King Abdulaziz University

Faculty of Science
Department of Biochemistry
Girls Section

Enzymes Lab
BIOC 231
 Table of Contents

Lab # Experiment name Page #

1 Comparison between Enzymes and non-biological Catalysts 3

The hydrolysis of sucrose by yeast β-Fructofuranosidase and

2 the determination of the produced sugars with Benedict 9
quantitative method

The determination of the optimum temperature

3 15
of salivary Amylase

4 Estimation of lipase activity 19

5 Indirect estimation for aldehyde oxidase 22

6 Indirect estimation of lactate dehydrogenase 25

The effect of hydrogen ion concentration on Catalase

7 28
enzyme

8 Indirect estimation of succinic dehydrogenase 33

The effect of substrate concentration and time on the

9 35
digestion of casein by trypsin

Enzyme inhibition
10 40
Non-competitive inhibition of salivary α-amylase

The effect of coenzyme

11 Nicotinamide adinine dinucleotide as the coenzyme for 45
lactate dehydrogenase

1
Experiment 1: Comparison between Enzymes and non-biological
Catalysts
Definition of enzymes: enzymes are biological catalysts. They greatly
enhance the rate of specific chemical reactions that would occur very
slowly.
Starch which the storage form of glucose in plant. Starch consist of
1- Amylose

1-4 α- glycosidic linkage

2- Amylopectin

1-6 α- glycosidic linkage

2
Contents of Saliva

 In animals, saliva is produced in and secreted from the salivary

glands. It is a fluid containing
 Electrolytes: (2-21 mmol/L sodium, 10-36 mmol/L potassium, 1.2-
2.8 mmol/L calcium, 0.08-0.5 mmol/L magnesium, 5-40 mmol/L
cloride, 2-13 mmol/L bicarbonate, 1.4-39 mmol/L phosphate)
 Mucus. Mucus in saliva mainly consists of mucopolysaccharides
and glycoproteins;
 Antibacterial compounds (thiocyanate, hydrogen peroxide, and
secretory immunoglobulin A)
 Various enzymes. The major enzymes found in human saliva are
alpha-amylase, lysozyme, and lingual lipase. Amylase starts the
digestion of starch before the food is even swallowed. It has pH
optima of 6.7-7.4. Human saliva contains also salivary acid
phosphatases A+B, N-acetylmuramyl-L-alanine amidase,
NAD(P)H dehydrogenase-quinone, salivary lactoperoxidase,
superoxide dismutase, glutathione transferase, glucose-6-phosphate
isomerase, and tissue protein. The presence of these things causes
saliva to sometimes have a foul odor.

Healthy people produce about 1.5 L of saliva per day.

Amylase:- found in two forms

1- α-amylase (in saliva and pancreatic juice) which is

endoglycosidase that attack starch randomly. Inactivated by the
acidity of the stomach.
2- β-amylase (from plant origin) which is exoglycosidase cleaves
maltose from the non-reducing end to produce β-maltose.
3
 Principle:

When we want to measure enzyme activity either we measure the

decrease in the substrate concentration or the increase in the product
concentration.

[S] E [ES] [P]

Starch Amylase Maltose

pH 6.4-7.2 Cl+ reducing sugar

Indicator I2 Indicator Fehling

Blue color Red copper oxide ppt

Hydrolysis of starch by Amylase reaction with I2

Starch blue
Soluble starch blue
Amylodextrin purple
Erthrodextrin red
Achrodextrin colorless
Maltose colorless

Hydrolysis of starch by non-biological catalysts

Starch Acid Glucose

4
Other uses of amylase in industry:
It is used in clarification of fruit juices. The turbidity present in natural
beverages is due primly to the presence of starch and cellulose molecules
too large to be completely soluble. Amylase hydrolysis these molecules
to glucose which are more water soluble.
Reagents:
Starch 1% solution in 0.3% aqueous sodium chloride, freshly prepared;
iodinated potassium iodide solution.
Dilute saliva 1ml: 10ml D.W, HCl 10% solution.
Method:
1- Transfer 1ml of distilled water to a test tube, 1ml of HCl solution
to another test tube and 1ml of dilute saliva sample to a third test
tube.
2- For each test tube prepare a series of 15 test tube, each containing
2ml of pale yellow iodine solution.
3- Place the first and the third test tubes in a water bath at 38C˚(check
the temperature by the thermometer)
4- Place the second test tube in a boiling water bath.
5- Prewarm starch solution in the water bath for 5min at 38C˚.
6- Add 5ml of starch solution to each of the three test tubes; stir the
content with glass rode.
7- Record the time of addition of starch and at the end of each minute
remove two drops reaction mixture using a test pipette, and add to
one of the tubes of iodine solution.
8- Compare the color developed in the sample.
9- Draw the relationship between the color developed and the time
required for the color to develop.

