Lactate
Dehydrogenase
Characterization


Angela
KC



Introduction:


 At
neutral
pH,
the
enzyme
lactate
dehydrogenase
(LDH)
catalyses
the

oxidation
of
NADH
coupled,
highly
exergonic
and
stereospecific
reduction
of

pyruvate
to
L‐lactate
as
shown
in
the
reaction
below
(Nelson
and
Cox.
2008)




 LDH
is
a
bisubstrate
enzyme
consisting
of
NAD+
as
the
hydrogen
carrying

coenzyme
and
either
lactate
or
pyruvate
as
the
second
substrate.
LDH
is
a

tetramer
of
Mr
140,000
that
consists
of
combinations
of
two
similar

polypeptides,
named
M
and
H,
for
muscle
and
heart
respectively.
These
two

types
of
polypeptides
make
up
different
combinations
to
make
the
LDH
tetramer,

M4,
M3H,
M2H2,
MH3
and
H4.
These
various
combinations
are
called
the
isozymes

of
LDH
(Zubay.
1988).

The
various
isozymes
of
LDH
have
different
functions

depending
on
the
nature
of
the
tissue
they
are
located
in.
The
M4
isozyme
in
the

muscle
shows
a
lesser
degree
of
LDH
inhibition
in
this
anaerobic
tissue
by
the

increased
concentrations
of
pyruvate.
Whereas,
the
H4
isozyme
in
the
aerobic

heart
tissues
show
a
greater
degree
of
LDH
inhibition
through
increases
in

pyruvate
concentrations.
Thus,
the
enzyme
activity
is
dependent
on
the
isozyme

type
(Zubay.
1988).



 To
characterize
these
isozymes,
their
kinetics
were
studied

experimentally
to
determine
differences
in
their
Michaelis
consonant
(Km)
and

maximum
velocity
(Vmax)
values
and
compare
them
to
standard
literature

values.
Enzyme
assays
of
varying
substrate
concentrations
were
prepared
to

evaluate
the
reconstituted
rabbit
M4
isozyme.
Three
kinetics
plots,
Michaelis‐
Menten,
Lineweaver‐Burke
and
Eadie‐Hofstee
of
the
resulting
absorbances
were

made
and
compared
to
that
from
the
plots
made
from
the
given
H4
absorbance

data.



 Further
exploration
of
LDH
kinetics
was
carried
out
by
studying
the

change
in
enzyme
assay
absorbances
at
a
constant
pyruvate
concentration
upon

the
addition
of
various
concentrations
of
the
pesticide
Kepone,
which
is
a
known

LDH
inhibitor.
It
competitively
inhibits
LDH
action
at
concentration
as
low
as

0.01mM
(Hendrickson
and
Bowden.
1975).


 SDS‐PAGE
was
used
to
find
out
the
M
subunit
molecular
weight
of
the
M4

isozyme
through
the
comparison
of
the
electrophoretic
mobility
via
Rf
values
of

the
subunit
to
that
of
the
standard
protein
masses.

Size
exclusion

chromatography
by
HPLC
was
then
used
to
determine
the
molecular
weight
of

the
isozyme
through
the
use
of
a
standard
curve.
The
comparison
of
the
values
of



 1

the
weights
for
the
subunit
and
the
total
protein
mass
was
expected
to
reiterate

the
fact
that
the
LDH
is
indeed
a
tetramer.


 Molecular
modelling
was
then
used
to
determine
the
distances
between

the
various
residues
and
substrates
in
the
active
site
to
verify
the
proposed
LDH

catalysis
mechanism.
The
structure
used
for
this
analysis
was
the
M
subunit
of

the
M4
isozyme
obtained
from
Protein
Data
Bank
of
the
Brookhaven
National

Lab.


 With
these
analytical
tools,
it
was
assumed
that
LDH
M4
isozyme
that
had

been
isolated
previously
and
purified
could
be
characterized
to
in
order
to

understand
the
enzyme
action
more.



Experimental
Procedures:

A.
Reconstitution
of
lyophilized
sample


 The
isolated
LDH
samples
4+5,
diluted
10X
with
the
ΔA340/min
of
0.1689

was
lyophilized
to
conserve
enzyme
activity
during
storage.
The
volume
of
the

sample
4+5
was
1.41mL.
Given
the
dilution
factors
and
the
absorbances,
the

enzyme
activity
was
calculated
to
be
65.25
units
of
enzyme/mL
(KC.
2011).


 After
storage,
the
enzyme
was
restored
by
adding
half
the
original

volumes
to
the
sample,
705μL
of
deionized
water
for
sample
4+5
a
week
prior
to

actual
use
to
allow
all
of
the
solidified
masses
to
go
into
solution.
Vortexing
or

shaking
was
avoided
to
conserve
enzyme
structures
and
thus
enzyme
function.



 During
the
experiment,
half
of
the
volume
of
the
reconstituted
sample

was
taken,
i.e.,
353.5μL
of
4+5
and
an
equal
volume
of
the
deionized
water
was

added
into
this
sample
to
get
to
the
original
concentration.
The
sample
was
then

diluted
1:10
and
its
enzyme
assay
was
conducted
to
record
its
ΔA340/min
to

ensure
that
the
activity
had
not
changed.
The
ΔA340/min
of
the
reconstituted
4+5

sample
was
0.1326,
which
gives
the
enzyme
activity
of
63.96
units/mL
(KC.

2011)

Thus,
sample
4+5
was
used
to
conduct
the
kinetics
experiments.



B.
Kinetics


 The
reconstituted
LDH
M4
sample
4+5,
with
ΔA340/min
of
0.1326
was

used
to
conduct
an
enzyme
assay
with
varying
concentrations
of
pyruvate

present
in
them.
The
absorbances
measured
were
to
be
used
to
plot
the

Michealis‐Menten,
Lineweaver‐Burke
and
Eadie‐Hofstee
plots
and
thus
calculate

the
Vmax
and
Km
values
from
each
of
these
plots.
The
Michaelis‐Menten
plot
is

also
used
to
describe
whether
there
is
substrate
(pyruvate)
inhibition
present
in

this
isozyme
or
not.
Table
1
lists
the
volumes
of
substrate,
enzyme
and
LDH
used

to
perform
the
assay.























