oxidation
reduction
>
catabolism
s
anabolism [ synthesis]
oxidation & reduction
catalyzed by
[break down]
are
both
enzymes
Biological chemistry: Practical -3
if
it has
oxidized
a
proton
if
used NADH
it'll
we
get
we
used NAD
it 'll
get
reduced
.
(Co)-ENZYME:NAD/NADH or
NADP/NADPH absorption and relevance
in enzyme activity measurement
-1
oxidized
,
reduced
,
µA☐+
µA☐µ
Significance: sensitive tool and specific
marker in diagnosis and research
Enzymes help in maintaining body physiology and
Metabolism
Clinical diagnosis: Diseases (diabetes, heart attack,
cancer, genetic diseases)
Tissue damage-release specific isoenzymes from heart,
muscles, liver, kidney, brain etc
Design/target for drug action and metabolism: enzyme
inhibitors or activators
Toxicities: CO/CN/pesticides/nerve gases/drug overdose
cyanide
or
You
in
can
find which
response
enzymes
because
test
get
affected
Enzymes
and poisons
tissue &
of
better damage diagnosis
are
they're
liver
enzymes
to drugs
a
more
s
specific
check liver
di
-
agonistic
specific
enzymes
protein
,
fatal lipid
specific
more
particular
for
reaction for
example
,
,
metabolism
diabetic
will be
patients
enzymes
affected
enzymes of the cardiac
muscles will be released in the
in
fool than total
sugar
are
,
hearfattack-n.it
That's what the
Dr
.
said feels weird
,
tho
Enzymes
cofactor
e-
most of the time
vitamins or
their deraiitive
are
Biological catalysts which facilitate specific reactions in the
body
Always a protein (may contain metals, ions, carbohydrates,
lipid and nucleic acid as a support structure
Co-enzymes: organic molecules (ATP, NAD/NADH,
NADP/NADPH, CoA) which support the catalytic function of
the enzymes and often used up in the reaction (which can
be measured)
Co-enzymes are synthesized in the tissues and are mostly
derived from vitamins
ATP NADYNADH
,
,
NADPYNADPH
,
CoA
are
all vitamin derivative
Coenzymes
are
always present
required
not
or
Enzymes have active site (pockets) for
substrate/coenzyme binding
coenzyme
are
needed
because
sometimes the binding of substrate
to the enzyme is not optimum
Co-enzymes may also bind at
another sites on enzyme proteins
Enzyme
Substrates and Coenzymes are utilized in
the catalysis but not the
enzyme
'
substrates sometimes don't fit
the enzyme pocket site
enzyme
felicitate the binding of the
on
substrate
,
and allow it to
sometimes participate
in
co
-
fit and
,
the reaction
As
time
decrease
goes
as
by
,
they
concentration
form the
of
substrate
product Groduct concentration
increases
Enzyme velocity
Enzyme activity is commonly expressed by the
intial rate (V0) of the reaction being catalyzed.
is not measured
enzyme activity
at the equilibrium it's measured
before that / because there will be
,
a
rate of change]
When substrates and products are in
equilibrium S
P The
the
to tell
way
IV
whether
properly
an
is
enzyme is working
its defective (could be higher
'
or
or
lower than IU)
Enzyme activity = moles
of substrate converted to
activity
product per unit time (IU) internationally
The
-
standardized value
of
enzyme
that is followed
international unit
Enzyme
activity
moles of
is
the rate at which
substrate form the product
over
time
To
Enzyme reactions
i
2
make
a
an
enzyme
substrate
is
reaction land
measure
it)
required
calculate enzyme
activity
find the amount of sub1-rate lost
find the amount of product formed
to
:
When a substrate (a molecule-e.g.-glucose) is
metabolized by an enzyme it is converted to a product
(another molecule-e.g.-glucose-phosphate).
Depend on the pH, temperature. Most of the enzymes
are active at pH7.4 and 300 -37OC.} Normally these the conditions for the bestof theperformance
enzyme
system
There will be an equilibrium when enzyme reaches at
steady state, when substrate and product formation is
in equilibrium.
Enzymes reach at maximum velocity (Vmax) when
substrate is at the saturating concentration (all
enzymes molecules are bound to the substrate).
,
buffer
is
needed
are
Measurement of enzyme activity by
change in coenzyme concentrations
Measure the rate of disappearance of the substrates
Measure the rate of appearance of the products
Example: measurement of NAD or NADH levels is based on their absorption at
specific wavelength (UV range, ~200-380 nm)
•
removes
the H+ from lactic acid and transfer it to
NAD
-1
Enzyme (lactate dehydrogenase-LDH)
oxidized form
reduced form
reduced
CH3COCOOH + NADH2
(Pyruvic acid)
+
"
"
died
it
,
CH3CHOHCOOH + NAD
•
is
the
-
(Lactic acid)
If
Experiments:
to find the
enzyme
activity
find
pain
the concentration of either of these two
-
-
Today you will measure and analyze the absorption spectra of NAD and NADH
an
oxygen sensitive enzyme
enzyme
,
senses
the
oxygen
working
level
exercise
muscle pain
or
the
in
too fast
too much
and shortness of breath
why?
we
all
the time
,
body
develop
we
muscle
,
:
due
to lactic acid build
shortness of breath
:
due to
0,
up
debt [0
,
is
convert
needed to
pyruvate
Usually colorless solutions absorb light in UV range (below 400 nm) and colored
solutions at the 400 nm and above (visible range)
you will demonstrate that NADH has an absorption max at 340nm (Table 1) and
the absorption is proportional to the concentration of NADH (Table 2). NAD, on
the other hand, will not have any significant absorption at 340 nm.