5
Lab report:
At the end of the experiment we notice the following differences:
Biological catalysis Non-biological catalyst
α- amylase HCl
Work under mild conditions Work under drastic conditions
Required in small amount Required in high concentration
High rate Low rate
Highly specific Unspecified
Protein in nature, has all Not protein in nature, do not
chemical and physical properties have chemical and physical
of protein properties of protein

References:
1- Plummer, D. An introduction to practical biochemistry.
McGraw-HILL, london. 1978
2- Harvey,R and Champe,P. Lippincott biochemistry,
london.2005

6
Result:
Draw the relation between the color developed and the time required
for the color to developed
Time
Color
Enzyme HCl D.W
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17

7
Experiment 2: The hydrolysis of sucrose by yeast β-
Fructofuranosidase and the determination of the produced sugars
with Benedict quantitative method

Principle:
β-Fructofuranosidase is a glycosidase found in yeast. It catalyses the
hydrolysis of sucrose to glucose and fructose. The enzyme is also known
as invertase or sucrase, but these names are no longer used.
The substrate sucrose is a non-reducing sugar, where as the products
formed are both reducing sugar. Therefore the reaction can be followed
by the estimation of the quantity of reducing sugar formed. Between the
several methods which can be used for such estimation, Benedict
quantitative method was utilized.
Benedict quantitative reagent is composed of:
1- Copper sulphate: to provide the oxidizing Cu+2ions.
2- Sodium carbonate: to provide the alkaline medium necessary for
the formation of the highly reactive reducing sugar 1-2 endediol.
3- Sodium citrate: combines with Cu carbonate to prevent its
precipitation by forming a slightly soluble complex with cupric
ions (Cu+2ions). This complex dissociates slowly to give a
sufficient supply of Cu+2 ions.
4- Potassium thiocyanate (KSCN): reacts with cupric ions to give
Cu(SCN)2
5- Potassium ferrocyanide (K4Fe(CN)6): prevents the re-oxidation of
the formed cuprous thiocyanate (CuSCN) to cupric thiocyanate.

8
The reaction takes place as fallow:
1- The enolization of reducing sugar in alkaline medium to give a
highly reactive reducing compound, which is 1-2 endediol.

2- Formation of cupric carbonaate

Na2CO3 + CuSO4 CuCO3 + Na2SO4

3- Formation of sodium cupric citrate complex:

4- Ionization of sodium cupric complex

9
 5- Reaction of KSCN with Cu+2ions:
Cu+2 + KSCN Cu(SCN)2
Cupric thiocynate (blue)
6- reduction of Cu(SCN)2 by 1,2 enediol to cuprous thiocynate:

boil
Cu(SCN)2 CuSCN
Sugar (cuprous thiocyanate, white ppt.)

Method:
A- Hydrolysis of sucrose by yeast β-Fructofuranosidase
Prepare five tubes containing the following mixtures
Tube 1 2 3 4 5
Sucrose 0.3M 10 ml 8 ml 6 ml 4 ml 2 ml

D.W 0 2 ml 4 ml 6 ml 8 ml
Buffer pH 4.5 6 ml 6 ml 6 ml 6 ml 6 ml
Pre-incubate at 37C˚ for 5 min
Yeast 4 ml 4 ml 4 ml 4 ml 4 ml
suspension
Incubate for 15 min
1% NaOH 2 ml 2 ml 2 ml 2 ml 2 ml
Final conc. of 150 120 90 60 30
sucrose

10
Note:
The yeast must be added to each tube at a constant time intervals, i.e. tube
1 at time 0, tube 2 at 2min etc. This will enable the incubation time to be
measured exactly and ensures that each tube is incubated for the same
time. Incubate each tube for exactly 15 min. Stop the reaction by the
addition of 2ml of 1% sodium hydroxide. This will be at 15 min for tube
1, 17 min for tube 2 and so on.

B- Determination of the hydrolyzed sucrose solution by Benedict

method
1- Place the sugar solution of hydrolyzed sucrose from the previous
experiment in burette.
2- Measure 5ml of Benedict quantitative reagent into 100 ml conical
flask add approximately 1g of anhydrous sodium carbonate and
few pieces of porcelain. Heat the mixture vigorously.
3- Run in the sugar solution slowly from the burette until a bulky
white precipitate is formed. Continue the titration by adding the
sugar solution drop by drop until the last trace of blue or green has
disappeared.
4- Record the volume of the sugar required to titrate 5 ml of benedict
reagent. This volume will be your titer number.