 2

Table
1:
The
volumes
of
reactants
in
the
enzyme
assay
(3mL)

Vol of
22.7mM Vol of KPhos Vol of NADH
[Pyruvate] in Pyruvate buffer, pH 7 (5mg/mL) Vol of LDH
assay (mM) (µL) (µL) (µL) (µL)
0.025 3.30 2936.7 50 10
0.050 6.60 2933.4 50 10
0.10 13.2 2926.8 50 10
0.25 33.0 2907.0 50 10
0.50 66.1 2873.9 50 10
1.0 132 2807.8 50 10
2.5 330 2609.6 50 10
5.0 661 2279.2 50 10


The
Vmax
and
Km
values
for
the
muscle
LDH
isozyme
were
then

compared
with
that
of
the
pre‐obtained
data
for
the
heart
LDH
isozyme
using
the

three
above‐mentioned
plots
to
account
for
differences.


 The
exploration
kinetics
study
was
extended
by
observing
the
effects
of

the
pesticide
kepone
(chlordecone)
on
LDH
activity.
A
constant
pyruvate

concentration
of
0.10mM
was
used
to
conduct
an
enzyme
assay
against

increasing
kepone
concentrations.
The
absorbance
data
obtained
from
the

assays
were
plotted
to
give
a
velocity
versus
kepone
concentration
graph
to

determine
the
type
of
inhibition
presented
by
kepone.




Fig
1:
The
structure
of
Kepone



Table
2:
The
volumes
of
reactants
in
the
enzyme
assay
for
Kepone
(3mL)

Kphos Vol of
buffer, 95% 22.7mM 0.01M Vol NADH
pH 7, ethanol Pyruvate pyruvate Kepone (5mg/mL)
Tube (mL) (µL) (mM) (µL) [Kepone] (µL) (µL)
1 2.937 0 0.1 13.2 0 0 50
2 2.637 300 0.1 13.2 0 0 50
3 2.637 298.5 0.1 13.2 0.005 1.5 50
4 2.637 297 0.1 13.2 0.01 3 50
5 2.637 292.5 0.1 13.2 0.025 7.5 50
6 2.637 285 0.1 13.2 0.05 15 50
7 2.637 270 0.1 13.2 0.1 30 50
8 2.637 225 0.1 13.2 0.25 75 50
9 2.637 150 0.1 13.2 0.5 150 50
10 2.637 0 0.1 13.2 1 300 50










 3

C.
Molecular
weight
determination

1.
SDS
Gel
Electrophoresis


 SDS‐PAGE
was
performed
on
the
pure
sample
4+5
to
calculate
the
value

of
the
subunit
weight
of
M
by
comparing
the
electrophoretic
mobility
of
the
pure

sample
band
to
that
of
the
standard
protein
samples.

SDS‐PAGE
(sodium

dodecyl
sulfate
polyacrylamide
gel
electrophoresis)
separates
protein
subunits

based
on
their
size,
which
can
then
provide
us
with
information
about
their

molecular
weight.
SDS
is
an
anionic
detergent
that
gives
the
protein
a
uniform

negative
charge
by
covering
it,
so
they
subunits
move
towards
the
anode
through

the
gel,
thereby
separating
through
their
mass.
SDS
is
able
to
do
so
by

dissociating
the
native
proteins
into
their
linear
forms
of
subunits
with
the
help

of
reducing
agents
like
dithiothreotol
(DTT)
that
disrupts
the
disulfide
bridges.

The
acrylamide
gel
used
acts
as
a
porous
sieve
that
lets
the
smaller
molecules

travel
faster
than
the
larger
ones,
thus
the
bands
that
travel
the
farthest
in
the

gel
are
the
lighter
protein
masses.


 The
crude,
dialyzed
and
pure
samples
were
run
in
this
SDS‐PAGE
along

with
sigma
LDH,
NE
Biolab
protein
standards
and
bovine
serum
albumin
(BSA)

at
~140V.
A
3X
reducing
sample
SDS
sample
buffer
(RSB)
containing
10μL
of
30X

reducing
agent,
DTT
to
100μL
of
3X
SDS
sample
buffer
that
contains
bromphenol

blue,
tracking
dye,
SDS
and
glycerol
to
make
the
sample
heavier.
The
gel
was

loaded
in
the
sequence
shown
in
table
2.



Table
3:

The
volumes
of
samples,
water
and
RSB
added
in
each
lane
in
the
gel.

Sample
volume Deionized
Lane (µL) RSB (µL) water (µL)
1 Blank - 5 -
Crude
2 (~1mg/mL) 1 10 19
3 Sigma LDH 6 10 14
NE Standard
4 I 20 - -
5 Pure (4+5) I 20 - 10
BSA
6 (1mg/mL) 4 10 16
Pure
7 (4+5)II 20 - -
NE Standard
8 II 25 10 -
ASPD
9 (~1m/mL) 1 10 19
10 Blank - 5 -



 About
23μL
of
each
sample
was
loaded
into
their
respective
lanes
and
the

gel
was
run
until
the
dye
was
about
0.5cm
from
the
bottom
of
the
gel.
The
gel

was
then
stained
with
Coomassie
blue
stain
for
about
half
an
hour,
rinsed
with

water
and
then
destained
twice
for
15
minutes
for
each
destaining
process.
The

gel
was
then
photographed
and
the
bands
in
each
lane
measured
for
the

distances
that
they
had
traveled
from
the
wells.



 4


 The
NE
standards
had
their
BSA
and
triosephosphate
isomerase
(TPI)

present
in
double
the
concentration
than
other
standard
proteins,
because
it
is

expected
that
the
molecular
weight
of
the
subunit
will
be
within
this
range
and
it

is
easier
to
determine
the
identity
of
each
band
through
lane
6
BSA.
Through
the

measurement
of
the
distances
that
the
protein
units
seen
as
bands
have
traveled

and
the
total
distance
traveled
by
the
dye
itself,
an
Rf
calculation
was
conducted

using
the
formula,
Rf=
(distance
of
protein
migration)/(distance
of
tracking
dye

migration).
A
standard
curve
was
then
generated
using
the
two
NE
standard
Rf

data
to
extrapolate
the
weight
of
LDH
subunit
using
the
equation
generated
from

the
line
of
best
fit
of
the
log
of
standard
molecular
weight
versus
Rf
plot.
This

was
expected
to
give
the
subunit
molecular
mass
for
M.




