! We will use the Bradford reagent,
which is a solution of Coomassie
blue G250 in ethanol/phosphoric
acid. This is less tricky than the
text’s recommendation of FolinCiocalteau.
! The product sheet for Sigma’s
Bradford reagent is provided on the
course web site. We will use a
19 variant of the standard procedure A.
Bradford Assay
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Marion Bradford published and
patented the assay. Bradford, M. M.
(1976) Anal. Biochem. 72: 248-254.
(This is one of the most heavily cited
scholarly articles of all time).
Based on a shift in the absorbance
maximum of Coomassie brilliant
blue G-250 upon binding to arginine
side chain (red form of dye
converted to a more blue form).
Two chemical bases for the λmax
shift:
–
–
–
20
Acidic dye is added to protein, λmax of
the dye shifts from 465 nm to 595 nm.
Dye binds to basic and aromatic amino
acids especially Arg.
Detergents and alkaline pHs interfere
with the dye’s colour shift.
Coomassie Brilliant Blue
and Mechanisms of Protein Staining and
Bradford-Assay
Coomassie brilliant blue
G-250
Precautions for
Chromophorogenic assays
ORIGIN
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The name Coomassie was first used in the late 19th century, adopted from the town of Coomassie (modern-day
AtKumasi
acidic
pH,
the
Ns
protonated,
the
sulfonates
in Ghana),
as a trade
name
of theare
dye manufacturer
Levinstein Ltd. for
two similar
triphenylmethane dyes
used as acid ionized,
wool dyes. The twonet
blue dyes
were then firstis
produced
1913 by Max is
Weiler
based in Elberfeld,
remain
charge
+1 incolour
red.
Germany. Today, the term ‘Coomassie ’ is a registered trademark of Imperial Chemical Industries.
xy’, while only Coomassie only
G250 and one
Coomassie
there are approx.
dyes called
AtOverall,
neutral
pH 40the
Ns‘Coomassie
are deprotonated,
is R250
play a crucial role in biochemical analyses. During the last years, however, most authors referred to these dyes
+ve,
molecule is an an anion. Molecule is green with
simply as ‘Coomassie ’ without specifying which dye is actually meant.
-1cm-1.
ε~
Mwas
The 43,000
term ‘250’ originally
used for denotation of the purity of the
dye. The suffix ‘G’ in ‘Brilliant Blue G250’ was added to describe the
Binding
stabilizes
the anion, and
slightly greenish to
colourprotein
of the blue dye. The
suffix ‘R’ in ‘Brilliant Blue
R250’
is
an
abbreviation
for
‘red’
as
the
blue
colour
of
the
dye
has
slight reddish
produces the blue-green form aeven
when free dye
tint. Coomassie Brilliant Blue G-250 differs from Coomassie Brilliant Blue R-250
molecules
remain
cationic
(red).
by the addition of two methyl groups.
Brilliant Blue G250
Initially used to dye wool (keratin).
TM
!
TM
TM
!
TM
TM
!
TM
!
!
TM
BACKGROUND OF COLOUR CHANGES
The colour of the two dyes depends on the acidity of the solution and on its binding status to amino acids or
peptides. At a pH of less than 0 the dye has a red colour with an absorption maximum at a wavelength of 470 nm.
At a pH of around 1 the dye is green with an
absorption maximum at 620 nm while above
pH 2 the dye is bright blue with a maximum at
595 nm.
The different colours result from the differently
charged states of the dye molecule, corresponding to the amount of positive charges at the three nitrogen atoms
present, while the two sulfonic acid groups are normally always negatively charged.
• At a pH of around zero, all three nitrogen atoms are positively charged, thus the dye will be a cation with an
+
overall charge of +1, being in the red form.
• In the green form (pH of approx. 1) the dye will have no net overall charge (+2 and -2).
+Arg
-
!
R-250 lacks two
methyl groups
!
1
Structure of Coomassie brilliant blue
http://en.wikipedia.org/wiki/
Coomassie_Brilliant_Blue
G-250.
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The reaction must be limited ONLY
by the compound to be measured.
(Every molecule of compound is
counted)
A linear relationship must be
demonstrated for the absorbance and
the reactant that forms the dye.