Note:
1- The end point must be determined while the mixture is still
boiling. When the mixture is not boiling atmospheric oxidation
occurs and the green color returns.
2- The addition of sodium carbonate to the titration mixture results in
the liberation of CO2, which prevents atmospheric oxidation.

11
 3- If the mixture bumps or it becomes too concentrated during
titration, remove it from the heater, boil 10 ml water in atest tube
and add it to the reaction mixture. Heats the mixture until it boils
again and continues the titration.
4- The tip of the burette must be over the mouth of the flask while
mixture is titrated.

Calculation:

The concentration of the sugar in each tube can be calculated from the
following sugar equivalent equation
M * V = M′ * V′
The equivalents for a number of sugars are given as follow, but they only
applied if the above conditions are strictly adhered to.
25 ml of Benedict’s reagent is equivalent to 50 mg of glucose
53 mg of fructose
68 mg of lactose
74 mg of maltose
49 mg of hydrolyzed sugar
Since we used 5ml of Benedict reagent which is equivalent to 9.8 mg of
hydrolyzed sugar
5ml benedict = 9.8 mg
5ml benedict = Titer no. ml
9.8 mg of sugar = Titer no. ml

X mg of sugar = 1 ml
X mg of sugar/ ml = 1 *9.8 /titer No. *dilution factor
Dilution factor= Final volume / Initial volume

12
References:
3- Plummer, D. An introduction to practical biochemistry.
McGraw-HILL, london. 1978
4- Harvey,R and Champe,P. Lippincott biochemistry,
london.2005

Result:
Fill the table below and plot a relationship between the substrate and
product concentration.
Tube no. [S] Titer number [P]
mM ml mg/ml
1
2
3
4
5

13
 Experiment 3: The determination of the optimum temperature
of salivary Amylase

The optimum temperature: is the temperature at which an enzyme

shows maximum catalytic activity. This temperature can be determined
by following the increase and the decrease in the activity of salivary α-
amylase. α-amylase catalyses the hydrolysis of α-1-4 linkage of starch
with the production of maltose. The reaction is followed by measuring the
increase in the reducing sugar, the product. The reagent used is 3,5-
dinitrosalicylate which is reduced in alkaline medium to 3-amino-5-
nitrosalicylic acid. The yellow color produced is measured at 540nm.

14
Materials:
1- phosphate buffer (0.1M, pH6.7)
2- Starch substrate (0.5% in phosphate buffer)
3- Sodium chloride NaCL (1%)
4- Sodium hydroxide NaOH (2N). Sodium potassium tartarate.
Dissolve 150g of sodium potassium tartarate in 250ml distilled
water.
5- 3,5- Dinitrosalicylic acid. Dissolve 5g of 3,5- Dinitrosalicylic acid
in 100ml of 2N sodium hydroxide. Prepare the 3,5- Dinitrosalicylic
acid by mixing (4) and (5) and complete them to 500ml with
distilled water.
6- Amylase enzyme, dilute 1ml saliva to 100ml distilled water.

Procedure:
Since the goal of the experiment is to determine the optimum
temperature for amylase, the same experimental steps will be done
at the following temperatures: 0 C˚, 10 C˚, 20 C˚, 37 C˚, 40 C˚,
60 C˚, 70 C˚. for each student prepare 3 tubes and add the
following reagents.

15
 Tube no. 1 2 3 (Blank)

Substrate (ml) 2.5 2.5 2.5

Starch 5%
Buffer (ml) 1 1 1
Phosphate
1 1 1
Activator (ml)
NaCL
Pre heat at the chosen temperature for 10 min
Enzyme (ml) 0.5 0.5
D.W (ml) - - 0.5
Incubate for 15 min. at the chosen temperature then stop the reaction
by adding 2N NaOH
Inhibitor (ml) 0.5 0.5 0.5
NaOH
The next step is to determine the amount of reducing sugar produced
in each tube by using 3,5, dinitrosalicylate reagent.
Reagent (ml) 0.5 0.5 0.5
Heat the tube in boiling water bath for 5min. cool and read the
absorbance of the colored product at 540nm.

Note: all tubes must be cooled to the same temperature before reading
on the spectrophotometer, since the extinction is sensitive to
temperature change.

16
Results:
Plot a curve of the change in the initial reaction (V) against
temperature.

What is the optimum temperature for the enzyme α-amylase?

