Transcript BCHM2072/2972 2004 THEORY of PRACTICAL PAPER MCQs 18 - 33

BCHM2072/2972
2004 THEORY of PRACTICAL PAPER
MCQs
18 - 33
Enzymic methods (questions 18 to 33).
The alternative methodology for estimating ketone bodies
in urine and serum is to use the enzyme β-hydroxybutyrate
dehydrogenase (3-HBDH). This enzyme catalyses the
reversible oxidation of β-hydroxybutyrate to acetoacetate,
with the concomitant reduction of NAD+. The reaction is
shown below.
OH
H3C
CH
O
3-HBDH
H2
C
COOH
-hydroxybutyrate
+ NAD+
H3C
C
H2
C
Acetoacetate
COOH
+ NADH
Measuring the level of acetoacetate in biological samples: The reaction will
proceed quantitatively in the direction of β-hydroxybutyrate formation at pH 7 in
the presence of excess NADH (NADH:acetoacetate of at least 2:1). Under
these conditions the conversion of acetoacetate to β-hydroxybutyrate is >98 %.
The progress of the reaction can be monitored by measuring the amount of
NADH in the reaction. NADH and not NAD+ absorbs strongly at 340 nm.
Measuring the level of β-hydroxybutyrate in biological samples: At an
alkaline pH (pH 9.5) the conversion of β-hydroxybutyrate to acetoacetate is
favoured. β-hydroxybutyrate, in the presence of a large excess of NAD+ is
oxidized to acetoacetate. Again the reaction is monitored by absorbance at 340
nm. To ensure this reaction proceeds to completion in this direction a trapping
reagent, hydrazine, is also added. Hydrazine will react with the acetoacetate
forming a hydrazone derivative.
To improve the sensitivity of the β-hydroxybutyrate reaction: Your
research team has developed a synthetic derivative of NAD (syn-NAD) which
has much stronger absorbing properties in the reduced state to NADH. The
absorption maximum for syn-NADH is 360 nm, giving an extinction coefficient
of 200 mM-1cm-1, whereas NADH has an extinction coefficient of 6 mM-1cm-1.
Like NAD, oxidized syn-NAD has no absorbance at 360 nm. Syn-NAD has the
same affinity for 3-HBDH as natural NAD. With either cofactor both the forward
and reverse reaction are completed within 30 min under the conditions
described.
The measurement of acetoacetate in samples.
Acetoacetate + NADH  β-hydroxybutyrate + NAD+
You begin by setting up the acetoacetate assay as
in the table below.
Component
Volume (ml)
5 X buffer pH 7.0
200
NADH
50
Standard acetoacetate
0 - 200
3-HBDH (1 U/ml)
10
water
Made up to 1000 ml
18. What is the most appropriate [NADH] stock
Given that the working range of the spectrophotometer is
0.1 to ~1.2, what would be the most appropriate
concentration of the stock NADH solution to use in the
assay described in the table above (ε = 6 mM-1cm-1)?
A. 0.2 mM
B. 0.2 μM
C. 1 mM
D. 4 mM
E. 60 mM
During the reaction, the NADH is going to be used – and
the absorbance of the cuvette will DECREASE
The absorbance needs to start at an absorbance of
about 1.2 (higher would be beyond the useful range of
the spec, lower would not give us enough substrate to
fuel the reaction
If a 1 mM NADH solution has an absorbance of 6, an abs
of 1.2 would be given by an [NADH] of 0.2 mM
0.2 mM is 0.2 umol/ml – so there’s 200 nmol NADH in
the cuvette to begin with.
This came from 50 ul stock… so the stock must be 4
nmol/ul = 4 mM
19. What is the maximum measurable amount of
acetoacetate
Again, using the working range of the spectrophotometer of
0.1 to ~1.2, what is the maximum amount of acetoacetate
the assay above can accurately measure (final volume 1
ml), using NADH? Hint: NADH must be in excess; NADH:acetoacetate of
at least 2:1
A. 200 nmoles
From the last question, the [NADH] starts at 0.2 mM
Which is 0.2 umol/ml
B. 100 nmoles
So, in the 1 ml cuvette, there’s 200 nmol NADH to start
with
C. 0.2 nmoles
So we can have 100 nmol of AcAc
D. 0.1 nmoles
E. 10 nmoles
A
Absorbance 340 nm
1.2
1.0
0.8
0.6
0.4
0.2
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
10
20
Time (min)
30
0
C
1.4
10
20
Tim e (m in)
1.2
1.0
0.8
0.6
0.4
0.2
0.0
30
D
1.2
Absorbance 340 nm
Which graph (A-E)
below shows the
time course for
standards for this
reaction?
B
1.4
0.0
Absorbance 340 nm
You perform a time course
over 30 min with 5 different
concentrations of the
acetoacetate, measuring the
absorbance at 340 nm every 5
min. From this you obtain a
standard curve.