2.
Size
exclusion
chromatography
by
HPLC


 Data
for
size
exclusion
chromatography
was
obtained
in
order
to

determine
the
total
molecular
weight
of
the
LDH
tetramer,
and
to
confirm
the

fact
that
the
enzyme
is
indeed
a
tetramer.
Size
exclusion
chromatography
or
gel

filtration
chromatography
is
can
separate
molecules
of
different
sizes
through

their
differential
diffusion
into
the
gel
pores.
The
larger
molecules
do
not
enter

the
pores
of
the
packing
gel
unlike
the
smaller
ones,
and
are
eluted
faster

through
the
fluid
volume
as
a
result.



 Thus,
the
smaller
molecules
spend
more
time
passing
through
the

stationary
phase
packing
material
of
spherical
gel
beads
of
hydrophilic
bonded

phase
silica
in
the
column;
while
the
large
molecules
travel
easily
through
the

mobile
phase.
HPLC
was
used
to
speed
up
this
elution
process
so
that
microliter

quantities
of
protein
injected
into
the
column
would
elute
through
the
column

faster
and
be
detected
by
the
UV
detector
at
280nm.



 20μL
of
standard
LDH
sample
was
injected
into
the
column
and
the

absorbance
was
recorded
for
15
minutes.
The
same
was
done
with
20μL
of
LDH

sample.
The
standard
plot
of
log
of
molecular
weights
versus
retention
time
of

the
given
standard
protein
masses
were
used
to
calculate
the
molecular
weight

of
LDH
through
the
given
data
for
LDH
retention
time.



 The
molecular
mass
for
LDH
tetramer
obtained
from
this

chromatography
can
be
compared
with
that
of
the
M
subunit
obtained
from
SDS‐
PAGE
to
confirm
that
LDH
is
in
fact
a
tetramer.
Table
3.
Shows
the
data
obtained

for
the
standard
protein
size‐exclusion
chromatography.



Table
4:
HPLC
protein
standard
data

Log of
Molecular molecular
Protein Rt/min weight weight
Thyroglobulin 7.28 670,000 5.82607
Bovine gammaglobulin 8.018 158,000 5.19866
Rabbit ovalbumin 8.938 44,000 4.64345
Bovine myoglobulin 9.56 17,000 4.23045
Cyanocobalamine 11.738 1,350 3.13033










 5

D.
Molecular
modeling


 The
M
polypeptide
chain
of
LDH
M4
isozyme
from
Brookhaven
National

Lab
obtained
via
Protein
Data
Bank
was
used
to
study
the
possible
mechanism
of

action
of
LDH
using
molecular
modeling.
The
structure
of
LDH
was
studied
and

the
distance
between
amino
acid
residues
and
substrates
were
measured
to

understand
the
mode
of
LDH
action.


 The
flexible
loop
that
enfolds
the
substrate
and
coenzyme
in
the
active

site,
made
of
residues
#98‐120,
was
identified
and
each
of
the
amino
acid
was

listed
down.
Distances
between
other
various
active
site
residues
and
functional

groups
present
in
the
coenzyme
NADH
and
the
substrate
analog
oxamate
were

also
measured
to
study
the
interactions
within
the
active
site.
The
substrate

analog
oxamate
was
used
instead
of
pyruvate
to
arrest
the
enzyme
to
prevent

further
conformational
changes
that
lead
towards
product
release.






Fig
2:
Pyruvate
and
oxamate
structures





Results
and
Discussion:

A.
Reconstitution
of
lyophilized
sample


 Prior
to
lyophilisation,
the
ΔA340/min
for
sample
4+5
was
0.1689
for
a

1:10
diluted
sample.
Thus,
giving
the
enzyme
activity
of
81.45
units
of

enzyme/mL.
When
this
lyophilized
sample
was
reconstituted
by
addition
of

deionized
water,
the
ΔA340/min
obtained
for
the
same
dilution
was
0.1326,

which
gave
the
enzyme
activity
of
63.96
units/mL.
The
percentage
of
activity

recovered
after
reconstitution
was
78.53%.



 It
shows
that
although
lyophilisation
is
a
useful
technique
to
store
and

recover
enzyme
activity
it
is
not
a
complete
activity
conservation
technique.
As

preserving
the
proteins
in
a
stable
form
is
necessary
for
their
efficacy,
various

optimization
methods
in
addition
to
lyophilisation
should
be
carried
out.
An

experiment
showed
that
polyethylene
glycol
(PEG)
and
lactose
had
a
stabilizing

effect
on
the
LDH
during
the
screening.
The
experiment
also
showed
that
the

temperature
of
freezing
and
rates
of
sublimation
also
affected
the
relative

stability
of
the
protein
structure
(Grant
et.al.
2009).

Another
experiment
showed

that
addition
of
PEG
8000
successfully
recovered
90%
of
enzyme
activity
after

LDH
reconstitution,
unlike
the
60‐80%
standard
activity
recovery
of
LDH
after

lyophilization
(Mi
and
Wood.
2004).
Thus,
as
the
percentage
recovery
falls
under

the
range,
the
simple
lyophilization
was
considered
to
be
successful
enough
in

this
case.








 6

B.
Kinetics


 From
the
analysis
of
kinetics
of
LDH
M4
isozyme
of
rabbit
muscle,
the

following
graphs
for
Michaelis‐Meten,
Lineweaver‐Burke
and
Eadie‐Hofstee

were
prepared.

Similar
graphs
were
made
for
the
preobtained
data
for
the
H4

rabbit
isozyme
of
LDH.
The
Km
and
Vmax
were
calculated
from
each
of
these

graphs
for
the
two
isozymes
and
listed
under
table
5.





