COURSE BOOK OF

CHEMISTRY 2
(BIOCHEMISTRY)
Department of Biochemistry
Benha University, Agriculture College
PRACTICAL
 PRACTICAL BIOCHEMISTRY
Course name Practical Biochemistry
Teacher in charge Ahmed Mahmoud Hassan Mohamed
Department/ College Biochemistry / Agriculture
Contact Ahmed Mahmoud / [email protected]

Course link at the University

Course overview: In two or three constructive paragraphs mention the importance and
the necessity of this course

Biochemistry can be defined as the science concerned with the chemical basis of life.
The cell is the structural unit of living system, thus biochemistry can also be described as the
science concerned with the chemical constituents of living cells and with the reactions and
processes they undergo. By this definition, biochemistry encompasses large areas of cell
biology, of molecular biology, and of molecular genetics.

Course objective: in two or three paragraphs mention the aims of the course and the
main points students should have learned by the end of the course.

We learn the students the general and specific tests for determine the normal
subjects of biochemistry also the abnormal one. That related with diseases.
The Topics :
I CARBOHYDRATES
1- General tests: Molisch, Benedict, Fehling and Barfoid.

2- Specific tests: Seliwanoff.

3- Tests for polysaccharide: (Iodine, and Hydrolysis of polysaccharide ).

II LIPIDS
1- General tests: (Solubility test of lipids, Grease spot, Emulsification of lipids).

2- Specific test: (Copper acetate, iodine, Iodine number determination,

Acid value, Saponification of oils and fats, Saponification value.)

III PROTEINS
1- General tests:-
a- Protein composition test.
b- Precipitation of proteins; By salts of heavy metals, By alkaloidal reagents. By
neutral salts: Half saturation and Complete saturation, and By alcohol.
c- Coagulation of proteins by heat.
d- Biuret’s test, Ninhydrin’s test.

2- Specific tests for Amino acids: ( Milon’s test, Xanthoproteic’s test, Lead acetate
test, and sakaguchi’s test.

IV Enzyme

1- General information.
 Practical of Chemistry 2
BIOCHEMISTRY
AC 0102
Introduction:
The good practical worker will therefore seek to obtain accurate
and precise measurements at the bench. Errors may be random, or
caused by carelessness or inaccurate instruments. To reduce such
random errors which are individually unpredictable, take a large
number of measurements and calculating the average value.

Accuracy:
Accuracy is defined as the degree of conformity to the truth and
expressed as absolute error.
Absolute error = experimentally measured value – true value

Precision:
Precision is defined as the degree of agreement between
replicate experiments and expressed as standard deviation.
Precision does not mean accuracy, since measurements may be highly
precise but inaccurate due to a faulty instrument or technique.

Biological variation
An additional factor to be considered when working with
material derived from living matter is biological variation. A physical
quantity such as the refractive index of a liquid, for example, may be
measured and the value obtained compared with the correct figure,
but for biochemical measurements there is rarely a single value which
can be considered as correct, but a range of
so-called normal values. This means that if an animal is healthy and
free from stress then the value of say a serum constituent should be
within the normal range.

Standards and blanks:

To obtain a value as accurate as possible from an estimation,
errors must be reduced to a minimum. This can be done by careful
working and the use of standard solutions.
Standard solutions of the substance to be estimated should be
included with any test even when a calibrated instrument and standard
reagents are used. This provides a useful check on the accuracy of a
method since the measured figures should fall within the acceptable
limits of the true values. Ideally the standard solution should be
treated in an identical manner to the fluid under investigation.
A standard curve can then be constructed showing the variation of the
quantity measured with concentration. Values obtained for the test
solution should fall within the range of the standard curve and the
value of the test can then be read. Control solutions of body fluids are
now commercially available and are used in clinical laboratories as a
check on methods.
Blank solutions should be included in any measurements. The same
volume of distilled water replaces the substance to be estimated and
the blank is then treated in exactly the same way as the test and
standard. Any value obtained for the blank is, of course, subtracted
from the value for the test and standard in the final calculation, since
the blank value is due to the reagents used and not the substance
under investigation.
Standard pH solutions:
The pH meter is calibrated before use by means of a standard
solution. The meter should be calibrated with a solution whose pH is
close to that under test and several convenient standards are given
below.