1.0
0.8
0.6
0.4
0.2
0.0
0
10
20
Tim e (m in)
30
0
10
20
Tim e (m in)
30
Start at Abs 1.2 and go
down
E
1.4
Absorbance 340 nm
21. Time Course
Absorbance 340 nm
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
10
Tim e (m in)
20
30
At lowest abs (when all
AcAc in the top standard
has run out) is 0.6
(showing that there’s still
100 nmol of NADH
available
Measuring the amount of β-hydroxybutyrate.
β-hydroxybutyrate + NAD+  acetoacetate + NADH
Below are the assay components for the β-hydroxybutyrate
assay, using NAD.
Component
5 x glycine buffer containing hydrazine, pH 9.5
60 mM NAD+
Standard -hydroxybutyrate
3HBDH (1 U/ml)
Volume (ml)
200
50
0 - 200
10
H2O
Made up to 1000 ml
22. What [β-hydroxybutyrate] gives absorbance of 0-1.2
What concentration of standard β-hydroxybutyrate would
generate a standard curve with an absorbance range
between 0 and ~1.2, when measured at 340 nm?
A.10 mM
B. 0.2 mM
Same logic as before: at the end of the reaction, all the
NAD will have gone to NADH and an abs of 1.2 would
be given by a 0.2 mM solution
C. 0.2 μM
Since one NAD reacts with one bOHBu, thin initial
bOHBu in the top standard should be 0.2 mM
D. 1.0 mM
This 200 nmol will have come from 200 ul
So the stock bOHBU is 1 nmol/ul = 1 umol/ml = 1 mM
E. 1.0 μM
23. What [components] to change for syn-NAD
You are planning to use the same assay components, simply replacing
the 60 mM NAD+ solution with a solution of the syn-NAD derivative of a
similar concentration. However, you would like to set up the new
standard curve with an absorbance range from 0 to 1.0 at 360 nm. To
do this, what component(s) would you need to decrease in
concentration in the new assay?
Enzyme still necessary to take reaction to completion
A. β-hydroxybutyrate dehydrogenase
B. Syn-NAD
Still need syn-NAD to be in excess to drive coversion to
completion
C. Standard β-hydroxybutyrate
D. A & B
E. A, B & C
A smaller amount of bOHBu is required because a tiny
concentration gives a big absorbance. If we keep the
bOHBu the same as it was before, the final absorbances
will be too high
Use the figures below (A-E) to answer questions 24 to
30.
You have worked up the new assay, using the syn-NAD
derivative and the β-hydroxybutyrate standard. The ideal
standard curve should have an absorbance range of 0 to 1
when measured at 360 nm. The following time courses (AE) above were produced during the course of your
investigations.
Which was the result of:
Reactions not reaching
completion, but otherwise OK
B
0.8
0.7
Absorbance 360 nm
0.4
0.3
0.2
0.1
0.0
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0
10
20
30
40
0
10
Tim e (m in)
C
Perfect!
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
10
20
30
10
E
Absorbance 360 nm
0.7
0.6
bOHBu0.5running out
prematurely
0.4
0.7
0.6
0.5
0.4
0.1
0.0
Series3
Series5
0
10
Series2
Series4
20
0.0
0
Series1
0.2
0.2
0.1
E
0.3
0.3
20
Tim e (m in)
Tim e (m in)
40
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0
40
0.8Tim e (m in)
0.8
30
D
Perfect! Even faster than C
0.9
0.8
0
20
Tim e (m in)
Absorbance 360 nm
Absorbance 360 nm
1.0
Absorbance 360 nm
The graphs right (A-E)
have been obtained
with the new assay,
using syn-NAD. Series
1-5 are increasing
concentrations of
β-hydroxybutyrate.
Absorbance 360 nm
0.5
Bunching at higher bOHBU
Running out of syn-NAD A
30
40
40
20
Tim e (m in)
30
40
24. Pipette dispensing 30% too little
Using a pipette to dispense the standard
β-hydroxybutyrate, which consistently dispenses 30% less
volume?
bOHBu running out
prematurely
Option E
25. Adding 2x enzyme
Adding twice as much enzyme to each of the assay tubes?
Very fast completion
Option D
26. Using old enzyme
Using an old preparation of the β-hydroxybutyrate
dehydrogenase enzyme, lacking much of the original
activity?
Getting there slowly
but surely
Option B
27. Adding 2x syn-NAD
Adding twice the volume (100 ul instead of 50 ul) of the
syn-NAD solution to each of the assay tubes?
Syn-NAD is already in
excess
Won’t make synNADH any faster
Option C
28. Using impure standard
Using an impure preparation of the β-hydroxybutyrate
standard?
Not enough bOHBU
Option E
29. Adding 2 nmoles syn-NAD
Adding 2 nmoles syn-NAD to each assay tube?
Without calculation,
tempted to say “the
one where the synNAD is running out”!