Graph
1:
Michaelis
Menten
for
muscle
LDH
(M4)

0.02500


Vmax


0.02000

V
as
D
[NADH]/min
(mM/min)


0.01500


0.01000


0.00500

Km


0.00000

0
 1
 2
 3
 4
 5

[pyruvate]
mM







 7

 250.00

Graph
2:
Lineweaver­Burke
LDH
muscle
(M4)


200.00


150.00

1/V



y
=
4.9884x
+
29.417

100.00
 R²
=
0.99868


50.00


0.00

0
 5
 10
 15
 20
 25
 30
 35
 40
 45

1/[S]
mM­1







Graph
3:
Eadie­Hofstee
Muscle
LDH
(M4)

0.02500


0.02000

mM
NADH/min


0.01500


0.01000


y
=
‐0.1417x
+
0.03

0.00500
 R²
=
0.98538


0.00000

0
 0.02
 0.04
 0.06
 0.08
 0.1
 0.12
 0.14
 0.16
 0.18
 0.2

V/[S]





 8

 
Graph
4:
Michaelis
Menten
for
LDH
heart
(H4)

0.03000


V
max


0.02500


0.02000

V
as
D
[NADH]/min


0.01500


0.01000


0.00500

Km


0.00000

0
 1
 2
 3
 4
 5
 6
 7
 8
 9
 10
 11

[pyruvate]
mM







 9

 Graph
5:
Lineweaver
LDH
heart
(H4)

80


70
 y
=
0.3774x
+
35.148

R²
=
0.99707

60

1/V
mM­1
min


50


40


30


20


10


0

0
 5
 10
 15
 20
 25
 30

1/
[pyruvate]
mM­1






0.03

Graph
6:
Eadie­Hofstee
LDH
Heart
(H4)


0.025


0.02

mM
NADH/min


y
=
‐0.0107x
+
0.0284

R²
=
0.99577

0.015


0.01


0.005


0

0
 0.1
 0.2
 0.3
 0.4
 0.5
 0.6
 0.7

v/[S]
min­1
 




 



 10

Table
5:
A
comparative
kinetics
data
for
different
plots
for
the
two
isozymes
of
LDH


Michaelis- Lineweaver-
Menten Burke Eadie-Hofstee
Muscle LDH
V max (mM/min) 0.023 0.034 0.030
Km (mM) 0.09864 0.1696 0.1417
Heart LDH
V max (mM/min) 0.028 0.028 0.028
Km (mM) 0.0114 0.0107 0.0107



 Earlier
studies
show
that
the
Km
values
for
the
M4
and
H4
LDH
isozymes

were
observed
to
be
0.35mM
and
0.10mM
respectively
(Stambaugh
and
Buckley.

1967).

The
experimental
values
however
were
much
lower
than
the
expected

literature
values
suggesting
that
the
enzyme
was
more
active
than
expected.
The

average
Km
for
M4
isozyme
is
0.1038mM
compared
to
the
0.35mM
and
that
for

H4
is
0.0107mM
compared
to
0.10mM.
This
might
have
been
the
case
because

the
enzyme
active
site
might
have
been
more
accessible
during
the
structural

destabilization
that
occurred
during
and
after
lyophilization.



 Like
the
LDH
heart
data,
LDH
muscle
followed
Michaelis‐Menten
kinetics

from
the
pyruvate
concentrations
of
0.025mM
to
0.50mM
after
which
it
showed

the
effects
of
negative
feedback
generated
due
to
the
larger
production
of
the

product
lactate.
This
shows
the
regulatory
mechanisms
that
are
present
in
the

metabolic
cycles
in
living
beings
by
which
pyruvate
inhibits
the
dehydrogenase

(Zubay.
1988).


 Similarly,
heart
LDH
data
also
followed
Michaelis‐Menten
kinetics
until

0.513mM.
After
that
concentration
of
pyruvate,
the
curve
slopes
downward

instead
of
reaching
the
saturated
plateau
level
due
to
feedback
mechanisms

induced
by
lactate
formation.



 These
isozymes
have
different
kinetics,
as
they
are
present
in
different

tissue
types
that
have
different
energy
requirements
and
level
of
pyruvate

production
and
use.
The
muscle
is
active
and
hence
anaerobic,
so
produces
a
lot

of
pyruvate
that
is
converted
into
lactate.
Thus,
M4
shows
less
substrate

inhibition
than
H4
isozyme.

The
heart
LDH
is
present
in
the
aerobic
heart

tissues,
and
most
of
the
pyruvate
goes
into
Kreb’s
cycle
to
produce
more
energy,

and
hence
LDH
more
readily
inhibited
by
the
pyruvate
(Zubay.
1988).


 The
analysis
of
LDH
M4
kinetics
in
presence
of
the
pesticide
kepone
is

given
below.



Table
6:
Data
for
the
change
in
velocity
of
the
reaction
with
increasing
Kepone

concentrations

[Kepone] V in
mM ΔA/min mM/min
0 0.0919 0.01477
0.0050 0.0682 0.01096
0.010 0.0659 0.01059
0.025 0.0436 0.00701
0.050 0.0223 0.00359
0.100 0.0177 0.00285

 



 11

 Graph
7:
Effect
of
increasing
[Kepone]
on
LDH

activity

0.016


0.014


0.012

V
in
mM/min


0.01


0.008


0.006


0.004


0.002


0

0
 0.02
 0.04
 0.06
 0.08
 0.1

[Kepone]
mM





From
graph
7
it
is
seen
that
Kepone
competitively
inhibits
the
enzyme

LDH
at
concentrations
even
as
low
as
0.005mM.
Although
structurally
Kepone
is

distinctly
different
from
pyruvate,
it
successfully
prevents
the
oxidation
of
NADH

by
LDH
muscle
isozyme.
Increasing
Kepone
concentrations
is
also
seen
to

drastically
reduce
the
enzyme
activity
due
to
the
competitive
nature
of
the

inhibition
induced.




C.
Molecular
weight
determination

1.
SDS
Gel
Electrophoresis

The
results
from
SDS‐PAGE
and
size
exclusion
chromatography
by
HPLC

were
used
to
determine
the
total
molecular
weight
of
the
LDH
enzyme
as
well
as

the
each
of
the
subunit
weights
for
the
enzyme.
SDS‐PAGE
was
conducted
and
a

standard
curve
was
generated
to
calculate
the
mass
of
each
of
the
LDH
subunits.

Size
exclusion
chromatography
by
HPLC
was
conducted
to
calculate
the
total

molecular
weight
of
the
LDH
enzyme
and
in
doing
so
it
was
used
to
determine

that
the
enzyme
consists
of
four
subunits
as
anticipated.