References:
1- Plummer, D. An introduction to practical biochemistry.
McGraw-HILL, london. 1978
2- Harvey,R and Champe,P. Lippincott biochemistry,
london.2005
3- Boyer, R. Concepts in biochemistry. John Wiley and sons, New
york. 2002

17
 Experiment 4: Estimation of lipase activity

Lipase is a pancreatic enzyme secreted into the small intestine. It

catalyses the hydrolysis of triacylglycerols to free fatty acids and glycerol
as follow:

The release of fatty acids in the solution will cause decrease in the pH and
the rate of the reaction may be followed by:
1- Noting the change of pH with time.
2- Titration the liberated free fatty acids with standard alkali using a
suitable indicator
3- By continues titration using an automatic apparatus, (pH-state)
which keeps the pH constant and at the same time plots a curve of
titer number against time.
Method 2 has been adapted foe this experiment. The librated free fatty
acids at different enzyme concentrations will be titrated with 0.05 N
NaOH. Since we are using oils as substrates CaCl2 is used as emulsifying
agent for two reasons:
1- to increase the surface area
2- to decrease the surface tension, thus the oil drop is effetely attacked
with the enzyme.

18
Materials:
1- Lipase (1g%)
2- Chloroform (10%)
3- Fresh oil as the substrate
4- Calcium chloride
5- Sodium hydroxide 0.05 N

Procedure
Prepare 6 tubs which contain the following:
Tube 1 2 3 4 5 B
Oil 2 2 2 2 2 2
Substrate
(ml)
CaCl2 1 1 1 1 1 1
Mix well
D.W 8 6 4 2 0 10
Lipase 2 4 6 8 10 -
(ml)
Incubate in a water bath 37C˚
Enzyme 0.02 0.04 0.06 0.08 0.1 -
concentration

Titrate the liberated fatty acids with NaOH noting the time of the titration
should not exceed 10 min.

19
Result
Draw a graph of enzyme concentration against ml of NaOH has taken. Is
your curve hyperbolic or liner, comment?
References:
4- Plummer, D. An introduction to practical biochemistry.
McGraw-HILL, london. 1978
5- Harvey,R and Champe,P. Lippincott biochemistry,
london.2005
6- Boyer, R. Concepts in biochemistry. John Wiley and sons, New
york. 2002

20
 Experiment 5: Indirect estimation for aldehyde oxidase

Principle:
Milk contains the enzyme aldehyde oxidase which catalyses the oxidation
of variety of aldehydes to acids. The reaction proceeds anaerobically and
can be demonstrated by a suitable hydrogen acceptor such as methylene
blue. The reaction is performed via an intermediate hydrated aldehyde as
follow:

Methylene blue, which acts as a cofactor changes in color from blue to

colorless, thus the progress of the reaction can be noted.
The reaction is conveniently carried out in a tube designed for the study
of anaerobic reactions, Thunberg tube.

Materials:
1- methylene blue, 0.02g%
2- Fresh cows milk, different trade marks.
3- Neutral formaldehyde, 0.5 % (by titration with NaOH using ph.ph
as indicator.

21
Procedure:
Prepare 4 Thunberg tubes. If the tubes are not available use ordinary test
tubes covered with parafilm while the substrate is added by a syringe.
Perform the following steps:
Tubes 1 2 3 4
Fresh milk 5 5 5 5
enzyme (ml)
Boiling water bath 1 min 5min - -
M.B 1 1 1 1
Indicator (ml)
Formaldehyde 1 1 1 -
Substrate (ml)
All tubes in water bath 40C˚
Note the time of complete decolorization

References:
7- Plummer, D. An introduction to practical biochemistry.
McGraw-HILL, london. 1978
8- Harvey,R and Champe,P. Lippincott biochemistry,
london.2005
9- Boyer, R. Concepts in biochemistry. John Wiley and sons, New
york. 2002

22
Result

Tube Time of comment

NO. declorization
1
2
3
4

Which tube decolorize first?

Is methylene blue is the only substance that is reduced?

23
 Experiment 6: Indirect estimation of lactate dehydrogenase

Lactic acid produced during anaerobic glycolysis can be converted to

pyruvic acid with the aid of the enzyme lactate dehydrogenase when
oxygen becomes available. The hydrogen acceptor NAD+ accepts the
hydrogen atoms from the lactic acid and the pyruvic acid molecule
results. Part of the produced pyruvic acid enters the citric acid cycle after
being converted to acetyl coA. The remainder of the pyruvic acid is
converted into glycogen.

Acetyl coA
Citric acid cycle
Lactate dehydrogenase
CH3CHOHCOOH CH3COCOOH
Pyruvic acid
glycogen

In this experiment, yeast will be used as a source of lactate

dehydrogenase. The reaction will be followed by allowing methylene
blue dye to function in place of the natural hydrogen acceptor NAD+. As
methylene blue is reduced it becomes colorless.