Buffer Solutions:
A buffer solution is one that resists pH change on the addition of
acid or alkali.
Buffer consisted of weak acid + its salt (acetic acid + sodium acetate)
or weak base +
its salt (ammonium hydroxide + ammonium chloride).
Such solutions are used in many biochemical experiments where the
pH needs to be accurately controlled.
 Qualitative assay of carbohydrates
Objective:
To characterize carbohydrates present in the unknown solution
on the basis of various chemical assays.
Theory:
Carbohydrates are polyhydroxy aldehydes and ketones or
substances that hydrolyze to yield polyhydroxy aldehydes and
ketones. Aldehydes (– CHO) and ketones (=CO) constitute the major
groups in carbohydrates.
Carbohydrates are mainly divided into monosaccharides,
disaccharides and Polysaccharides. The commonly occurring
monosaccharides includes glucose, fructose, galactose, ribose etc. The
two monosaccharides combined together to form disaccharides which
include sucrose, lactose and maltose. Starch and cellulose fall into
the category of polysaccharides which consists of many
monosaccharide residues.

1. Molisch’s Test:
This test is specific for all carbohydrates, Monosaccharide
gives a rapid positive test, Disaccharides and polysaccharides react
slower.
Principle:
The test reagent dehydrates pentoses to form furfural and
dehydrates hexoses to form 5- hydroxymethyl furfural. The furfurals
further react with α-naphthol present in the test reagent to produce a
purple product.

Ribose 5-(hydroxymethyl )furfural

Method:
• Add 2 drops of the α-naphthol solution (5% in ethanol, prepare
fresh) to 2 ml of test solution in a test tube.
• Carefully, pour about 1 ml of conc. H2SO4 down the side of the tube
so as to form two layers.
• Carefully observe any colour change at the junction of the two
liquids.
• Repeat the test, using water instead of the carbohydrate solution.

2. Fehling’s Test:
This forms the reduction test of carbohydrates. Fehling’s
solution contains blue alkaline cupric hydroxide solution, heated with
reducing sugars gets reduced to yellow or red cuprous oxide and is
precipitated. Hence, formation of the yellow or brownish-red colored
precipitate helps in the detection of reducing sugars in the test
solution.
Preparation of Fehling's solution A:
Dissolve 35g of Cu2SO4.7H2O in water and make up to 500ml
Preparation of Fehling's solution B:
Dissolve 120 g of KOH and 173 g of Sod. Pot. Tartarate (Rochelle
salt) in water and make up to 500 ml
Fehling’s reagent: Equal volumes of Fehling A and Feling B are
mixed to form a deep blue solution.
Note: If you do not have sodium potassium tartarate, it can prepared
using tartaric acid as described below.
Method:
• Mix equal volumes of Fehling's solution A and B.
• Add 5 drops of the test solution (glucose, fructose,
and sucrose solution) to the mixed Fehling's
solution and boil.
Results
Glucose solution Orange-brown color is appeared. Fructose
solution Orange-brown color is appeared. Sucrose solution No
change.

Discussion:
Fehling's tests for aldehydes are used extensively in
carbohydrate chemistry. A positive result is indicated by the
formation of a brick red precipitate. Like other aldehydes, aldoses are
easily oxidized to yield carboxylic acids. Cupric ion complexed with
tartrate ion is reduced to cuprous oxide.
The cupric ion (Cu++) is complexed with the tartarate ion. Contact
with an aldehyde group reduces it to a cuprous ion, which the
precipitated as orange-brown Cu2O.
The sucrose does not react with Fehling's reagent. Sucrose is a
disaccharide of glucose and fructose. Most disaccharides are reducing
sugars, sucrose is a notable exception, for it is a non-reducing sugar.
The anomeric carbon of glucose is involved in the glucose- fructose
bond and hence is not free to form the aldehyde in solution.
On the other hand, glucose, a reducing sugar, reacts with
Fehling's reagent to form an orange to red precipitate.
Fehling's reagent is commonly used for reducing sugars but is
known to be not specific for aldehydes. For example, fructose gives
a positive test with Fehling's solution too, because fructose is
converted to glucose and mannose under alkaline conditions. The
conversion can be explained by the keto-enol tautomerism.
The reduction of Fehling solution using fructose is not only to
be attributed to the fact that the ketose is isomerized into an aldose.
The treatment of fructose with alkali - e.g. Fehling solution - causes
even decompostion of the carbon chain. More products with
reducing capability are formed.

Note:
Fehling's test takes advantage of the ready reactivity of
aldehydes by using the weak oxidizing agent cupric ion (Cu2+) in
alkaline solution. In addition to the copper ion, Fehling's solution
contains tartrate ion as a complexing agent to keep the copper ion in
solution. Without the tartrate ions, cupric hydroxide would precipitate
from the basic solution. The tartrate ion is unable to complex
cuprous ion Cu+, so the reduction of Cu2+ to Cu+ by reducing
sugars results in the formation of an orange to red precipitate of
Cu2O. Copper-tartrate-complex
CuSO4 + NaOH Cu(OH)2 + Na2SO4
Cu(OH)2 + HO-CH-COONa O-CH-COONa + 2H2O
HO-CH-COOK Cu
O-CH-COOK