 The
subunit
weight
LDH
M4
isozyme
was
expected
to
be
35,000
Da
as

described
by
earlier
SDS‐PAGE
analysis
(Javed,
et.al.
1995).
Another
study

suggests
that
the
subunit
molecular
weight
is
conserved
among
different
species

(Fosmire
and
Timasheff.
1972).



 During
the
analysis
of
the
SDS‐PAGE
data,
the
BSA
sample
had
Rf
of

0.2222
(1.0cm)
and
0.4667
(2.10cm),
so
the
0.2222
Rf
was
omitted
in
both
the

standard
and
the
BSA
data.
Thus,
the
standard
curve
of
graph
8
and
the
equation

of
the
line
generated
were
used
to
calculate
the
subunit
weight
of
M
for
the
LDH

muscle
M4
isozyme
as
34,906
Da.





 12

Table
7:
The
distance
travelled
by
each
band
in
the
lanes
3­8
and
the
calculated
Rf

Dye migration = 4.5cm
Sigma Standard Pure Standard Pure
LDH/cm 1/cm LDH/cm BSA/ cm 2/cm LDH/cm
3.3 0.8 3.3 1 0.8 3.3
3.5 1 3.5 2.1 1 3.5
1.2 1.2
1.4 1.45
1.6 1.6
2.1 2.1
2.45 2.5
2.9 2.9
3.5 3.5
4.15 4.15

Dye migration = 4.5cm, Rf = band migration/dye migration

Sigma Standard 1 Pure LDH Standard
LDH Rf Rf Rf BSA Rf Rf Pure Rf
0.7333 0.1778 0.7333 0.2222 0.1778 0.7333
0.7778 0.2222 0.7778 0.4667 0.2222 0.7778
0.2667 0.2667
0.3111 0.3222
0.3556 0.3556
0.4667 0.4667
0.5444 0.5556
0.6444 0.6444
0.7778 0.7778
0.9222 0.9222





Figure
3:
The
SDS­PAGE
gel
for
LDH
migration
with
standards



 13

 Graph
8:
The
standard
SDS­PAGE
curve
showing

the
line
of
best
fit
from
BSA
to
TPI


6.0000


5.0000


4.0000

y
=
‐0.8579x
+
5.2102

Log
of
MW


R²
=
0.98908

3.0000


2.0000


1.0000


0.0000

0
 0.1
 0.2
 0.3
 0.4
 0.5
 0.6
 0.7
 0.8
 0.9
 1

Rf





2.
Size
exclusion
chromatography
by
HPLC




Graph
9:
Standard
curve
for
size
exclusion

chromatography
by
HPLC

7.00000


6.00000

Log
of
molecular
weight


5.00000


4.00000


3.00000

y
=
‐0.5936x
+
10.012

2.00000
 R²
=
0.98942


1.00000


0.00000

7
 7.5
 8
 8.5
 9
 9.5
 10
 10.5
 11
 11.5
 12

Retention
time
(Rt)
in
min







 14

 

Figure
4:
The
peaks
for
size
exclusion
chromatography
by
HPLC



As
the
size
exclusion
chromatography
by
HPLC
calculations
gives
the
total

molecular
weight
of
LDH
(4
subunit)
to
be
140,605
Da,
the
Rf
value
of
0.7778

with
the
subunit
molecular
weight
of
34,906
Da
and
the
one
with
0.7333,
giving

38,114.5
must
be
the
accurate
migration
bands
of
the
LDH
subunits.
As
the
band

with
the
Rf
0.7778
was
much
darker
than
the
one
at
0.7333,
it
can
be
assumed

that
the
heavier
variant
of
the
subunit
is
present
at
a
lower
concentration
than

the
lighter
one.
Thus,
the
LDH
subunit
of
molecular
mass
34,906
Da
can
be



 15

regarded
as
the
value
of
each
of
the
LDH
subunit
mass,
giving
4
X
34,905
=

139620
Da
as
the
anticipated
total
LDH
mass
which
is
comparable
to
140,605
Da

from
size
exclusion
data.
Previous
studies
give
the
total
molecular
weight
of
LDH

as
142,000
Da
(Fosmire
and
Tinasheff.
1972).


Hence,
it
can
be
assumed
that
both
of
the
experimental
procedures
were

used
efficiently
to
calculate
the
total
molecular
weight
and
subunit
molecular

weight
for
LDH.




D.
Molecular
modeling


 The
residues
of
the
active
site
were
labeled
and
determined
via
Protein

Data
Bank
viewer
for
the
M4
LDH
isozyme
as
listed
in
table
12.
The
M4
structure

of
interest
was
the
M
polypeptide
chain
with
NADH
and
the
pyruvate
analog,

oxamate.
The
structure
and
the
mode
of
action
of
the
active
site
on
each
of
the

subunits
of
LDH
are
known
to
be
conserved
while
the
amino
acid
resides
are

known
to
be
different
among
different
LDH
samples.
It
has
a
bound
NADH
in
its

active
site
as
the
coenzyme
that
is
oxidized
to
NAD+
during
the
conversion
of

pyruvate
to
lactate
in
a
reaction
that
is
strongly
favoured
in
the
forward

direction.
In
this
reaction
the
central
carbon
of
pyruvate
(‐C=O)
is
known
to

undergo
change
from
sp2
conformation
to
an
sp3
conformation
of
lactate.



Fig
5:
NAD+




 The
LDH
enzyme
active
site
is
known
to
bind
the
NADH
before
the

binding
of
the
actual
substrate
pyruvate.
The
C‐4
atom
of
the
nicotinamide
ring
is

accessible
to
associations
with
the
reactive
carbon
of
pyruvate.
As
the
enzyme

binds
the
NADH,
it
undergoes
conformation
change
visible
through
the

association
of
the
adenine
part
of
the
ring
and
Asp
53.
The
distance
was
found
to

be
3.526
Å
for
Asp
52
and
the
ribose
part
of
the
adenine
ring
showing
that
the

binding
of
NADH
to
the
enzyme
brings
the
conformational
change,
pulling
the

amino
acid
residues
closer
to
the
NADH.
Similarly,
Arg
101
forms
an
ion
pair

with
the
pyrophosphate
group
of
NADH.
The
distance
between
–NH2
of
Arg
99

and
the
nearest
oxygens
of
the
phosphate
in
NADH
was
found
to
be
2.978
Å
and



 16

4.706
Å.
This
ion
pair
formation
is
regarded
as
the
main
association
in
the
mobile

loop
that
brings
about
further
conformational
changes
in
the
active
site
that

promotes
the
enzyme
activity
on
pyruvate.
The
mobile
loop
movement
functions

a
lid
with
two
hinges
that
closes
down
the
active
site
and
begins
the
enzymatic

reactions.
The
movement
of
the
mobile
loops
brings
about
a
conformational

change
of
around
11
Å.
Also,
Asp
30
is
known
to
hold
the
NADH
in
place.