Conduct the experiment in such a way
that the readings corresponding to
unknown samples fall within the
reading that make up the standard
curve.
If necessary, make dilutions of the
unknown. Do this BEFORE conducting
the reaction.
Amino acids that absorb
strongly in the UV.
Second Experiment:
2- Direct absorbance
measurement on a protein
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!
!
23
We will exploit the strong absorbance
of UV radiation by tryptophan (Trp)
and tyrosine (Tyr) side chains in a
protein.
Each protein species has a
characteristic 3D structure that places
its various Trp and Tyr side chains in
unique environments and causes
them to have extinction coefficients
that vary quite widely.
However if a protein is denatured to a
‘random coil’ all the side chains are
exposed to the medium and behave
as if they were all simply amino acids
dissolved in that medium.
Garrett and Grisham, 3rd ed. Fig. 4.15
24
UV-absorbing amino acids
A typical protein:
Lysozyme
6 Trp and 3 Tyr.
25 2ZYP.pdb
26
UV-absorbing amino acids
Denatured protein
!
!
In a denaturing medium, the
extinction coefficient of the protein at
280 nm can be approximated as the
sum of the contributions of the Trps
and the Tyrs:
εprotein = nTrp • εTrp + nTyr • εTyr
We will use the protein lysozyme from
chicken egg white. the amino acid
sequence of this protein is known1 :
LYS
ARG
ASN
THR
TYR
ASP
PRO
VAL
MET
THR
!
Trpand
and33Tyr.
Tyr.
!! 66Trp
!
27
Some are buried, others are stacked.
VAL
HIS
TRP
GLN
GLY
GLY
CYS
ASN
ASN
ASP
PHE
GLY
VAL
ALA
ILE
ARG
SER
CYS
ALA
VAL
GLY
LEU
CYS
THR
LEU
THR
ALA
ALA
TRP
GLN
ARG
ASP
ALA
ASN
GLN
PRO
LEU
LYS
VAL
ALA
CYS
ASN
ALA
ARG
ILE
GLY
LEU
LYS
ALA
TRP
GLU
TYR
LYS
ASN
ASN
SER
SER
ILE
TRP
ILE
LEU
ARG
PHE
THR
SER
ARG
SER
VAL
ARG
ARG
ALA
GLY
GLU
ASP
ARG
ASN
ASP
SER
ASN
GLY
ALA
TYR
SER
GLY
TRP
LEU
ILE
ASP
ARG
CYS
ALA
SER
ASN
SER
TRP
CYS
THR
GLY
CYS
ARG
MET
LEU
PHE
THR
CYS
ASN
ALA
ASN
LYS
LEU
LYS
GLY
ASN
ASP
ASN
ILE
SER
GLY
GLY
In our denaturing medium, at 280 nm
εTrp = 5690 M-1cm-1 and
εTyr = 1280 M-1cm-1.
28 J Biol Chem. 1963 Aug;238:2698-707
The experiment
We will determine the concentration
of a lysozyme solution indirectly, by
first determining the concentration of
an aliquot of that solution that we
dilute into denaturing conditions. We
do that because under denaturing
conditions, we can calculate the
extinction coefficient because we
know the Trp and Tyr content. This
extinction coefficient enables us to
determine the concentration.
! From the dilution factor we will
calculate the concentration of the
parent native solution.
! The calculated concentration and the
measured absorbance at 280 nm will
then be used to calculate the native
protein’s extinction coefficient at 280
29 nm.
!
Experiment 1, Precautions for
Chromophorogenic assays
!
!
!
!
30
The reaction must be limited ONLY
by the compound to be measured.
(Every molecule of compound is
counted)
A linear relationship must be
demonstrated for the absorbance and
the reactant that forms the dye.
Conduct the experiment in such a way
that the readings corresponding to
unknown samples fall within the
reading that make up the standard
curve.
If necessary, make dilutions of the
unknown. Do this BEFORE conducting
the reaction.
The assay conditions
!
!
!
!
!
!
31
The Bradford concentrate contains
methanol and phosphoric acid .
These are potentially hazardous.
How might this formulation be
changed for reduced danger ?
How will you handle it ?
How will you dispose of your
reactions ?
Standard has a concentration of ≈2
x 10-2 mM. (Check with your T.A.)