2OH- + Cu++ + HO-CH-COONa

HO-CH-COOK
R-CHO + Cu++ R-COOH + Cu+
Cu+ + OH- CuOH W.∆B. Cu2O
Red ppt
3. Benedict's test:
Benedict modified the Fehling's test to produce a single solution
which is more convenient for tests as well as being more stable than
Fehling's reagent.
Preparation of Benedict's reagent:
Dissolve 173 g of sodium citrate and 100 g sodium carbonate in
about 800 ml of warm water. Filter through a fluted filter paper into a
100 ml measuring cylinder and make up to 850 ml with water.
Meanwhile dissolve 17.3 g of copper sulfate in about 100 ml of water
and make up to 150 ml. Pour the first solution into a 2-liter beaker
and slowly add the copper sulfate solution with stirring.
Principle:
The copper sulfate (CuSO4) present in Benedict's solution reacts
with electrons from the aldehyde or ketone group of the reducing
sugar. Reducing sugars are oxidized by the copper ion in solution to
form a carboxylic acid and a reddish precipitate of copper (I) oxide.

Method:
1. Add 5 drops of the test solution to 2 ml of Benedict's reagent
and place in a boiling water bath for 5 min. Orange-brown color
is appeared.
2. Compare the sensitivity of Benedict's and Fehling's test, using
increasing dilutions of 1% glucose.
3. Both fehling's and benedict's test are used as a test for the
presence of reducing sugars such as glucose, fructose, galactose,
lactose and maltose, or more generally for the presence of
aldehydes (except aromatic ones). It is often used in place of
Fehling's solution.
CuSO4 + Na2CO3 CuCO3 + Na2SO4
CH2-COONa
CuCO3 + HO-C-COONa H2CO3 +
CH2-COONa
CH2COONa NaOOC-CH2 CH2-COONa
++ - HOH
HO-C-COONa + Cu + 2OH NaOOC-C O C-COONa
CH2-COONa NaOOC-CH2 CH2-COONa
++ +
R-CHO + Cu R-COOH + Cu
Cu +
+ OH -
CuOH W.∆B. Cu2O
Red ppt
4. Barfoed’s Test:
Barfoed's test is used to detect the presence of monosaccharide
(reducing) sugars in solution. Barfoed's reagent, a mixture of
ethanoic (acetic) acid and copper (II) acetate, is combined with the
test solution and boiled. A red copper (II) oxide precipitate is formed
will indicates the presence of reducing sugar. The reaction will be
negative in the presence of disaccharide sugars because they are
weaker reducing agents. This test is specific for monosaccharides.
Due to the weakly acidic nature of Barfoed's reagent, it is reduced
only by monosaccharides.
Preparation of Barfoed's reagent:
Dissolve 13.3 g of copper acetate in about 200 ml of water and add
1.8 ml of glacial acetic acid.
Method:
• Add 1 ml of the test solution to 2 ml of Barfoed's reagent.
• Boil for 1 min and allow to stand.

5. Iodine Test:
This test is used for the detection of starch in the solution. The
blue black colour is due to the formation of starch-iodine complex.
Starch contain polymer of α-amylose and amylopectin which
forms a complex with iodine to give the blue black colour.

Iodine forms colored adsorption complexes with polysaccharides,

starch gives a blue color with iodine, while glycogen and partially
hydrolyzed starch react to form red- brown colors.
Method:
Acidify the test solution (1% starch, glycogen or cellulose) with dilute
HCl, then add two drops of iodine (0.005 N in 3% KI) and compare
the colors obtained with that of water and iodine.

6. Seliwanoff’s test:
It is a color reaction specific for ketoses. When conce: HCl is
added. ketoses undergo dehydration to yield furfural derivatives
more rapidly than aldoses. These derivatives form complexes with
resorcinol to yield deep red color. The test reagent causes the
dehydration of ketohexoses to form 5-hydroxymethylfurfural. 5-
hydroxymethylfurfural reacts with resorcinol present in the test
reagent to produce a red product within two minutes. Aldohexoses
reacts so more slowly to form the same product.
 Results
Test Monosaccharide Disaccharide Polysaccharide
Glucose Fructose Sucrose Lactose Starch Dextrin

1. Molisch

2. Fehling

3. Benedict
4. Barfoed
5. Iodine
6.Seliwanoff
Lactose

Milk sugar or 4-O-β-D-galactopyranosyl-D-glucose.

This reducing disaccharide is obtained as the α-D anomer (see
formula, where the asterisk indicates a reducing group); the melting
point is 202°C (396°F). Lactose is found in the milk of mammals to
the extent of approximately 2–8%. It is usually prepared from
whey, which is obtained by a by-product in the manufacture of
cheese.

