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UDEE 2134 ENZYMOLOGY 1

YEAR 2 SEMESTER 3

Name : Hemalatha Kannan

Student ID : 09ADB07509

Partners name: Mohana Maradamuthu

Sanmugapriya Elamparuthi

Suganthi Pakianathan

Mirohsha Mohan

Faculty : Science

Course : Biochemistry

Session : 2009/2010

Lecturer : Dr Chang

Date : 15th March 2011

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 Determination of malate dehydrogenase activity in plant tissues.cc

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1. To gain exposure of purifying an extracted enzyme.

2. To determine the activity of an enzyme that has been purified.

3. To know the function of malate dehydrogenase in TCA cycle

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Malate dehydrogenas (MDH) is an enzyme in the citric acid cycle that catalyzes the
conversion of malate into oxaloacetate (using NAD+) and vice versa (this is a reversible
reaction). Malate dehydrogenase is not to be confused with malic enzyme, which catalyzes
the conversion of malate to pyruvate, producing NADPH.

Malate dehydrogenase is also involved in gluconeogenesis, the synthesis of glucose

from smaller molecules. Pyruvate in the mitochondria is acted upon by pyruvate carboxylase
to form oxaloacetate, a citric acid cycle intermediate. In order to get the oxaloacetate out of
the mitochondria, malate dehydrogenase reduces it to malate, and it then traverses the inner
mitochondrial membrane. Once in the cytosol, the malate is oxidized back to oxaloacetate by
cytosolic malate dehydrogenase. Finally, phosphoenol-pyruvate carboxy kinase (PEPCK)
converts oxaloacetate to phosphoenol pyruvate

MDH is an oxidoreductase which utilizes the reduced form of nicotinamide adenine

dinucleotide (NADH) for reduction of oxaloacetate to L-malate. During this reaction, NADH
is oxidized to NAD. NADH exhibits absorbance maximum at 340 nm whereas at this
wavelength NAD has a negligible absorbance. Hence, the decrease in absorbance at 340 nm
due to the utilization of NADH provides a convenient parameter for following the progress of
the reaction catalysed by MDH'c

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Äc Plant material: Freshly harvested leaves

Äc Acid washed river sand: suspend the sand in I M HC1 and keep it dor several hours
with occasional stirring. Drain out the acid and wash it thoroughly with distilled water
until it is completely free of acid.
Äc Refrigerated centrifuge,
Äc UV-V IS spectrophotometer,
Äc 37°C water bath
Äc Extraction buffer: 50 mM Jmidazole-HCI buffer containing 10 mM dithiothreitol,
20mM MgCl2 and 2 mM EDTA.
Äc Assay buffer: 50 mM Tris-HCI, pH 8.0, containing I mM EDTA 2 ml of 6 mM
NADH in Tris-HC1 buffer, pH 8.0 ( store in ice)
Äc 2 ml of 0.3 M oxaloacetate in distilled water, neutralize by adding NaHCO3 until gas
bubbles ceased to evolve.
Äc Polyvinylpyrrolidone (PVP

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1)c Firstly, the enzyme extract was prepared by homogenized 0.5 g of leaves at 0 - 4°C in
a chilled mortar and pestle.
2)c After that, small amount of acid washed sand was used as an abrasive in the presence
of 1% PVP and 10 ml of ice-cold extraction buffer.
3)c The buffer was added small amount each time.
4)c Then, the leaf homogenate was centrifuged at 10, 000 x g for 20 minutes in a
refrigerated centrifuge.
5)c The supernatant was decanted slowly and was used as the enzyme preparation.
6)c The volume of the supernatant was note and kept in ice until the enzyme assay is
performed.
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1)c 1.5 ml of the assay buffer was pipette into a small test tube.
2)c Then, 0.1 ml of the enzyme extract was added to it and incubated in 37° C water bath
for 5 minutes.
3)c The entire content was pour into a quartz cuvette, followed by adding 50 µl of
NADH, mix by inversion and placed it immediately in the spectrophotometer set at
340 mn.
4)c The initial reading and the follow of decrease in absorbance at 15 seconds intervals of
up to 3 minutes was note. These readings serve as the control.
5)c After that, the above reaction was repeated by adding 50 µl of oxaloacetate and the
absorbance as above for 3 minutes was recorded.
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(NADH + Enzyme extract (NADH + Oxaloacetate +

(Abs Treatment ± AbsControl)
+ assay buffer)
assay buffer)