(
>
wavelength
"
NAD
in
&
other
NADH
words
,
are
using
colorless
solutions that
wavelengths
,
above 400
nm
means
that both will only
absorb
(which show color ) will be useless
light
in
since
both
UV
range
are
1<400
colorless
.
nm
)
,
.
to ATP]
shows that NADH has
Experiments-A
absorption
NADH
is
not NAD
-1
,
that
a
characteristic
means
that
measurable
Charat eristic absorption means that
detectable change in concentration will
a
be
observed under little/small enzyme
activity
Pipette the required volume of NAD and NADH
solutions in tubes and add water as suggested
Set up the spectrophotometer at 300 nm and
read the NAD and NADH absorption against
water as a blank.
Change the wavelength as suggested and read
the absorption again. Continue to read up to 400
nm
Plot a graph using wavelength at X-axis and
absorption at Y-axis
from
300
-
400
nm
Video
TABLE 1 Compare the absorption maxima of NADH and NAD at 300 to 400 nm wavelengths
Results:
Wavelength nm
NADH Absorption
NAD Absorption
300
0.675
0.296
320
0.771
0.151
330
0.9981
0.114
340
1.128 Maximum peak
0.080
350
1.116
0.058
360
0.976
0.043
370
0.709
0.034
380
0.405
0.024
400
0.064
0.003
Result: NADH absorption at
340 nm is Maximum
Because NADU has
at
340
nm
,
which
a
characteristic absorption it
,
is
the best
wavelength
is
to
measurable
measure
+
the
enzyme
max
.
activity
absorption
is
NAD absorption is
decreasing and
minimum in this range
No
characteristics
absorption
,
it decreases
in
fact
NADH/NAD Absorption
A
b
s
o
r
p
t
i
o
n
Max
.
NADH absorption at 340 nm
^
•
NAD absorption
300
320
340
360
380
400 nm
Confirms that
produce
Experiment-B
more
and
light absorption
This
as
reaction
more
increases
experiment tries
to
it will
enzyme proceeds
concentration NADH thus
,
.
prove
& concentration of
whether as a result of
of NADH will be produced
the
the
absorption
prove
,
relationship between
enzyme
It also tries
.
enzyme reaction increased
or
not
,
to
concentration
.
Pipette the desired volumes of NADH solution 1100am)
in the tubes and add water to make final
volume 1.0ml.
max for NADH) and read each tubes against
water as a blank.
Plot a graph using NADH concentrations at Xaxis and absorption(readings) at Y-axis.
TABLE 2
Plot a graph of NADH concentrations on X-axis with absorption at Y-axis
Tube number
÷=¥÷µ -100,8¥
☐F-
-
DF
New
conc
=
20
=
.
-
cone
.
=
.
=
1020¥
1
G-
=
=
50
c.
100
=
=10¥¥
=
10am
Cave
c÷= 109¥91
-
-
sa
3
4
c.
200
=
-
=
a-
5
=
"÷='Y¥o%-
400
0.045
10 uM
0.090
20 uM
0.161
30 uM
0.249
40 uM
0.334
÷
.
absorption
increases
which
that the
active
,
enzyme
means
is
and
NADH (product)
30µm
=v¥=
=
40µm
**Calculation: Original conc. of NADH is 100µM divided by dilution
factors (total volume= 1.0ml): e.g.CC
Tube #1 dilution factor is 20 (50 µl in
1000 µl (1ml)). Therefore in tube #1 the NADH conc. Is 100/20=5 µM Or
use N1V1=N2V2 (where N1 is 100uM NADH, V1 is volume taken
final volume 1.0 ml or 1000ul)
concentration
increases,
20AM
a- "
300
5 uM
As
5µm
2
GV ,
Absorption at
340nm
folds
dd☐÷nc
New
Volume of NADH Concentration**
( L)
of NADH (µm)
In 1.0ml
u
is
formed
Absorption at 340nm
B:NADH absorption at 340 nm
9
Almost
5
10
20
30
a
linear
relationship
40 uM NADH
Questions
Why was it considered unnecessary to
investigate the absorption of NAD and NADH
between 400 and 800 nm?
What differences did you notice between the
two (NAD/NADH) absorption spectra?
How would you apply your results to clinical
activity
practice?
Because, NAD
ig
you
That's
could
"
say
colorless i. they won't be visible
É NADH
that light won't be absorbed at that range
why only
are
e.
< 400
nm
will be
investigated
since
light
will
NADH showed
absorption
+
NAD
kept
Shows
enzyme
Also ( clinical)
:
diagnostic relevance for example cardiac enzymes
these experiments
help pinpointing the diagnosis
For
,
can
on
which tissue
is
releasing
,
in
this said
enzyme
.
even
at
> 400
nm
.
at
a
decreasing
a
.
absorbed
.
characteristic absorption
340
didn't show
an
get
with
peak
the
absorption
nm
characteristic absorption
and