Lactate dehydrogenase
CH3CHOHCOOH CH3COCOOH
Pyruvic acid

Methylene Methylene
Blue Blue
(blue) (coloreless)

Materials:

24
 - yeast suspension
- 5% sodium lactate solution
- 0.1% methylene blue
- Water bath 37˚C
- Boiling water bath

Procedure:
- Lable three clean test tubes as a, b and c as followed
- Make sure that you shake the bottle of yeast suspension before
removing your sample
Test tube A B C
Yeast 2ml 2ml 2ml
suspension Yeast
suspension
Pre heated for
10 min in
boiling water
bath and cooled
to 37˚C before
being used
Sodium lactate 10 drops 10 drops
Methylene blue 1 drop 1 drop 1 drop
Continue to add methylene blue drop wise ( mixing after each drop) until
each solution becomes a uniform light blue in color
Mix and place in water bath 37˚C
Observe the tubes after 10 min. note any color changes and record your
observations

Results
A B C

25
References:
1- Plummer, D. An introduction to practical biochemistry.
McGraw-HILL, london. 1978
2- Harvey,R and Champe,P. Lippincott biochemistry,
london.2005
3- Boyer, R. Concepts in biochemistry. John Wiley and sons, New
york. 2002

26
 Experiment 7: The effect of hydrogen ion
concentration on Catalase enzyme

Catalase is a common enzyme found in living organisms’ red blood cells

and yeasts are good sources of catalase. Its functions include catalyzing
the decomposition of hydrogen peroxide to water and oxygen. Catalase
has one of the highest turnover rates for all enzymes; one molecule of
catalase can convert 6 million molecules of hydrogen peroxide to water
and oxygen each minute. Catalase is a tetramer of 4 polypeptide chains
which are at least 500 amino acids in length. Within this tetramer there
are 4 porphyrin haem (iron) groups which are what allows it to react with
the hydrogen peroxide. Its optimum pH is at a neutral level.

H2O2 catalase---> H2O + O2

Hydrogen peroxide is formed as a waste product of metabolism in many

living organisms. It is toxic and must be quickly converted into other, less
dangerous, chemicals.

27
 Effect of pH on enzyme activity

www.biomedcentral.com/1471-2180/2/5/figure/F3

Principle:
Iodide anion I-2 is a weak reducing agent that will react with an oxidizing
analytes to produce iodine
K2I + H2O2 I2 + starch
(ind)

The iodine may be titrated with Na2S2O3 to determine the H2O2

concentration

Na2S2O3 + I2 Na2S4O6 + 2I-

28
Materials:
1- hydrogen peroxide 0.005M
2- phosphate buffer of different values 3.8, 4.8, 6.8 and 8
3- Sulfuric acid 2N
4- Potassium iodide 10%
5- Sodium molybdate 1%
6- Sodium thiosulfate 0.005M
7- Starch 1%
8- Enzyme source 10% yeast and 1% blood collected in EDTA tube
with ice cold distilled water, the solution must be kept cold
throughout the experiment.

29
 Procedure:
Tube 1 2 3 4
H2O2 15 15 15 15
Substrate
(ml)
Selected 10 10 10 10
buffer (ml) pH 3.8 pH 4.8 pH 6.8 pH 8
Pre-incubate the flask in ice bath for 5 min
Enzyme (ml) 1 1 1 1
Incubate exactly for 5 min
H2SO4 10 10 10 10
Inhibitor (ml)

Note: with each selected buffer make a blank instead of the

enzyme add 1ml D.W.
The decrease in hydrogen peroxide is now determined as follow:
1- Add 10ml of KI and, 1ml of sodium molybdate 1% to the above
mixture in the same flask.
2- Let the mixture stand for 20 min
3- Titrate the liberated iodine with 0.005 M sodium thiosulfate.
4- When the yellow color of the iodine begins to fade, add 3 drops of
starch to act as an indicator.
5- Continue the titration until the purple color of the starch
disappears.

30
Results:
Draw a curve of the pH against the titer no. ,enzyme activity
What is the optimum pH for the catalase.
Note:
Titrt. No. = Titer B – Titer T

References:
1- Plummer, D. An introduction to practical biochemistry.
McGraw-HILL, london. 1978
2- Harvey,R and Champe,P. Lippincott biochemistry,
london.2005
3- Boyer, R. Concepts in biochemistry. John Wiley and sons, New
york. 2002

31
Experiment 8: Indirect estimation of succinic dehydrogenase

The citric acid cycle functions primarily to provide hydrogen atoms for
the electron-transport chain. The hydrogen atoms are picked up from the
citric acid cycle and carried to the electron transport chain by several
coenzymes. FAD, one of these coenzymes. Transports the hydrogen
atoms generated in citric acid cycle when succinic acid is dehydrogenated
to form fumaric acid.

