Download BIOCHEMISTRY LAB CHE-554 First portion: A chromophorogenic

First portion:
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 ! or a standard curve
are used to relate the A to the starting
compound’s concentration. (The
standard curve in-essence yields a !).
! 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 assay
2 type ‘A’ from that sheet.
!
BIOCHEMISTRY LAB
CHE-554
Experiment #1
Spectrophotometry
Professor Testa
Making standard samples -1
Precautions for
Chromophorogenic assays
!
!
!
!
!
3
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
reaction 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
conduction the reaction.
!
!
The active ingredient is Coomassie
blue G250, which binds primarily to
arginine residues.
The sigma reagent is used as
follows: 3.0 ml reagent + 0.1 ml
solution to be tested.
First generate a series of known
concentrations of the standard
protein solution as follows:
!
4
Vol. of Protein stdd. soln. (µl)
Vol. of buffer (µl)
100
0.0
80
20
60
40
40
60
20
80
0
100
Reading the standard
samples
Making standard samples -2
!
!
!
5
In your notebook, be sure that you
have a name for each sample
included in your table and that this
name is used to label the
corresponding tube.
Make these samples up in test
tubes that have sufficient capacity
for the addition of 3 ml of the
Bradford reagent and subsequent
mixing without spilling.
Add 3 ml Bradford reagent to each
tube. DO NOT dip a pipette tip into
the master bottle, pour a little
reagent out of the bottle and
dispense from this aliquot. If there
is left-over and it is not
contaminated, pass it along to a
colleague for use.
!
!
6
Mix each sample and then wait at
least 5 min. but no longer than 45
min. (Observe changes in your
samples as a function of time, and
take photographs.)
Read A595 and record these results.
Making the unknown
samples
!
!
Adapt the protocol detailed
above to make a series of five
samples of the unknown, plus a
‘null’ sample.
React these with Bradford
reagent as above and read them
as above.
Questions
!
!
!
!
!
7
8
Why do we test a whole series of
standard samples instead of just
one ?
Why do we include a null sample ?
Why do we test a whole series of
dilutions of our unknown sample
instead of just one ?
What is a potential pitfall of making
a standard curve from one species
of protein as a basis for determining
the concentration of a different
protein ?
What are potential hazards of
working with the Bradford reagent
and why ?
Second Portion:
Direct absorbance
measurement on a protein
!
!
!
9
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.
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:
!protein = nTrp • !Trp + nTyr • !Tyr
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
!
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
!Trp = 5690 M-1cm-1 and
!Tyr = 1280 M-1cm-1.
10 1 Canfield (1963) ‘The Amino Acid Sequence of Egg White Lysozyme‘
Chem. 238:2698-707
J Biol
Questions to be answered
in your theory section and
pre-lab.
!
!
!
!
11
What is the extinction coefficient of
denatured lysozyme at 280 nm in our
denaturing medium ? (Show your
work.)
What potential hazards are
associated with working with
guanidinium HCl ?
Does this material contain HCl ?
Look at the MSDS for guanidinium
HCl available on the course web site
and assess this material’s toxicity by
comparing its LD50 with those of
glucose and aspiring, which are also
posted.
The experiment
!
!
!
!
12
The implication is that under
denaturing conditions, we can
determine the concentration of any
protein if we know its Trp and Tyr
content. We will use this strategy to
determine the concentration of a
lysozyme solution.
This denatured solution will be
derived from a native (folded) solution
of lysozyme by a known dilution.
Thus you will calculate the
concentration of the parent native
solution.
The calculated concentration and the
measured absorbance at 280 will
then be used to calculate the native
protein’s extinction coefficient at 280
nm.
Protocol, continued
Protocol
!
!
!
!
13
Measure the A280 of a solution of
native (folded) lysozyme provided
by your T.A. (This will be in 20 mM
phosphate buffer at pH 6.5).
Use a quartz cuvetted for this
measurement.
Recover the native lysozyme from
the cuvette. Dilute 2.5 ml of the
native lysozyme with 7.5 ml of 8 M
guanidinium hydrochloride in the
same phosphate buffer.
What will be the final concentration
of guanidinium hydrochloride ?
By what factor will the denatured
lysozyme concentration be related
to the original concentration of
native lysozyme ?
!
!
!
!
14
As a pedagogical exercise, we will
pretend we only have one quartz
cuvette each. Thus you will need to
re-use the cuvette you used for the
native lysozyme sample.
Transfer 3 ml of denatured lysozyme
to the cuvette and measure its A280
(first measurement).
Discard this solution and transfer a
second 3 ml aliquot of denatured
lysozyme to the cuvette and
measure its A280 (second
measurement).
Discard this solution too and transfer
a second 3 ml aliquot of denatured
lysozyme to the cuvette and
measure its A280 (third
measurement).
Post-lab thoughts
Questions
!
!
!
!
How do your three readings for the
A280 of denatured lysozyme
compare ?
Should you use the average of these
values ?
What value should you use and how
will you know if it is valid ?
Use your calculated extinction
coefficient for denatured lysozyme to
calculate the concentration of
denatured lysozyme.
! Now calculate the lysozyme
concentration of the original native
solution of lysozyme.
! Use this and the A280 you measured
for native lysozyme to calculate the
extinction coefficient of native
15 lysozyme.
!
!
!
16
How does the extinction coefficient
you determined for native
lysozyme compare with published
values ? (look up at least one, and
provide your source. Remember
that lysozyme from different
sources can have different
extinction coefficients, you are
looking for hen egg white
lysozyme. Hen is also known as
Galus galus.
Compare your measured
absorbances for the unknown with
those measured by a classmate
(say who). Discuss the magnitude
and possibles causes of any
differences.
Also answer questions 2, 3, 4 from
our text, experiment 1(page 24).