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Review of Practicals
BIOC2101
Background
Parts A, B and C all use an assay for
protease activity
The substrate is casein
The enzyme is a pancreatic protease
Background
The protease hydrolyses internal bonds
within the casein to produce smaller
peptides
This reaction can be stopped by adding
TCA, which denatures and precipitates
the proteins (enzyme and large casein
molecules)
Centrifugation ensure only the small
peptides remain in the supernatant
Background
Bradford's reagent is used to measure
the amount of peptides present in the
supernatant
The reagent produces an intense blue
colour with all peptides and proteins
(measured at 620 nm)
Part A
Part A
Substrate and
buffer
RO water
Trypsin inhibitor
Trypsin
Trypsin
Reactions stopped
Bradford assay performed
Part B
Substrate and
buffer
RO water
Trypsin inhibitor
chymotrypsin
chymotrypsin
Part B
Trypsin inhibitor does not inhibit the
protease activity of chymotrypsin
Reactions stopped
Bradford assay performed
Part C
Chymotrypsinogen is a zymogen
Chymotrypsinogen is activated by
trypsin
Part C
chymotrypsinogen
trypsin
trypsin
0 min 5 min 10 min
inhibitor
Casein added
Reactions stopped
Bradford assay performed
Glycolysis(Prac,cal(
Part C
The longer the incubation, the more
chymotrypsinogen is activated
The trypsin inhibitor stopped the
zymogen from being activated and
prevents trypsin from acting on the
casein
The more chymotrypsin is present, the
more casein will be digested
GLUCOSE
ATP
Provided with:-"
- Dialysed rat skeletal muscle extract, containing all the
enzymes for glycolysis and other soluble cytoplasmic
enzymes, but no mitochondrial enzymes and no
metabolites/cofactors."
"
- Assay system for lactate"
"
Expt A investigated what cofactors are necessary for
glycolysis (fructose-1,6-bisP --> lactate)"
"
Expt B investigated the rate limiting step in glycolysis
(glycolytic intermediate --> lactate)"
GLUCOSE
ATP
Experiment A
ADP
G-6-P
F-6-P
ATP
F-6-P
ATP
ADP
F-1,6-bisP
DHAP
ADP
F-1,6-bisP
GAP
Expect to find
that ADP, Pi
and NAD+ are
all required for
rapid glycolysis"
NAD+ + Pi
NADH + H+
2 x 1,3-bisPG
ADP
ATP
2 x 3-PG
DHAP
NAD+ + Pi
ATP
2 x 3-PG
2 x 2-PG
H2O
2 x PEP
ADP
NADH
ATP
2 x PYR
H2O
2 x PEP
ADP
"
NAD+
lactate
GLUCOSE
ATP
F-6-P
ATP
ADP
F-1,6-bisP
NAD+ + Pi
NADH + H+
2 x 1,3-bisPG
ADP
ATP
2 x 3-PG
2 x 2-PG
H2O
2 x PEP
ADP
GLUCOSE
ATP
lactate
ADP
G-6-P
"
"
lactate
"
Experiment A
F-6-P
ATP
ADP
F-1,6-bisP
DHAP
2 x PEP
ADP
"
NADH NAD+
ATP
2 x PYR
Typical Results"
"
Addition !
!A"
Water
"
"0.02"
NAD+/ADP/Pi "0.45"
NAD+/ADP"
"0.10"
NAD+/Pi "
"0.02"
ADP/Pi
"
"0.04"
""
NADH NAD+
ATP
2 x PYR
"
Experiment A
ADP
G-6-P
GAP
GAP
NADH + H+
2 x 1,3-bisPG
ADP
2 x 2-PG
DHAP
Experiment A
ADP
G-6-P
ATP
2 x PYR
"
NADH NAD+
lactate
"
GLUCOSE
ATP
GLUCOSE
ATP
Experiment B
ADP
G-6-P
F-6-P
ATP
F-6-P
ATP
ADP
F-1,6-bisP
ADP
F-1,6-bisP
DHAP
GAP
Start with:-"
Glucose"
F-6-P"
F-1,6-bisP"
GAP"
1,3-bisPG"
+ all necessary
cofactors"
NAD + Pi
NADH + H+
2 x 1,3-bisPG
ADP
ATP
2 x 3-PG
2 x 2-PG
H2O
2 x PEP
ADP
NADH
ATP
2 x PYR
DHAP
GAP
2 x 2-PG
H2O
2 x PEP
ADP
"
NAD+
ADP
F-1,6-bisP
NADH
"
pyruvate"
2 x 2-PG
"
NADH NAD+
"
How(can(lactate(dehydrogenase(catalyse(reac,ons(in(the(
opposite(direc,ons(during(rst(glycolysis(and(then(the(lactate(
assays?(
Glycolysis"
NADH + H+"
DHAP
NAD+
lactate
Experiment B
ADP
G-6-P
ADP
F-1,6-bisP
ATP
2 x PYR
"
lactate"
GLUCOSE
ATP
F-6-P
ATP
2 x PEP
ADP
Typical Results"
"
Addition !A"
Water
"0.02"
Glucose "0.08"
F-6-P"
"0.15"
F-1,6-bisP "0.45"
GAP "
"0.50"
1,3-bisPG "0.02"
NADH NAD+
ATP
2 x PYR
F-6-P
ATP
DHAP
NAD+ + Pi
ATP
2 x 3-PG
Experiment B
ADP
G-6-P
rate
limiting
step"
NADH + H+
2 x 1,3-bisPG
ADP
lactate"
GLUCOSE
ATP
Experiment B
ADP
G-6-P
Relatively low pH
of 7.4 (high [H+])
lactate" favours lactate
formation"
NAD+"
Lactate assay"
Relatively high pH
of 9.4 (low [H+])
NAD+" NADH + H+"
lactate"
pyruvate" favours pyruvate
formation"
reacts non-enzymically
with hydrazine"
ATP
2 x PYR
lactate
"
Separa,on(Techniques(
3 types of chromatography:-"
"
Partition (thin layer)"
"
Permeation (gel filtration)"
"
Ion exchange"
"
All involve the differential distribution of
molecules between a stationary phase
and a mobile phase"
TLC(separa,on(of(amino(acids(
The Mobile phase is the solvent (n-butanol/acetic acid/
water).!