Hydrolysis Test:
This test is used to convert sucrose (non-reducing
disaccharide) to glucose and fructose (reducing mono saccharides).
Principle:
Sucrose is the only non-reducing disaccharide so it does not
reduce the Cu++ solution (Bendict's and Fehling's test) because the
glycosidic bond is formed between the two hemiacetal bonds. So
there is no free aldehydic or ketonic group to give potitive reducing
properties. This bond can be hydrolysed and the individual
components of sucrose (glucose + fructose) are then able to give
positive reducing test.
Method:
• 6ml of a sucrose solution is placed in two test tubes.
• Add two drops of concentrated hydrochloric acid (HCl) to only
one tube.
• Heat the tubes in boiling water bath for 15 minutes.
 LIPIDS
Determination of triglycerides
Esters of glycerol and fatty acids are known as glycerides. The
trihydric alcohol glycerol can be esterified to give mono-, di-, and
triglycerides. The fatty acids may be the same or different. On
saponification, free glycerol and fatty acids are obtained:

Naturally occuring glycerides are called fats or oils depending on

whether they are solid or liquid at room temp.
Animal fat is made up largely of triglycerides containing fully
saturated fatty acids with straight chains and an even number of
carbon atoms.
Methods for the quantitation of plasma triglycerides include chemical
and enzymatic methods. The chemical methods require solvent
extraction of the plasma to solublize triglycerides and to denature and
remove protein. The extract is treated with an adsorbent material to
remove phospholipids and interfering substances; isopropanol extracts
are treated with a zeolite mixture or with alumina, and chloroform
extracts are treated with silicic acid. Once isolated and purified,
triglycerides are quantitated by either chemical or enzymatic reactions
directed against their glycerol component.

In the chemical methods: glycerol is released from triglycerides in

the purified extracts by saponification with alcoholic potassium
hydroxide. The glycerol is then oxidized to formaldehyde by sodium
periodate. The formaldehyde is reacted with a chromotropic-sulfuric
acid mixture to form a product that absorbs at 570 nm.

Qualitative tests of lipids:

1- Solubility test:
Principle:
Fats are not dissolved in water due to their nature, non-polar
(hydrophobic), but it is soluble in organic solvents such as
chloroform, benzene, and boiling alcohol. Different lipids have
ability to dissolve in different organic solvent. This property enable
us to separate a mixture of fat from each other for example,
undissolve phosphatide lipid in acetone; undissolve of cerebroside,
as well as sphingomyline in the ether.
Method:
• Place 0.5ml of oil in 6 test tubes clean, dry containing 4ml of
different solvents (acetone, chloroform and ether and ethanol,
cold ethanol and hot water),
• Shake the tubes thoroughly, then leave the
solution for about one minute,
• Note if it separated into two layers , the oil are not
dissolve; but if one layer homogeneous transparent formed ,
oil be dissolved in the solvent.

2- Saponification test:
Triacyl glycerol can be hydrolyzed into their component
fatty acids and alcohols. This reaction can also be carried out in the
laboratory by a process called saponification where the hydrolysis is
carried out in the presence of a strong base (such as NaOH or KOH).
Principle:
Saponification is a process of hydrolysis of oils or fat with
alkaline and result in glycerol and salts of fatty acids (soap) and can be
used the process of saponification in the separation of saponifiable
materials from unsaponified (which are soluble in lipid). The process
of saponification as follows:

Soap can be defined as mineral salts of fatty acids. The soap is

soluble in water but insoluble in ether. Soap works on emulsification
of oils and fats in the water as it works to reduce the attraction surface
of the solution.
Method:
• Place 2 ml of oil in a large
test tube (or flask).
• Add 4 ml of alcoholic potassium hydroxide (preferably add
little small pieces of porcelain to regulate the boiling point).
• Boil the solution for 3 minutes. After this period, make sure
it is perfectly saponification process, by taking a drop of the
solution and mix with the water if oil separated indicates
that the non-completion of the saponification. In this case,
continued to boil until all the alcohol evaporates.
• Take the remaining solid material (soap) and add about 30
ml of water and keep it for the following tests.
• Shake the solution after it cools and
noted to be thick foam.

3- Copper acetate test:

This test is used to distinguish between oil or neutral fat
and fatty acid saturated and unsaturated.
Principle:
The copper acetate solution does not react with the oils (or fats),
while saturated and unsaturated fatty acids react with copper acetate
to form copper salt.
Copper salt formed in the case of unsaturated fatty acids can only be
extracted by petroleum ether.