 In
the
active
site,
Tyr
85
is
known
to
act
with
the
adenine
of
the
NADH.

The
distance
measured
here
was
4.006
Å
for
Tyr
83
and
of
the
–OH
group
in
Tyr

and
the
N,
next
to
–NH2
in
the
adenine
ring.
Showing
that
there
is
indeed
a
close

interaction
between
the
two
that
is
not
as
strong
as
the
other
mobile
loop

interactions
with
NADH.



 Arg
171
is
known
to
hold
the
pyruvate
in
place
through
association
with

the
carboxylic
acid
or
pyruvate.
Here,
a
pyruvate
analog,
oxamate
is
used
to

arrest
the
enzyme
in
a
position
with
its
mobile
loop
closed
but
without
the

occurrence
of
any
forward
reaction
that
leads
to
product
formation.
Arg
169
–
NH2
was
found
to
be
3.141
Å
and
2.797
Å
from
the
oxygen’s
of
the
COO‐
part
of

oxamate.



 Association
of
Arg
109
–NH2
with
the
central
carbonyl
oxygen
of
pyruvate

was
also
an
indicator
of
the
substrate
being
in
association
with
the
active
site.

The
distance
between
Arg
106
and
the
central
oxygen
was
found
to
be
2.866
Å.



 His
195
N
in
the
ring
and
the
central
carbonyl
oxygen
of
pyruvate
were

also
known
to
interact
during
the
enzyme
action.
The
distance
was
measured
to

be
2.702
Å
from
His
193.



 There
was
also
association
seen
between
the
NADH
and
the
pyruvate

analog
as
shown
by
the
distance
between
the
C4
of
the
nicotinamide
ring
of

NADH
and
the
central
carbon
of
oxamate.
The
distance
was
found
to
be
3.201
Å.



 As
the
substrate‐binding
pocket
lies
deep
in
the
enzyme
active
site,

making
the
site
accessible
to
pyruvate
is
thus
done
through
the
association
of

NADH
with
the
mobile
loop
residues
that
allow
the
pyruvate
to
first
enter
and

the
hinge
the
active
site
close
once
the
pyruvate
is
held
by
the
active
site

residues.
In
the
active
site
the
pyruvate
is
in
association
with
the
catalytically

important
His
195
and
Arg
109
that
stabilize
the
polar
transition
states.
Arg
171

in
the
site
solvates
the
negatively
charged
carboxyl
group
of
pyruvate.



 Association
of
NADH
with
the
enzyme
activates
the
mobile
loop
(residues

98‐110)
causes
the
closing
of
the
loop
over
the
active
site
and
Arg
109
is
brought

in
close
association
with
the
substrate,
while
water
is
expelled
and
pyruvate
and

NADH
are
brought
close
together
to
form
an
association
that
leads
to
catalysis

and
lactate
formation
(McClendon
et.al.
2005).



 The
measured
distances
between
active
site
and
mobile
loop
residues

with
the
coenzyme
and
the
substrate
are
all
within
2‐4
Å

indicating
that
there

are
indeed
associations
among
the
residues
and
the
NADH
and
pyruvate
along

with
the
association
of
NADH
and
pyruvate
themselves.




 17

 

Fig
6:
The
active
site
of
the
enzyme
LDH
with
bound
oxamate



Conclusion:


 The
experimental
procedure
followed
a
sequential
characterization
of
the

rabbit
muscle
(M4)
isozyme.
Although
not
optimized,
the
reconstitution
of
the

lyophilized
sample
gave
a
78.53%
recovered
sample
that
was
considered
as
the

pure
sample
of
LDH
with
an
activity
of
63.96
units
of
enzyme/mL.



 The
data
for
kinetic
studies
of
the
two
isozymes,
M4
and
H4
were

conducted
using
enzyme
assays
and
three
kinetic
curves,
Michealis‐Menten,

Eadie‐Hofstee
and
Lineweaver
Burke.
The
average
Km
for
M4
was
found
to
be

0.1038mM
and
for
H4
was
0.0107mM.
The
values
for
Km
were
much
lower
than

anticipated,
which
might
be
the
possible
result
of
destability
during
not

optimized
lyophilization
procedures
that
make
the
active
site
more
accessible
to

the
substrate
or
due
the
fluctuations
in
pH
and
temperature
(Place
and
Powers.

1984).

 


 Kepone
on
the
other
hand,
as
anticipated
showed
kinetics
of
competitive

inhibition,
starting
at
concentrations
as
low
as
0.005mM.



 SDS‐PAGE
and
size
exclusion
chromatography
gave
the
subunit
molecular

weight
and
total
tetramer
weight
of
of
LDH
M4
to
be
34,906Da
and
140,605Da

respectively.
These
experimental
data
were
consistent
with
prior
obtained
data.


 Through
molecular
modeling
the
enzyme
was
seen
to
efficiently
transfer
a

hydride
ion
from
the
pro‐R
face
of
the
reduced
nicotinamide
group
of
NADH
to

the
central
carbonyl
carbon
of
pyruvate
to
turn
it
to
lactate.
Initial
association
of

the
enzyme
active
site
was
seen
to
be
present
before
the
association
of
the



 18

substrate
with
the
active
site
residues.
The
distances
measured
were
within
4
Å

and
showed
that
Tyr
85,
Asp
53
and
Arg
101
hold
the
NADH
in
place,
while,
Arg

169,
Arg
109
and
His
195
held
the
pyruvate
analog.
There
was
also
an

association
observed
between
the
NADH
and
the
pyruvate.
Thus,
the
mobile
loop

was
seen
to
undergo
conformational
changes
that
enclosed
the
enzyme
active

site
and
brought
the
coenzyme
and
the
substrate
close
together
to
undergo

catalysis.