!
The Stationary phase is water associated with
cellulose fibres."
"
More polar molecules partition into the stationary
aqueous phase and do not migrate far from the origin.
(e.g. lysine and glutamic acid)"
"
Less polar molecules are more soluble in the organic
mobile phase and migrate further. (e.g. leucine)"
What(is(the(func,on(of(the(cellulose(in(
the(TLC?(
Results(of(the(Gel(ltra,on(Expt.(
Yellow ferricyanide
forms a diffuse slow
moving band!
Red/brown
hemoglobin forms a
much tighter fast
moving band!
Why(does(the(Rf(change(in(dierent(
solvents?(
Different solvents may be more or less polar, affecting
partitioning between the stationary and mobile phases."
"
Different solvents may change the ionisation of amino
acids and therefore their distribution between polar
stationary and less polar mobile phases."
"
e.g. glutamate is less polar in an acidic solvent than in
a basic solvent."
-OOC-CH
+
2-CH2-CH(NH3 )COO "
HOOC-CH2-CH2-CH(NH3+)COO-"
HOOC-CH2-CH2-CH(NH3+)COOH"
Gel(ltra,on(
The Mobile phase is the buffer which passes through
the chromatography column.!
!
The Stationary phase is water (buffer) immobilised in
pores in the gel filtration particles, that is accessible to
relatively small molecules, but not to large molecules."
"
Larger molecules will pass through the column faster
than smaller molecules (the opposite of conventional
filtration)."
Ion(exchange(chromatography(
The Mobile phase is the buffer which passes through
the chromatography column.!
!
The Stationary phase is ionised groups immobilised
on the ion exchange particles."
"
Molecules with net charges opposite to those on the ion
exchange column will form ionic bonds and bind tightly
to the stationary phase. Molecules with no net charge
(or with a net charge the same as that on the ion
exchange column) will pass through with the mobile
phase."
Ion(exchange(chromatography(
Ion(exchange(chromatography(
At pH 2.0!
"
Glycine has a net charge of ~+0.5 "
"
"+H3N-CH2-COO- and +H3N-CH2-COOH"
"
Arginine has a net charge of ~+1.5"
"
"+(NH2)2C-NH-CH2-CH2-CH2-CH(NH3+)-COO-"
Ion(exchange(chromatography(of(proteins(
Ion exchange chromatography is an important
technique for separating and purifying proteins.!
"
The pI (isoelectric point or isoelectric pH) is the pH at
which a protein has an overall charge of zero."
"
This means that at pH values <pI, a protein will have an
overall +ve charge, and bind to an ion exchange column
which has -ve charged groups."
"
At pH values >pI, the protein will have an overall -ve
charge and bind to an ion exchange column which has
+ve charged groups."
Glucose(Tolerance(Test(Prac,cal(
"
and "+(NH2)2C-NH-CH2-CH2-CH2-CH(NH3+)-COOH"
"
BOTH will bind, but the arginine will elute much more
slowly."
Glucose(Tolerance(Test(Prac,cal(
16
14
12
Patient 1
10
Patient 2
Patient 3
Patient 4
Patient 5
normal!
onset of
diabetes!
untreated
diabetic!
0
0
30
60
90
120
150
180
Time (min)
What(other(tests(might(assist(the(
diagnosis?(
16
14
12
Patient 1
10
Patient 2
Patient 3
Patient 4
Patient 5
normal!
No fast!
2
0
0
30
60
90
120
Time (min)
150
180
Urinary [glucose]"
"
Plasma [insulin]"
"
Glycosylated hemoglobin"
Fasting is required to
ensure a normal
initial fasted blood
glucose"