Option A
When 2 nmol syn-NAD all used up, the
[syn-NADH] will be 2 nmol/ml = 2 uM
Since 1 uM syn-NADH gives abs of 0.2,
a 2 uM soln would give abs of 0.4
30. Measuring at too low a wavelength
Measuring the absorbances at 340 nm instead of 360 nm?
Absorbance never
gets quite as high as
expected
Option E
The following information refers to questions 31 to 33.
Once you have worked up the assay using standard β-hydroxybutyrate, you then try the
assay on a number of urine samples. These samples have been collected from well
nourished individuals and from volunteers who have been on the Atkin’s diet for one
month. The level of all ketone bodies in urine increases on this diet.
Urine is a complex biological sample which may contain compounds which interfere with
this procedure. There are a number of compounds known to do this:
1) Ascorbic acid (vitamin C) at high concentrations can slowly reduce NAD+
non-enzymically.
2) Vitamin B supplements sometimes result in the excretion of urinary metabolites
which strongly absorb at 340 - 360 nm.
3) The β-hydroxybutyrate dehydrogenase enzyme can be inhibited competitively by
a number of other aldehyde and ketone containing compounds, which may be
present in urine. The presence of sufficient amounts of these compounds can
interfere with the reaction.
To overcome the problem of potential interference you have decided to assay 10 and 20
μl of each urine sample with and without a known amount of β-hydroxybutyrate standard,
termed a spike. You have measured the absorbance of the assays, containing the two
volumes of urine, at various times (0, 10, 20, 30, 40 min) throughout the course of the
reaction.
In a duplicate set of assays you have added a known amount of β-hydroxybutyrate
(*enough to produce a 0.4 change in absorbance) and incubated these spiked tubes for
40 min. The results are presented below.
**The control urine contains no β-hydroxybutyrate. A “no enzyme blank” was also
measured for each sample.
Sample #
**control
A
B
C
D
E
Assay components
Enzyme
Volume
(ml)
urine (ml)
0
10
10
10
10
20
0
10
10
10
10
20
0
10
10
10
10
20
0
10
10
10
10
20
0
10
10
10
10
20
0
10
10
10
10
20
Absorbance 360 nm
0 min
10 min
20 min
30 min
40 min
0.01
0.01
0.02
0.04
0.04
0.09
0.08
0.08
0.15
2.1
2.1
>3
0.05
0.05
0.06
0.02
0.02
0.04
0.01
0.02
0.04
0.04
0.18
0.37
0.08
0.29
0.58
2.1
>3
>3
0.05
0.10
0.16
0.08
0.24
0.48
0.01
0.02
0.04
0.04
0.18
0.37
0.08
0.45
0.91
2.1
>3
>3
0.05
0.15
0.27
0.16
0.48
0.96
0.01
0.02
0.04
0.04
0.18
0.37
0.08
0.45
0.91
2.1
>3
>3
0.05
0.21
0.36
0.24
0.56
1.12
0.01
0.02
0.04
0.04
0.18
0.37
0.08
0.45
0.91
2.1
>3
>3
0.05
0.26
0.46
0.32
0.64
1.28
40 min +
spike*
0.01
0.42
0.44
0.04
0.58
0.77
0.08
0.85
1.31
2.1
>3
>3
0.05
0.46
0.66
0.32
1.04
1.68
B:
Spike
works,
soassay
assay
deals
with
bOHBu
fine.
Final
abs
proportional
to
A:
D:
C:
E:
Spike
The
Everything
Thespike
works,
spike
isworks,
just
so
not
maxed
fully
soeffective,
the
deals
out.
bOHBu
Must
with
sobOHBu
be
assay
something
something
isfine.
fine.
isFinal
compromising
Urine
in the
abs
gives
urine
isisproportional
increase
that
thegives
bOHBu
inato
abs
massive
with
amount
ofurine
urine
put
in.
Zero
enzyme
gives
lowpresent.
absorbance.
Soall
allBit
appears
good.
amount
assay.
absorbance
time even
Final
of
when
abs
– even
put
partially
there
in.
when
Zero
is
proportional
no
there
enzyme
enzyme
is nogives
present.
enzyme
to starting
low
absorbance.
amount of urine.
So
appears
slow??good.
31. Which has vitamin C
Which urine sample (A-E) has been collected from
someone who takes large doses of vitamin C?
Something that causes a slow, but steady, rise in
absorbance – even in the absence of enzyme
Option E
32. Which has vitamin B
Which urine sample (A-E) has been collected from
someone on vitamin B supplements?
Something that shows a strong absorbance when
urine is added, even in the absence of enzyme
Option C
33. Which interferes with enzyme
Which urine sample (A-E) contains a compound which
interfers with the β-hydroxybutyrate dehydrogenase
reaction?
Something that causes a slowing of the rate, with
the reaction not really reaching completion even
after 40 min
Option D