Method:
• Take three test tubes put 1 / 2 g of each sample and then
added 3 ml of petroleum ether and an equal volume of a
solution of copper acetate.
• Shake the tube and leave it for some time.
• In the case of olive oil notice that petroleum ether upper layer
containing the dissolved oil and appears colorless, aqueous
solution remains blue in the bottom.
• In the case of oleic acid the upper layer of petroleum ether
becomes green as a result of copper oleate. The lower layer
becomes less in blue.
• In the case of stearic acid notice that the petroleum ether
upper layer remains colorless, while consists of pale green
precipitate of copper stearate at the bottom.
 Proteins
The aim of this practical session is to:
1. Obtain a simplified knowledge about protein structure.
2. Practically apply this knowledge by performing some protein color
and precipitation reactions.

Practical
Using the provided solutions of albumin (egg white) and gelatin
(animal collagenous material), perform the following:
A. General tests
B. Color reactions
C. Precipitation reactions

A. General tests for proteins

1- Biuret test:
Principle:
The biuret reagent (copper sulfate in a strong base) reacts with
peptide bonds in proteins to form a blue to violet complex known as
the “biuret complex”. N.B. Two peptide bonds at least are required for
the formation of this complex.

Biuret assay

Method:
(a) Add 2 ml of protein solution
to 1 ml of NaOH.
(b) Add one drop of Fehling's A.
(c)Shake this solution carefully
and then you see purple
color.
B. Color reactions of proteins
 1- Reduced sulfur test:
Principle:
Proteins containing sulfur (in cysteine and cystine) give a black
deposit of lead sulfide (PbS) when heated with lead acetate in alkaline
medium.
Procedure & observation:
1- To 1 ml of protein solution in a test tube, add 2 drops of 10%
sodium hydroxide solution and 2 drops of lead acetate.
2- Mix well and put in a boiling water bath for few minutes; a black
deposit is formed with albumin and gelatin gives negative result.

2- Xanthoproteic acid test:

This test is used to differentiate between aromatic amino acids
which give positive results and other amino acids.
Amino acids containing an aromatic nucleus form yellow nitro
derivatives on heating with concentrated HNO3. The salts of these
derivatives are orange in color.
Principle:
Concentrated nitric acid reacts with the aromatic rings that are
derivatives of benzene giving the characteristic nitration reaction.
Amino acids tyrosine and tryptophan contain activated benzene rings
which are easily nitrated to yellow colored compounds. The aromatic
ring of phenyl alanine dose not react with nitric acid despite it
contains a benzene ring, but it is not activated, therefore it will not
react.
Caution:
Concentrated HNO3 is a toxic, corrosive substance that can
cause severe burns and discolor your skin. Prevent eye, skin and
cloth contact. Avoid inhaling vapors and ingesting the compound.
Gloves and safety glasses are a must; the test is to be performed in a
fume hood.
Procedure & observation:
1- To 2 ml of protein solution in a test tube, add 2 drops of
concentrated nitric acid.
2- A white precipitate is formed and upon heating in a boiling water
bath, it turns yellow with “tyrosine” and orange with the essential
amino acid “tryptophan” indicating a high nutritive value.
3- Millon’s test:
This test is specific for tyrosine, the only amino acid containing
a phenol group, a hydroxyl group attached to benzene ring.
Principle:
In Milon's test, the phenol group of tyrosine is first nitrated
by nitric acid in the test solution. Then the nitrated tyrosine
complexes mercury ions in the solution to form a brick-red solution
or precipitate of nitrated tyrosine, in all cases, appearance of red color
is positive test.
Note: all phenols (compound having benzene ring and OH attached to
it) give positive results
in Millon’s test.
Procedure & observation:
1- To 2 ml of protein solution in a test tube, add 3 drops of Millon’s
reagent.
2- Mix well and heat directly on a small flame.
3- A white ppt is formed with albumin and casein (but not gelatin);
the ppt gradually turns into brick red.

4- Hopkins-Colé test:
Principle:
Hopkins-Colé reagent (magnesium salt of oxalic acid) gives
positive results with proteins containing the essential amino acid
“tryptophan” indicating a high nutritive value.
Procedure & observation:
1- To 1 ml of protein solution in a test tube, add 1 ml of Hopkins-
Colé reagent and mix well.
2- Incline the test tube and slowly add 1 ml of concentrated H2SO4
on the inner wall of the test tube to form 2 layers.
3- Put the test tube in a boiling water bath for 2 minutes.
4- A reddish violet ring is formed at the junction between the 2
layers with albumin and casein; gelatin gives negative results.
 C. Precipitation reactions of proteins
1. Precipitation by heavy metals:
Principle:
Heavy metals (e.g. Hg2+, Pb2+, Cu2+) are high molecular
weight cations.
The positive charge of these cations counteracts the negative charge of
the carboxylate group in proteins giving a precipitate.
Procedure & observation:
1- To 1 ml of protein solution in a test tube, add 1 drop of lead
acetate; a white ppt is obtained.
2- To 1 ml of protein solution in a test tube, add 1 drop of 10%
copper sulfate; a blue ppt is obtained.

