Lab 6-Fumarase - preparation and assay

Objectives

 To determine the specific enzyme activity of fumarase through enzyme activity and
protein assay
 To differentiate the enzyme activity of boiled and unboiled samples.

Introduction

Fumarate hydratase, also known as fumarase is an enzyme found in both the cytoplasm and
mitochondria of all eukaryotes. In mitochondria, fumarase is involved in generating energy for
the cell through a metabolic pathway called the Krebs cycle. Fumarase catalyses the reversible
hydration of fumarate to malate. The reaction catalysed by fumarase is critical for cellular
energetics as a part of the tricarboxylic acid cycle, which produces reducing equivalents to
drive oxidative ATP synthesis (Mescam et al., 2011). Malate is then converted to
oxaloacetate by malate dehydrogenase, a reversible reaction requiring vitamin B3 as
NAD+ Although the equilibrium of this reaction strongly favours malate formation, net flux is
usually toward oxaloacetate formation since this compound, together with the other product of
the reaction (NADH), is removed continuously in further reactions (Engelking, 2010).

There are a few distinct differences in the chemical and physical properties of fumarate and L-
malate, allowing fumarase to be assayed in several ways. The most convenient method is a
continuous assay, in which changes in fumarate concentration are measured
spectrophotometrically between 250 and 300 mμ (Hill and Bradshaw, 2003). The activity of
fumarase is extremely sensitive to temperature and to the concentration and type of anion in
the assay mixture. The purification method gives a higher yield than the earlier methods, and
requires only a short time to obtain pure enzyme. As much as 100 mg of crystalline fumarase
may be obtained per kilogram of cow heart muscle, an increase of 5 to 6 times the yield given
by the other methods. The low yields given by earlier methods, appear to have resulted from
the loss of 60-70% of the fumarase content of heart muscle when the tissue was washed with
water prior to extraction of fumarase with buffer. The enzyme shows a bell-shaped dependence
on pH, which suggests that fumarase possesses, both an acidic and basic functional group in its
active site (Hill and Bradshaw, 2003). In this experiment, a fumarase preparation is obtained
from heart tissue. After partial purification, enzyme activity is assayed by observing the rate of
disappearance of fumarate, which is estimated by permanganate titration method.
 Results

Table 1: Volumes of permanganate used in titration

Tube no. 1 2 3 4 5

Initial volume of 0.1N

50.00 46.50 40.60 35.70 29.50
permanganate solution (mL)

Final volume of 0.1N

46.50 40.60 35.70 29.50 22.50
permanganate solution (mL)

Volume of 0.1N permanganate

3.50 5.90 4.90 6.20 7.00
solution used in titration (mL)

Table 2: Number of moles of fumarate converts to malate

Tube no. 1 2 3 4 5

Volume of 0.1N permanganate

3.50 5.90 4.90 6.20 7.00
solution used in titration (mL)

Initial number of mole of

_ _ _ _ 133
fumarate (μmol)

Number of mole of fumarate

remaining (reacted with 66.6 112 93.3 118
permanganate) (μmol)

Number of mole of fumarate

66.4 21 39.7 15
converted to malate (μmol)

Actual number of mole of

fumarate converted to malate 45.4 24.7
(μmol)
Calculations

1mL of 0.1M fumarate gives a titration value of 5.0-5.5mL 0.1N permanganate. Average
value of 5.0-5.5mL, which is 5.25mL is used.

Initial number of mole of fumarate, n = MV

7.00
= (0.1)(5.25 ÷ 1000)

= 1.33 x 10-4 mol

= 133 μmol

To calculate number of mole of fumarate remaining in each tube, n = MV is used where n is

the number of moles of fumarate remaining, M is the concentration of permanganate and V is
the volume of permanganate used.

Tube 1: n = MV Tube 2: n = MV
3.50 5.90
= (0.1)( ÷ 1000) = (0.1)( ÷ 1000)
5.25 5.25

= 6.66 x 10-5 = 1.12 x 10-4

= 66.6 μmol = 112 μmol

Tube 3: n = MV Tube 4: n = MV
4.90 6.20
= (0.1)( ÷ 1000) = (0.1)( ÷ 1000)
5.25 5.25

= 9.33 x 10-5 = 1.18 x 10-4

= 93.3 μmol = 118 μmol

Number of mole of fumarate converted to malate = Initial number of mole of fumarate -

number of mole of fumarate remaining.