Succinic dehydrogenase
HOOCH2CH2COOH HOOCCH=CHCOOH
Succinic acid Fumaric acid

FAD FADH2

Yeast will be used to supply the enzyme, and the reaction can be followed
outside the body by using the artificial hydrogen acceptor methylene
blue. The effect of malonic acid, a competitive inhibitor will also be
demonstrated in this experiment.

Materials:
- Yeast suspension
- 5% sodium succinate solution
- 10% sodium malonate solution
- 0.1% methylene blue solution

Procedure:
Lable four small, clean test tubes a,b,c and d

Test tube A (control) B C D

Yeast 2ml 2ml 2ml 2ml
suspension
Sodium 10 drops 5 drops

32
succinate
Sodium 10 drops 5 drops
malonate
Methylene 1 drops 1 drops 1 drops 1 drops
blue
Continue to add methylene blue drop wise ( mixing after each drop) until
each solution becomes a uniform light blue in color
Mix and place in water bath 37˚C
Observe the tubes after 10 min. note any color changes and record your
observations

Results:

Test tube A B C D

References:
1- Plummer, D. An introduction to practical biochemistry.
McGraw-HILL, london. 1978
2- Harvey,R and Champe,P. Lippincott biochemistry,
london.2005
3- Boyer, R. Concepts in biochemistry. John Wiley and sons, New
york. 2002

33
 Experiment 9 The effect of substrate concentration and time on the
digestion of casein by trypsin
Principle:
Proteins are long chains of amino acids linked by peptide bonds. During
hydrolysis these peptide bonds are broken. Depending upon the type of
enzyme used and processing conditions, smaller chains of amino acids
and different amino acid sequences are produced by the process.
Trypsin is a pancreatic enzyme (endopeptidase) which is secreted in the
pancreatic juice as trypsinogen. Catalyzes the hydrolysis of peptide bound
formed from the carboxyl groups of basic amino acids, L-Arginine and L-
Lysine.

Casein which is the major protein of milk is digested with trypsin. The
reaction can be followed by estimating the increase in the free amino
groups using formol titration.
Formaldehyde reacts with the amino groups to form methylol compounds
and the liberation of hydrogen ions. The solution will become more
acidic. The amount of alkali required to neutralize the increase in acidity

34
gives a measure of the amount of free amino groups formed from the
protein digestion. Formaldehyde is also very effectively stops any further
activity by reacting with the enzyme protein.
~NH3 + H.CHO ~NH.CH2OH + H+
(monomethylol)

~NH.CH2OH+H.C -N(CH2OH)2
(Dimethylol)

For an enzyme-catalysed reaction, there is usually a hyperbolic

relationship between the rate of reaction and the concentration of
substrate
www.ucl.ac.uk/~ucbcdab/enzass/substrate.htm
When the enzyme is saturaded with substrate the rate of the reaction is
independent of the substrate concentration. In other words is zero order
with respect to substrate. The velocity is dependent on the enzyme
concentration when there is sufficient substrate to saturate the enzyme
(first order)

35
 a- First order : at low substrate concentration the increase in velocity
is almost linear in relation to substrate concentration.
b- Zero order: The reaction rate is maximum when the enzyme is
saturated with substrate.
Materials:
1- Casein solution (1%, 2%, 4%, 6%, 8% at pH 8.5). Dissolve 4g
casein in 90ml of water containing 2ml of 0.1N sodium hydroxide.
Shake the suspension continuously and gently warm until
dissolved. Adjust the pH to 8.5 with HCL and make up to 100ml
with water.
2- Trypsin (4%). Curde trypsin prepared from pancreas is suitable.
3- Formaldehyde solution (40% w/v)
4- Phenolphthalein (0.255 in alcohol)
5- Sodium hydroxide (0.1N)

Procedure:
a- effect of substrate concentration
1- measure 5ml of formaldehyde into 5 small flasks.
2- Add one drop of phenolphthalein to each flask, the solution is
colorless.
3- Titrate with 0.1N sodium hydroxide until the mixture is faintly
pink. The color in all flasks should be the same.