References:



Fosmire,
G.
and
Timasheff,
S.
N.
1972.
Molecular
weight
of
beef
heart
lactate


dehydrogenase.

Biochemistry.
11
(13):
2455‐2460

Grant,
Y.,
Matejtschuck
P.
and
Dalby,
P.A.
2009.
Rapid
optimization
of
protein


freeze‐drying
formulations
using
ultra
scale‐down
and
factorial
design
of

experiment
in
microplates.
Biotechnology
and
Engineering.
105
(5):
957‐
964


Hendrickson,
C.M.
and
Bowden,
J.A.
1975.
The
in
vitro
inhibition
of
rabbit
muscle


 lactate
dehydrogenase
by
Mirex
and
Kepone.
J.
Agric.
Food
Chem.
23
(3):

407‐409

Javed,
M.,
Yousuf,
F.A.,
Hussain,
A.
N.,
Ishaq,
M.
and
Waqar,
M.
A.
1995.


Purification
and
properties
of
lactate
dehydrogenase
from
liver
of

Uromastix
hardwickii.
Comp.
Biochem.
Physiol.
111B
(1):
27‐34.

KC,
Angela.
2011.
Isolation
and
purification
of
the
enzyme
lactate


dehydrogenase.
3‐9.

McClendon,
S.,
Zhadin,
N.
and
Callender,
R.
2005.
The
approach
to
the
Michaelis


complex
in
lactate
dehydrogenase:
The
substrate
binding
pathway.

Biophys
J.
89
(3):
2024‐2032

Mi,
Y.
and
Wood,
G.
2004.
The
application
and
mechanisms
of
polyethylene
glycol


8000

on
stabilizing
lactate
dehydrogenase
during
lyophilization.
PDA
J.

Pharm.
Sci.
Technol.

58
(4):
192‐202

Place,
A.R.
and
Powers,
D.A.
1984.
Purification
and
characterization
of
the
lactate


dehydrogenase
(LDH‐B4)

allozymes
of
Fundulus
heteroclitus.
J.
Biol.

Chem.
259
(2):
1299‐1308

Place,
A.R.
and
Powers,
D.A.
1984.
Kinetic
characterization
of
the
lactate


dehydrogenase
(LDH‐B4)

allozymes
of
Fundulus
heteroclitus.
J.
Biol.

Chem.
259
(2):
1309‐1318

Sevnic,
A.,
Sari,
R.
and
Fadillioglu,
E.
2005.
The
utility
of
lactate
dehydrogenase


isoenzyme
pattern
in
the
diagnostic
evaluation
of
malignant
and


nonmalignant
ascites.
J
Natl
Med
Assoc.
97
(1):
79‐84.

Stambaugh,
R.
and
Buckley,
J.
1967.
The
enzymic
and
molecular
nature
of
lactic


dehydrogenase
subbands
and
X4
isozyme.
J.
of
Bio.
Chem.
242
(18):
‐
4059

Zubay,
G.
1988.
Biochemistry.
2




Supporting
information:



A.
Calculations
for
enzyme
activity
for
sample
4+5

Pre‐lyophilization:

ΔA340/min
=
0.1689



 19

Dilution
factor
=
10X

Corrected
ΔA340/min
=
1.689

ΔA/Δt
=
ε
b
(Δc/Δt)

1.689
=
6.22X
10^3
X
Δc

Δc=
2.715
X
10^‐4
Mmin‐1

LDH
in
assay
=
2.715
X
10^‐4
X
3.00
X
10^‐3
=
8.415
μmol
min‐1

LDH
enzyme
activity
=
81.45
units/mL



Post‐reconstitution:

ΔA340/min
=
0.1326

Dilution
factor
=
10X

Corrected
ΔA340/min
=
1.326

ΔA/Δt
=
ε
b
(Δc/Δt)

1.326
=
6.22X
10^3
X
Δc

Δc=
2.132
X
10^‐4
Mmin‐1

LDH
in
assay
=
2.132
X
10^‐4
X
3.00
X
10^‐3
=
6.396
μmol
min‐1

LDH
enzyme
activity
=
63.96
units/mL



Percentage
of
LDH
activity
recovered:

(63.96/81.45)
X
100%
=
78.53%



B.
Data
and
calculations
for
kinetics



Table
8:

The
data
for
LDH
muscle
isozyme


1/[S] V as
[S] mM (mM-1) ΔA/min ΔNADH/min 1/V V/[S]
0.025 40 0.027 0.00434 230.37 0.1736
0.050 20 0.049 0.00788 126.94 0.1576
0.10 10 0.081 0.01302 76.790 0.1302
0.25 4 0.118 0.01897 52.712 0.0759
0.50 2 0.144 0.02315 43.194 0.0463
1.0 1 0.141 0.02267 44.113 0.0227
2.5 0.4 0.124 0.01994 50.161 0.0080
5.0 0.2 0.106 0.01704 58.679 0.0034


For
Michaelis­Menten
of
muscle
LDH,

V=
Vmax
[S]/
[S]
+
Km

From
the
graph,
Vmax
=
0.02315
mM/min
~
0.023
mM/min

For
Km,
V=
0.01302mM/min
and
[S]=
0.10mM

V=
Vmax
[S]/
[S]
+
Km

0.01302=
0.02315
X
0.10
/
(0.10+
Km)

0.01302
Km
+
0.01302
X
0.10
=
0.02315
X
0.10

0.01302
Km
=
0.001302

Therefore,
Km
=
0.09864
mM




For
Lineweaver­Burke
data
of
muscle
LDH

The
equation
of
the
line
of
best
fit
is
y=
4.9884x
+
29.417
corresponding
to
the

equation
of
Lineweaver‐Burke
plot,

1/V
=
(1/Vmax)
+
(Km/Vmax
X
1/[S])