2. Precipitation by alkaloidal reagents:

Principle:
Alkaloidal reagents (e.g. tannate & trichloroacetate) are high
molecular weight anions. The negative charge of these anions
counteracts the positive charge of the amino group in proteins giving a
precipitate.
Procedure & observation:
1- To 1 ml of protein solution in a test tube, add tannic acid drop
wise until a buff ppt is obtained.
2- To 1 ml of protein solution in a test tube, add 1 ml of
trichloroacetic acid (TCA); a white ppt is obtained.
N.B. Precipitation of proteins by heavy metals and alkaloidal reagents
indicates the presence of both negative and positive charges and hence
the amphoteric nature of proteins.

3. Precipitation by denaturation:
a. Denaturation by heat (heat coagulation test):
Principle:
Heat disrupts hydrogen bonds of secondary and tertiary protein
structure while the primary structure remains unaffected. The protein
increases in size due to denaturation and coagulation occurs.
Procedure & observation:
1- Put 2 ml of protein solution in a test tube, incline it and heat to
boiling.
2- A permanent clotting and coagulation is obtained with albumin
only.
b. Denaturation by acids (Heller’s test):
Principle:
Nitric acid causes denaturation of proteins with the formation of
a white ppt (this differs from the nitration reaction in “xanthoproteic
acid test”).

Procedure & observation:

1- Put 2 ml of concentrated nitric acid in a test tube.
2- Incline the tube and slowly add 1 ml protein solution drop wise to
form a layer above the nitric acid layer.
3- A white ring is formed at the interface between the 2 layers.

4. Fractional precipitation by ammonium sulfate (salting out):

Principle:
Protein molecules contain both hydrophilic and hydrophobic
amino acids.
In aqueous medium, hydrophobic amino acids form protected areas
while hydrophilic amino acids form hydrogen bonds with surrounding
water molecules (solvation layer).
When proteins are present in salt solutions (e.g. ammonium sulfate),
some of the water molecules in the solvation layer are attracted by salt
ions. When salt concentration gradually increases, the number of water
molecules in the solvation layer gradually decreases until protein
molecules coagulate forming a precipitate; this is known as “salting
out”.
As different proteins have different compositions of amino acids,
different proteins precipitate at different concentrations of salt solution.
Procedure & observation:
1- To 2 ml of egg-white solution (containing both albumin &
globulin), add an equal volume of saturated ammonium sulfate
solution; globulin is precipitated in the resulting half saturated
solution of ammonium sulfate.
2- Separate globulin by centrifugation and recover the clear
supernatant.
3- Add ammonium sulfate crystals gradually to the clear supernatant
until full saturation occurs; another precipitate (albumin) is
obtained.
4- Separate albumin by centrifugation.
N.B. The reason for the precipitation of globulin and albumin at
different ammonium sulfate concentration could be that the solvation
layer around globulin is looser and thinner than that around albumin.
Therefore, globulin needs only half-saturated ammonium sulfate to
loose its solvation layer while albumin looses its solvation layer in a
fully saturated ammonium sulfate solution.
 Laboratory exercise:
Using the provided solutions of albumin and gelatin perform the tests
in the table below and write down your observations.
 Enzyme assay
Enzyme assays are laboratory methods for measuring enzymatic
activity. They are vital for the study of enzyme kinetics and enzyme
inhibition.
Enzyme units:
Amounts of enzymes can either be expressed as molar amounts, as
with any other chemical, or measured in terms of activity, in enzyme
units.
Enzyme activity = moles of substrate converted per unit time,
mol/min or mol/sec. Enzyme activity is a measure of the quantity of
active enzyme present and is thus dependent on conditions, which
should be specified.
The SI unit is the katal, 1 katal = 1 mol s-1, but this is an excessively
large unit.

UV/VIS Spectrophotometer.

Types of assay:
All enzyme assays measure either the consumption of substrate or
production of product over time.
A large number of different methods of measuring the
concentrations of substrates and products exist and many enzymes
can be assayed in several different ways. Biochemists usually
study enzyme-catalyzed reactions using four types of
experiments:
1. Initial rate experiments:
When an enzyme is mixed with a large excess of the substrate, the
enzyme-substrate intermediate builds up in a fast initial transient.
Then the reaction achieves a steady- state kinetics in which
enzyme substrate intermediates remains approximately constant
over time and the reaction rate changes relatively slowly. Rates are
measured for a short period after the attainment of the quasi-
steady state, typically by monitoring the accumulation of product
with time. Because the measurements are carried out for a very short
period and because of the large excess of substrate, the
approximation free substrate is approximately equal to the initial
substrate can be made. The initial rate experiment is the simplest to
perform and analyze, being relatively free from complications such as
back-reaction and enzyme degradation. It is therefore by far the most
commonly used type of experiment in enzyme kinetics.