0.25 - 0.001 - 0.017 0.016

0.50 - 0.001 - 0.030 0.029

0.75 -0.002 - 0.040 0.038

1.0 -0.002 - 0.048 0.046

1.25 -0.003 - 0.057 0.054

1.5 -0.003 - 0.065 0.062

1.75 -0.003 - 0.075 0.072

2.0 -0.004 - 0.080 0.076

2.25 -0.004 - 0.088 0.084

2.5 -0.005 - 0.096 0.091

2.75 -0.005 - 0.100 0.095

3.0 -0.006 Out of range

 


According to Beer Lamber¶s Law,

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οܿ ൌ
ß݈
¨A = slope,

R = light path length through the cuvette (1 cm)

ß = molar extinction coefficient of NADH (R

 ൈ ͳͲଷ )

ǻc = change of concentration of product

ǻA = slope from the graph

= (0.07-0.036) / (1.8-0.67)

= 0.03 /min

The amount of NADH used per min in enzyme assay is:

From the graph,

¨ Abs/min = 0.03/min

ß of NADH = R

 ൈ ͳͲଷ

Ͳ Ͳ͵Ȁ݉݅݊
οܿ ൌ  ൌ Ͷ 
ൈ ͳͲ ି଺ ‫ܯ‬Ȁ݉݅݊
R

 ൈ ͳͲ ଷ ‫ܯ‬-1ܿ݉-1 ȉ ͳܿ݉

Convert it into (µmol/ml)/min,

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 ൈ ͳͲ ି଺ ‫ܯ‬Ȁ‹

ൌ Ͷ 
 ൈ ͳͲ ି଺ ሺ‘Ž‡ȀŽ‹–‡”ሻȀ‹

ൌ Ͷ 
ൈ ͳͲ ି଺ ൈ ͳͲ ି଺ ሺµ‘ŽȀŽ‹–‡”ሻȀ‹

ൌ Ͷ 
ൈ ͳͲ ିଵଶ ሺµ‘ŽȀŽሻȀ‹

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Conversion into unit/ml,

= (4.82 x 10-12 (µmol/ml)/min) / 0.1 ml

= 4.82 x 10-11 unit/ml

Volume of the extraction enzyme = 4.4 ml.

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Weight of fresh leaves used = 0.5 g

Conversion into unit/ml,

= (4.82 x 10-12 (µmol/ml)/min) / 0.5 g

= 9.64 x 10-12 ȝmol/min/g

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In control, the slope of the graph is equivalent to 0. Therefore, it can state that there is no
enzyme activity or very less activity in it. This is due the lack of substrate or coenzymes in
the solution

  

In this experiment, the leaves was homogenized in the presence of PVP and ice cold
extraction buffer. The purpose is to extract the chlorophyll and some big pigment out of the
leaves. The ice cold buffer is used to maintain the pH of the enzyme in the leaves is kept
constant. After the centrifugation, the supernatant is collected because malate dehydrogenase
is present in it. When the enzyme is ready for the enzyme assay, the assay buffer was added
and the enzyme is incubated. The assay buffer is to provide the enzyme a slightly alkaline
condition whereas the incubation will activate the enzyme activity. The reaction is activated
by adding the NADH, the coenzyme, and the oxaloacetate, the substrate. In this experiment,
two graphs were plotted. From the first graph which is the control and treatment, the enzyme
activity was almost constant due to the absence of the oxaloacetate which will reduces to
malate in the present of NADH. Meanwhile, for the treatment graph which contains
oxaloacetate, the enzyme activity was mostly decrease. This can be conclude that the present
of NADH and oxaloacetate will cause the reduction to malate. For the second graph which is
the actual absorbance (treatment minus control), the enzyme activity was decreasing as the
present of NADH and oxaloacetate will cause the reduction to malate. The NADH used per
min in the enzyme assay was 4.82 x 10-12(µmol/ml)/min. Meanwhile, the enzyme activity in
units per ml of extrac was |
| x 10-10
R. Finally, the enzyme activity in units per gram
of fresh weight of leaves was 9.64 x 10-12 ȝmol/min/g.

  

The decreasing of absorbance in time is cause by the reduction of oxaloacetate with the
present of NADH to malate. The NADH used per min in the enzyme assay was 4.82 x 10-
12
(µmol/ml)/min. Meanwhile, the enzyme activity in units per ml of extract was |
| x 10-10

R. Finally, the enzyme activity in units per gram of fresh weight of leaves was 9.64 x
10-12 ȝmol/min/g.

Ô 

1.c http://en.wikipedia.org/wiki/Malate_dehydrogenase.Retrived on 22 March 2011.

2.c http://www.wikigenes.org/e/gene/e/947854.html. Retrieved on 22 March 2011.
3.c http://www.kikkoman.co.jp/bio/j/rinsyou/images/pdf/36_mdh-p.pdf. retrived on
March 2011.