 20

Therefore,
y‐intercept
=
1/Vmax


Slope
=
Km/
Vmax
=
4.9884

X‐intercept
=
‐1/Km

Let,
y=
0
=
4.9884x
+
29.417

‐29.417
=
4.9884x

Or,
x‐intercept
=
‐1/Km
=
‐5.897
mM‐1

Or,
Km
=
0.1696
mM

Slope
=
4.9884
=
0.1696/Vmax

Or,
Vmax
=
0.033999
mM/min
~
0.034
mM/min



For
Eadie­Hofstee
Muscle
LDH

The
equation
of
the
line
of
best
fit
is,
y
=
­0.1417x
+
0.03,
where,

V
=
(‐Km
X
V)/[S]
+
Vmax

Slope
=
‐Km
=
‐0.1417

Or,
Km
=
0.1417
mM

Y
intercept
=
Vmax
=
0.03
mM/min



Table
9:
The
data
for
LDH
heart
isozyme


1/[S] V as
[S] mM (mM-1) ΔA/min ΔNADH/min 1/V V/[S]
0.036 27.778 0.136 0.02186 45.735 0.60736
0.055 18.182 0.148 0.02379 42.027 0.43262
0.092 10.870 0.160 0.02572 38.875 0.27960
0.256 3.9063 0.169 0.02717 36.805 0.10613
0.513 1.9493 0.173 0.02781 35.954 0.05422
1.03 0.9709 0.140 0.02251 44.429 0.02185
2.49 0.4016 0.113 0.01817 55.044 0.00730
4.97 0.2012 0.093 0.01495 66.882 0.00301
9.98 0.1002 0.084 0.01350 74.048 0.00135


For
Michaelis­Menten
of
heart
LDH,

V=
Vmax
[S]/
[S]
+
Km

From
the
graph,
Vmax
=
0.02781
mM/min
~
0.028
mM/min

For
Km,
V=
0.02379mM/min
and
[S]=
0.055mM

V=
Vmax
[S]/
[S]
+
Km

0.02379
=
0.02781
X
0.055
/
(0.055+
Km)

0.02379
Km
+
0.02379
X
0.55
=
0.02871
X
0.055

0.02379
Km
=
0.000271

Therefore,
Km
=
0.011375
mM
~
0.0114mM



For
Lineweaver­Burke
data
of
heart
LDH

The
equation
of
the
line
of
best
fit
is
y=
0.3774x+
35.148
corresponding
to
the

equation
of
Lineweaver‐Burke
plot,

1/V
=
(1/Vmax)
+
(Km/Vmax
X
1/[S])

Therefore,

y‐intercept
=
1/Vmax


Slope
=
Km/
Vmax
=
0.3774

X‐intercept
=
‐1/Km

Let,
y=
0
=
0.3774x+
35.148

‐35.148=
0.3774x



 21

Or,
x‐intercept
=
‐1/Km
=
‐93.132
mM‐1

Or,
Km
=
0.0107
mM

Slope
=
0.3774=
0.0107/Vmax

Or,
Vmax
=
0.02835
mM/min
~
0.028
mM/min



For
Eadie­Hofstee
heart
LDH

The
equation
of
the
line
of
best
fit
is
y
=
­0.0107x
+
0.0284,
where,

V
=
(‐Km
X
V)/[S]
+
Vmax

Slope
=
‐Km
=
‐0.0107

Or,
Km
=
0.0107
mM

Y
intercept
=
Vmax
=
0.0284
mM/min



B.
Data
and
calculations
for
molecular
weight
determination

Table
10:
The
calculated
log
of
molecular
weight
and
Rf
for
standard
protein

samples

Molecular
Standard weight
protein (Da) Log of MW Rf
Myosin 212,000 5.3263 0.1778

MBP-b-
galactosidase 158,194 5.1992 0.2667

B-
galactosidase 116,351 5.0658 0.3167
Phosphorylase
B 97,184 4.9876 0.3556
BSA 66,409 4.8222 0.4667

Glutamic
dehydrogenase 55,561 4.7448 0.5500
Maltose
binding 42,710 4.6305 0.6444
Thioredoxin
reductase 34,622 4.5394 0.7778
Triosephos
Isomerase 26,972 4.4309 0.9222


From
the
graph
8,
the
equation
of
the
line
of
best
fit
is,


y
=
‐0.8579x
+
5.2102,
where
y
=
log
of
MW
and
x
=
Rf

Given
that
Rf
for
LDH
are
0.7333
and
0.7778,
it
is
possible
to
calculate
the
log
of

the
LDH
subunit
MW
and
hence
the
molecular
weight.


Y
or
log
subunit
MW
(3.30cm)=
‐0.8579
X
0.7333
+
5.2102
=
4.5811

LDH
subunit
MW
=
38,115.4
Da

Y
or
log
subunit
MW
(3.50cm)=
‐0.8579
X
0.7778
+
5.2102
=
4.5429

LDH
subunit
MW
=
34,906
Da













 22

Table
11:
The
log
of
the
MW
and
Rf
for
size
exclusion
standards

Log of
Protein Molecular molecular
standards Rt/min weight weight

Thyroglobulin 7.28 670,000 5.82607

Bovine
gammaglobulin1 8.018 158,000 5.19866
Rabbit ovalbumin 8.938 44,000 4.64345
Bovine
myoglobulin 9.56 17,000 4.23045
Cyanocobalamine 11.738 1,350 3.13033


From
the
size
exclusion
chromatography
by
HPLC,
the
retention
time
(Rt)

for
the
LDH
sample
was
found
to
be
8.194
min.


From
the
standard
curve,
the
equation
of
the
line
was
obtained,


y
=
‐0.5936x
+
10.012,
where
y=log
of
molecular
weight
and
x
=
retention

time.


Hence,
y=
log
of
LDH
molecular
weight
=
‐0.5936
X
8.194
+
10.012
=

5.148

Or,
molecular
weight
of
LDH
=
10^
5.148
=
140,605
Da.



Table
12:
The
amino
acid
residues
of
the
LDH
active
site

Residue Amino Residue Amino
# acid # acid
98 Ala 109 Leu
99 Arg 110 Val
100 Gln 112 Gln
101 Gln 113 Arg
102 Glu 114 Val
103 Gly 115 Asn
104 Glu 116 Ile
105 Ser 117 Phe
106 Arg 118 Lys
107 Leu 119 Phe
108 Asn 120 Ile




 23