2. Progress curve experiments:

In these experiments, the kinetic parameters are determined from
expressions for the species concentrations as a function of time. The
concentration of the substrate or product is recorded in time after the
initial fast transient and for a sufficiently long period to allow the
reaction to approach equilibrium. We note in passing that, while they
are less common now, progress curve experiments were widely used
in the early period of enzyme kinetics.

3. Transient kinetics experiments:

In these experiments, reaction behavior is tracked during the initial
fast transient as the intermediate reaches the steady-state kinetics
period. These experiments are more difficult to perform than either of
the above two classes because they require rapid mixing and
observation techniques.

4. Relaxation experiments:
In these experiments, an equilibrium mixture of enzyme, substrate and
product is perturbed, for instance by a temperature, pressure or pH
jump, and the return to equilibrium is monitored. The analysis of
these experiments requires consideration of the fully reversible
reaction. Moreover, relaxation experiments are relatively
insensitive to mechanistic details and are thus not typically used
for mechanism
identification, although they can be under appropriate conditions.

Enzyme assays can be split into two groups according to their

sampling method:
1. Continuous assays, where the assay gives a continuous reading of
activity.
2. Discontinuous assays, where samples are taken, the reaction
stopped and then the concentration of substrates/products determined.
Temperature-controlled cuvette holder in a spectrophotometer.
Continuous assays are most convenient, with one assay giving the
rate of reaction with no further work necessary. There are many
different types of continuous assays. Spectrophotometric assay:
In spectrophotometric assays, you follow the course of the reaction
by measuring a
change in how much light the assay solution absorbs. If this light is
in the visible region you can actually see a change in the color of the
assay, these are called colorimetric assays.
UV light is often used, since the common coenzymes NADH and
NADPH absorb UV light in their reduced forms, but do not in their
oxidized forms. An oxido-reductase using NADH as a substrate
could therefore be assayed by following the decrease in UV
absorbance at a wavelength of 340 nm as it consumes the coenzyme.

Direct versus coupled assays

Coupled assay for hexokinase using glucose-6-phosphate
dehydrogenase.
Even when the enzyme reaction does not result in a change in the
absorbance of light, it can still be possible to use a
spectrophotometric assay for the enzyme by using a coupled assay.
Here, the product of one reaction is used as the substrate of another,
easily-detectable reaction.

Fluorometric assay:
Fluorescence is when a molecule emits light of one wavelength after
absorbing light of a different wavelength. Fluorometric assays use a
difference in the fluorescence of substrate from product to measure
the enzyme reaction. These assays are in general much more
sensitive than spectrophotometric assays, but can suffer from
interference caused by impurities and the instability of many
fluorescent compounds when exposed to light.
An example of these assays is again the use of the nucleotide
coenzymes NADH and NADPH. Here, the reduced forms are
fluorescent and the oxidised forms non- fluorescent. Oxidation
reactions can therefore be followed by a decrease in
fluorescence and reduction reactions by an increase. Synthetic
substrates that release a fluorescent dye in an enzyme-catalyzed
reaction are also available, such as 4- methylumbelliferyl-β-D-
galactoside for assaying β-galactosidase.

Calorimetric assay:

Chemiluminescence of Luminol

Calorimetry is the measurement of the heat released or

absorbed by chemical reactions. These assays are very general,
since many reactions involve some change
in heat and with use of a microcalorimeter, not much enzyme or
substrate is required. These assays can be used to measure reactions
that are impossible to assay in any other way.
Chemiluminescent assay:
Chemiluminescence is the emission of light by a chemical reaction.
Some enzyme reactions produce light and this can be measured to
detect product formation. These types of assay can be extremely
sensitive, since the light produced can be captured by photographic
film over days or weeks, but can be hard to quantify, because not all
the light released by a reaction will be detected.
The detection of horseradish peroxidase by enzymatic
chemiluminescence (ECL) is a common method of detecting
antibodies in western blotting. Another example is the enzyme
luciferase, this is found in fireflies and naturally produces light from
its substrate luciferin.
Light Scattering assay:
Static Light Scattering measures the product of weight-averaged
molar mass and concentration of macromolecules in solution. Given a
fixed total concentration of one or more species over the
measurement time, the scattering signal is a direct measure of the
weight-averaged molar mass of the solution, which will vary as
complexes form or dissociate. Hence the measurement quantifies the
 stoichiometry of the complexes as well as kinetics. Light scattering
assays of protein kinetics is a very general technique that does not
require an enzyme.