Tube 1: 133 – 66.6 = 66.4 μmol Tube 2: 133 – 112 = 21 μmol

Tube 3: 133 – 93.3 = 39.7 μmol Tube 4: 133 – 118 = 15 μmol

 Actual number of mole of fumarate converted to malate in supernatant 1 = Number of mole
of fumarate converted to malate in test tube 1 - Number of mole of fumarate converted to
malate in test tube 2,

Actual number of mole of fumarate converted to malate in supernatant 1 = 66.4 - 21

= 45.4 μmol

Actual number of mole of fumarate converted to malate in supernatant 2 = Number of mole

of fumarate converted to malate in test tube 3 - Number of mole of fumarate converted to
malate in test tube 4,

Actual number of mole of fumarate converted to malate in supernatant 2 = 39.7 - 15

= 24.7 μmol

Table 3: Standard protein curve

Tube Blank 2mg 4mg 6mg 8mg

Standard protein (4mg/ml) - 0.5ml 1.0ml 1.5ml 2.0ml
Distilled water 3.0ml 2.5ml 2.0ml 1.5ml 1.0ml
Dilute Biuret reagent 4.0ml 4.0ml 4.0ml 4.0ml 4.0ml
Absorbance 0.000 0.069 0.108 0.200 0.264

Question 1 : Plot the protein standard curve

 Table 4: Protein concentration in samples

Sample S1 S2

0.5 0.5
Volume of enzyme solution used (ml)
0.157 0.100
Absorbance at 540nm
4.866 3.139
Amount of protein from standard curve (mg)
9.732 6.278
[protein] (mg/ml enzyme solution)

Calculations

To calculate the amount of protein from standard curve (mg), the equation y = 0.033x -
0.0036 obtained from the graph is used, where

y = absorbance at 540nm

x = amount of protein from standard curve (mg)

S1: y = 0.033x – 0.0036 S2: y = 0.033x – 0.0036

0.157 = 0.033x – 0.0036 0.100 = 0.033x – 0.0036

0.033x = 0.1606 0.033x = 0.1036

X = 4.866 X = 3.139

To calculate the [protein] (mg/ml enzyme solution), amount of protein from standard curve
(mg) is divided by volume of enzyme solution used (ml).

4.866 3.139
S1: = 9.732mg/ml S2: = 6.278mg/ml
0.5 0.5

Question 2: Enzyme activity (μmol/min)

𝐴𝑐𝑡𝑢𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑚𝑜𝑙𝑒 𝑜𝑓 𝑓𝑢𝑚𝑎𝑟𝑎𝑡𝑒 𝑐𝑜𝑛𝑣𝑒𝑟𝑡𝑒𝑑 𝑡𝑜 𝑚𝑎𝑙𝑎𝑡𝑒(𝜇𝑚𝑜𝑙)

Enzyme activity = 𝑇𝑖𝑚𝑒 𝑡𝑎𝑘𝑒𝑛 𝑓𝑜𝑟 𝑒𝑛𝑧𝑦𝑚𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦(min)

45.4 24.7
S1: = 9.08 μmol/min S2: = 4.94 μmol/min
5 5
Question 3: Specific enzyme activity (μmol/min/mg)

𝐸𝑛𝑧𝑦𝑚𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦(𝜇𝑚𝑜𝑙/𝑚𝑖𝑛)
Specific enzyme activity = 𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑓𝑟𝑜𝑚 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑐𝑢𝑟𝑣𝑒(𝑚𝑔)

9.08 4.94
S1: 4.866 = 1.866 μmol/min/mg S2: 3.139 = 1.574 μmol/min/mg

Question 4

Explain why Malate has negligible absorbance in the U.V. region compared to Fumarate. Use
structural diagrams for both molecules.

Figure 1: Structural diagram of malate Figure 2: Structural diagram of fumarate

Malate has a negligible absorbance in the UV region compared to fumarate due to the
difference in the structure comparing the both compounds. The hydration of fumarate to malate
is one of the steps in the citric acid cycle. Referring to figure 1 and 2 above, fumarate has a
conjugated double bond which greatly influence on the peak wavelength and absorption
intensities at UV region. According to the Beer’s Law, the molar absorption coefficient is the
measurement the intensity of light absorbed by a certain substance. The quantitative
spectroscopic determination of an absorbing molecule's concentration is greatly facilitated
when the species follows Beer's law. Molar absorption coefficient increases perpendicularly to
the absorption value, thus, a compound with a larger conjugated system contributes to larger
absorption peaks (Pretsch et al. 2009). The more convenient and rapid method is based on the
much larger absorption of ultraviolet light by fumarate than by L-malate. This effect can be
interpreted qualitatively in terms of the absorption properties of the conjugated electron system
in the fumaric acid molecule, which is absent in both L-malic acid and its corresponding anion.
Therefore, it can be concluded that a difference in the structure between malate and fumarate
causes malate to have a negligible absorbance in the UV region compared to fumarate.