36
 The enzymatic reaction
Conical no. 1 2 3 4 5
Casein 10 10 10 10 10
(substrate) ml 1% 2% 4% 6% 8%
Pre-incubate in water bath 37C˚ for 10 min
Trypsin 2 2 2 2 2
(enzyme) ml
Incubate in water bath 37C˚ for 6 min

1- Remove 5 ml of the mixture and add it to one of the five flasks

containing formaldehyde.
2- Add one drop of phenolphthalein and titrate with NaOH until a
faint but permanent color is obtained.
3- Repeat the same steps for each casein concentration.
Note: The final color at the end point should be the same in each case.
b- Effect of time
1- measure 5ml of formaldehyde into 5 small flasks.
2- Add one drop of phenolphthalein to each flask, the solution is
colorless.
4- Titrate with 0.1N sodium hydroxide until the mixture is faintly
pink. The color in all flasks should be the same.
Enzymatic reaction
1- measure 50ml of the casein solution into a conical flask and place
it in a water bath 37 C for about 10min
2- put about 7 ml of trypsin in a test tube and place in water bath 37C
for about 10 min
3- after about 10min. add 5ml of the trypsin solution to the conical
containing the casein and mix
37
 4- after 2,4,6,8 min. remove 10 ml of the reaction mixture and add it
to one of the conical flasks containing formaldehyde.
5- Add 5 ml of ph.ph to each flask and titrate with 0.1N NaOH untail
a faint pink color but permanent pink color is obtained.

References:
1- Plummer, D. An introduction to practical biochemistry.
McGraw-HILL, london. 1978
2- Harvey,R and Champe,P. Lippincott biochemistry,
london.2005
3- Boyer, R. Concepts in biochemistry. John Wiley and sons, New
york. 2002

38
 Experiment 10: Enzyme inhibition
Non-competitive inhibition of salivary α-amylase
Types of inhibition
1- Competitive inhibition
Competitive inhibition occurs when substrate (S) and inhibitor (I) both
bind to the same site on the enzyme. In effect, they compete for the active
site and bind in a mutually exclusive fashion.

The degree of inhibition depends on

the relative concentration of
substrate and inhibitor. Almost the
maximal velocity may be found in
the presence of the inhibitor if the
substrate concentration is high
enough. An example for such
inhibition the inhibition of the
enzyme succinate dehydrogenase by
malonic and maleic acids since
maleic acid compete with the
substrate (succinate) on the active
site.

2- Non- competitive inhibition

If a reversible inhibitor can bind to the enzyme at a site that is distinct
from the active site, it is described as a "noncompetitive inhibitor." In
pure noncompetitive inhibition, the inhibitor binds with equal affinity to
the free enzyme and to the enzyme-substrate (ES) complex.

39
 The enzyme –substrate- inhibitor complex
formed is unable to break down and the
inhibition effectively occurs by reduction of
the amount of the enzyme available. Increase
of the substrate concentration has no effect
on the degree of inhibition. Most non-
competitive inhibitors are not related
chemically to the substrate and the same
inhibitor may affect a number of enzymes.
Example for such inhibition:
The reaction of Heavy metal ion Ag+1 and
Cu+2. The reaction of cyanide with iron
prophyrin enzyme.

Principle:
α –amylase catalyses the hydrolysis of 1-4 links of starch with the
production of maltose. The reaction is followed by measuring the
increase in the reducing sugar. The reagent 3,5-dinitro-salcylate is
reduced in alkaline medium to 3-amino-5-nitrosalicylic acid. The
yellow colour produced is measured at 540nm.
The experiment is preformed with an activator NaCL and with a non-
competitive inhibitor HgCL2.

40
Materials:
1- Phosphate buffer (0.1M, pH 6.7).
2- Starch substrate with different concentrations (0.2%, .4%, .6%, .8%
and 1% in phosphate buffer).
3- Sodium chloride NaCl (1% w/v)
4- Mercuric chloride
5- Sodium hydroxide NaOH (2N)
6- Sodium potassium tartarate. 150g of sodium potassium tartarate
was dissolved in 250ml distilled water
7- 3,5-dinitrosalicylic acid. 5g of 3,5-dinitrosalicylic acid in 100ml of
2N NaOH. The dinitrosalicylate was prepared by mixing step 6
with step 7 and complete the volume to 500ml with distilled water.
8- Amylase enzyme was prepared by diluting 1ml of saliva to 25ml
with distilled water.