Discontinuous assay:
Discontinuous assays are when samples are taken from an
enzyme reaction at intervals and the amount of product production
or substrate consumption is measured in these samples.
Radiometric assay:
Radiometric assays measure the incorporation of radioactivity into
substrates or its release from substrates. The radioactive isotopes
most frequently used in these assays are 14C, 32P, 35S and 125I.
Since radioactive isotopes can allow the specific labelling of a single
atom of a substrate, these assays are both extremely sensitive and
specific. They are frequently used in biochemistry and are often the
only way of measuring a specific reaction in crude extracts (the
complex mixtures of enzymes produced when you lyse cells).
Radioactivity is usually measured in these procedures using a
scintillation counter.

Chromatographic assay:
Chromatographic assays measure product formation by
separating the reaction mixture into its components by
chromatography. This is usually done by high- performance
liquid chromatography (HPLC), but can also use the simpler
technique of thin layer chromatography. Although this approach can
need a lot of material, its sensitivity can be increased by labelling the
substrates/products with a radioactive or fluorescent tag. Assay
sensitivity has also been increased by switching protocols to improved
chromatographic instruments (e.g. ultra-high pressure liquid
chromatography) that operate at pump pressure a few-fold higher than
HPLC instruments (see HPLC#Pump_pressure).
Factors to control in assays
• Salt Concentration: Most enzymes cannot tolerate extremely high
salt concentrations. The ions interfere with the weak ionic bonds of
proteins. Typical enzymes are active in salt concentrations of 1-500
mM. As usual there are exceptions such as the halophilic (salt
loving) algae and bacteria.

• Effects of Temperature: All enzymes work within a range of

temperature specific to the organism. Increases in temperature
generally lead to increases in reaction rates. There is a limit to the
increase because higher temperatures lead to a sharp decrease in
 reaction rates. This is due to the denaturating (alteration) of protein
structure resulting from the breakdown of the weak ionic and
hydrogen bonding that stabilize the three dimensional structure of the
enzyme. The "optimum" temperature for human enzymes is
usually between 35 and
40 °C. The average temperature for humans is 37 °C. Human
enzymes start to denature quickly at temperatures above 40 °C.
Enzymes from thermophilic archaea found in the hot springs are
stable up to 100 °C. However, the idea of an "optimum" rate of an
enzyme reaction is misleading, as the rate observed at any
temperature is the product of two rates, the reaction rate and the
denaturation rate. If you were to use an assay measuring
activity for one second, it would give high activity at high
temperatures, however if you were to use an assay measuring
product formation over an hour, it would give you low activity
at these temperatures.

• Effects of pH: Most enzymes are sensitive to p H and have

specific ranges of activity. All have an optimum pH. The pH can
stop enzyme activity by denaturating (altering) the three dimensional
shape of the enzyme by breaking ionic, and hydrogen bonds. Most
enzymes function between a pH of 6 and
8; however pepsin in the stomach works best at a pH of 2 and
trypsin at a pH of 8.

• Substrate Saturation: Increasing the substrate concentration

increases the rate of reaction (enzyme activity). However, enzyme
saturation limits reaction rates. An enzyme is saturated when the
active sites of all the molecules are occupied most of the time. At the
saturation point, the reaction will not speed up, no matter how much
additional substrate is added. The graph of the reaction rate will
plateau.

• Level of crowding, large amounts of macromolecules in a

solution will alter the rates and equilibrium constants of
enzyme reactions, through an effect called macromolecular
crowding.
Protocol for Enzyme Assay
• Isolation of enzymes
• Different enzymes can be isolated from different sources. It
depends on the type of enzyme and the rich source of the required
enzyme.
• Adenosine triphosphatase (ATPase) can be isolated from cells
 and nervous tissues.
• Acetyl cholinesterase (AChE) can be isolated from red blood
cells.
• Butyrile choline esterase (BuChE) can be isolated from plasma.
• Hepatic soluble enzymes (GPT, GOT, AlP) can be isolated from
blood.
Tissues should be chopped, homogenized in buffer solution and then
centrifuged at certain round per minute (rpm).
Pellets or supernatant are taken as a source of enzyme. Pellets may
be suspended in buffer while supernatant is diluted with buffer.
In case of blood, sample should be centrifuged at 5000 rpm to
separate plasma and red blood cells (RBC’s)
References:

• Abousalah, K. and Alnaser, A., 1996, Principles of practical

biochemistry.
• Farid Shokry Ataya, 2007, Practical Biochemistry. AlRoshd
Publisher, Riyadh, Saudi Arabia.
• Milio, F. R. and Loffredo, W. M., 1995, Qualitative Testing for
Amino Acids and Proteins, modular laboratory program in
chemistry, REAC 448.
• Collection from internet researches.
• Dr. AHMED KHAMIS MOHAMED SALAMA (2013):
Practical BIOCHEMISTRY. Medical Laboratories Dept.,
Colleges compound at Zulf