Discussion

In this experiment, fumarase act as the catalyzing enzyme for conversion of fumarate
to malate. The preparation of fumarase was done by lab technician and kept in cool ice bath.
Total of 5 tubes consisting same amount of fumarate and phosphate buffer solution had been
warmed by immersed into water bath at 37℃ for 5 minutes. The advantage of adding phosphate
buffer into tubes is to maintain the pH of solution and enhance the enzyme activity (Moreau,
2017). 1% concentration of sodium chloride has been added into tube 5 and tube 5 act as
control. First supernatant and second supernatant were added into tube 1 and tube 3 whereas
tube 2 and tube 4 were added by boiled enzyme solution. Temperature plays an important role
in controlling enzyme activity, therefore tube 1,2,3,4 were placed into bath at optimum
temperature 37℃ immediately after addition of enzyme solution. TCA (trichloroacetic acid)
was added into tubes for precipitation of protein. After filtration of remaining fumarate,
titration was carried out by using 0.1N permanganate solution which oxidized the fumarate in
the solution until the first pink colour appeared (Sukalyan, 2009).

Based on the result, Tube 5 used the most titrant with 7.0 mL. This is because the
number of moles of fumarate is the highest as Tube 5 is a control and did not contain any
fumarase, therefore there was no conversion of fumarate to malate, it requires more
permanganate solution oxidizing the high amount of fumarate in the solution. Tube 1 and Tube
3 used 3.50mL and 4.90mL volume of titrant respectively. It is lower as compared to volume
of titrant used by Tube 2 and Tube 4 which are 5.90mL and 6.20mL respectively. Unboiled
enzyme which were added to Tube 1 and 3 have higher rate of enzyme activity as there are
more free enzyme catalyzing the reaction, therefore the remaining number of moles of fumarate
is lower and it requires lesser volume of titrant. Boiled enzyme in Tube 2 and 4 used higher
volume of titrant as boiling causes denaturation of active enzymes causing the rate of enzyme
activity is lower, hence, higher remaining number of moles of fumarate requires higher volume
of titrant. Enzyme activity is related directly to protein concentration. Higher protein
concentration means that there are more enzymes in the solution, high amount of enzymes
allows more substrates to bind with it and rate of enzyme activity is increases (Dev, 2018).
Based on Table 4, the protein concentration of S1 is higher than S2, therefore, higher number
of moles of fumarate converted to malate in S1 as compared to S2. Table 2 shows that actual
number of moles of fumarate converted to malate is 45.4 in S1 and 24.7 in S2 which S1 is
higher.

The biuret test is a chemical test used for detecting the presence of peptide bonds. Biuret
reagent which is made of sodium hydroxide (NaOH) and hydrated copper (II) sulphate,
together with potassium sodium tartrate, is added to chelate and thus stabilize the cupric ions.
The reaction of the cupric ions with the nitrogen atoms involved in peptide bonds leads to the
displacement of the peptide hydrogen atoms under the alkaline conditions. The Cu+ formed
during the biuret reaction with the chelate produces a violet-colour chelate complex (Rodger,
1999). The peak absorption for the ion in solution is at 540nm. The intensity of the color is
indicative of protein concentration. Enzyme solution from S1 and S2 shows different
absorbance value at 540nm wavelength. S1 has a higher absorbance value which is 0.157
compared to S2 with 0.100. Protein concentration affects the absorbance very similarly to path
length. If the protein concentration of solution is increased, then there are more molecules for
the light to hit when it passes through, more light is absorbed by molecules. Therefore, higher
absorbance value indicates higher amount of protein concentration in the solution. S1 has a
higher amount of protein with 4.866mg compared to S2 with 3.139.

Specific activity is a term used to measure the rate of reaction of an enzyme with a
substrate (Philips, 2019). It is a measure of the purity of an enzyme solution and is quoted as
units/mg. Based on result, specific enzyme activity of S1 is 1.866 μmol/min/mg and S2 is 1.574
μmol/min/mg. The purity of enzyme in S2 is higher than in S1. S2 has a higher efficiency of
enzyme fumarase that catalyze the conversion of fumarate to malate.

Conclusion

In conclusion, enzyme activity can be affected by several factors such as temperature, pH,
enzyme concentration, substrate concentration, and the presence of any inhibitors or activators.
Unboiled enzyme solution has a higher rate of enzyme activity due to higher amounts of free
enzymes present in the solution. The number of moles of fumarate converted to malate is higher
in unboiled enzyme compared to boiled enzyme. Denaturation of enzyme in boiled enzyme
solution causes lesser enzyme fumarase to convert fumarate to malate. Besides, the protein
concentration in S1 with 9.732mg/mL is higher than S2 with concentration of 6.278mg/mL.
This indicates the rate of enzyme activity in S1 is higher than on S2. The amount of protein
contained in S1 is higher, which is 4.866mg whereas amount of protein in S2 is 3.139mg. The
specific enzyme activity in S1 with 1.866 μmol/min/mg is higher than S2 with 1.574
μmol/min/mg. This shows that the purity of S1 is higher than S2.

References

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