41
Procedure:
For each starch concentration prepare 6 tubes and add the following
Tube B S+E+A B S+E+I B E+S
Starch - 2.5 - 2.5 - 2.5
Substrate(ml)
NaCl 1 1 - - - -
Activator(ml)
HgCl2 - - 1 1 - -
Inhibitor (ml)
D.W 3 - 3 - 3 -
Amylase - 0.5 - 0.5 - 0.5
Enzyme (ml)
Incubate all tubes in water bath 37 C˚ for 10 min
NaOH (ml) 0.5 0.5 0.5 0.5 0.5 0.5
Dinitrosalicylate 0.5 0.5 0.5 0.5 0.5 0.5
reagent (ml)
Heat the tubes in a boiling water bath for 5 min
Cool
Read at 540nm

References:
1- Plummer, D. An introduction to practical biochemistry.
McGraw-HILL, london. 1978
2- Harvey,R and Champe,P. Lippincott biochemistry,
london.2005
3- Boyer, R. Concepts in biochemistry. John Wiley and sons, New
york. 2

42
Results:
A- Draw a plot of different substrate concentration and enzyme +
substrate absorption and compare it with the behaviour of the
enzyme with the activator and with the inhibitor.
B- Draw the line-weaver Burk plot [1/S against 1/V] and show Km
and the Vmax.

Result:
A-Plot the increase in titer number against the casein
concentration
B- Draw a curve showing the relationship between time and
activity. Calculate the initial velocity.

43
 Experiment 11: The effect of coenzyme
Nicotinamide adinine dinucleotide as the coenzyme for lactate
dehydrogenase

Principle:

Enzyme consists of the protein and a combination of one or more parts

called cofactors. This enzyme complex is usually simply referred to
simply as the enzyme.

Cofactors: A cofactor is a non-protein substance which may be organic,

and called a coenzyme. The coenzyme is often derived from a vitamin.
Another type of cofactor is an inorganic metal ion called a metal ion
activator. The inorganic metal ions may be bonded through coordinate
covalent bonds. The major reason for the nutritional requirement for
minerals is to supply such metal ions as Zn+2, Mg+2, Mn+2, Fe+2, Cu+2,
K+1, and Na+1 for use in enzymes as cofactors. Nicotinamide is from the
niacin vitamin. The NAD+ coenzyme is involved with many types of
oxidation reactions including lactate dehydrogenas.

44
 Vitamin Coenzyme Function
nicotinamide adenine oxidation or hydrogen
niacin
dinucleotide (NAD+) transfer
flavin adenine oxidation or hydrogen
riboflavin
dinucleotide (FAD) transfer
pantothenic acid coenzyme A (CoA) Acetyl group carrier
vitamin B-12 coenzyme B-12 Methyl group transfer
thiaminpyrophosphate Aldehyde group
thiamin (B-1)
(TPP) transfer

Pyruvate forms a highly colored hydrazone with 2,4-

dinitrophenylhydrazine (DNP). An advantage for this reaction that
compound other than pyruvate in the reaction mixture do not absorb at
the wavelength used in the test. The amount of pyruvate converted to
lactate can therefore be measured.

Materials:

1- Sodium phosphate buffer (0.1M, pH 7.4)

2- Sodium pyruvate. Dissolve 0.5g in 100 ml of phosphate buffer. The
buffered substrate is prepared by diluting the above stock solution
1-50ml with buffer to give a concentration of 100mg/ml.
3- Reduced nicotinaminde adenine dinucleotide (10mg/ml). This is
prepared in phosphate buffer just before use.
4- 2,4-dinitrophenylhydrazine (2mM). Dissolve 40 mg in 9 ml of
concentrated hydrochloric acid and make up to 100ml with water.
5- Sodium hydroxide (0.4N).

45
6- Lactate dehydrogenase. Either serum of an appropriate dilution of
commercial rabbit muscle enzyme is suitable.

Procedure:

Prepare 4 test tubes incubated in a water bath at 37C˚ as follow:

Tube T1 T2 Control Blank

Pyruvate 1 1 1 -

Substrate
(ml)
Buffer (ml) - 0.1 0.2 1.2
NADH2 (ml) 0.1 - - -
Enzyme (ml) 0.1 0.1 - -
Incubate for 15min at 37C˚
DNP (ml) 1 1 1 1
Allow to stand in room temperature for 20 min. after the 20 min has
been completed add.
NaOH (ml) 10 10 10 10
After 10 min read the colour developed at 510nm against the blank.
References:
10- Plummer, D. An introduction to practical biochemistry.
McGraw-HILL, london. 1978
11- Harvey,R and Champe,P. Lippincott biochemistry,
london.2005
12- Boyer, R. Concepts in biochemistry. John Wiley and sons,
New york. 2002

46
Calculation:

Concentration of the unk. = T1abs. * control conc/control abs.

T2 abs. * control conc./control abs.

The amount of reacted pyruvate in the prescience of enzyme and

cofactor= conc. Control – conc T1

The amount of reacted pyruvate in the prescience of enzyme only

= conc. Control – conc T2

47