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Practical Principles
Practical Principles
Practical Principles
Resorcinol:
The reagent Resorcinol is a phenolic compound. Generally ketoses
are more rapidly dehydrated than aldoses, this is the base of this
experiment. First ketose like fructose, is dehydrated into
hydroxymethyl furfural by a strong acid like HCl. This derivative
reacts with resorcinol in a series of condensation reaction to
produce a deep cheery red coloured complex. Thio-urea assists in
stabilizing this colour. This reaction is specific for fructose, the
intensity of colour is directly proportional to the concentration of
fructose. The optical density is measured at 530nm.
Phenol H2SO4:
Phenol sulphuric acid can aid in detection of virually all classes of
carbohydrates, it can also help in identifying all glycopolymers and
glycol-conjugates. It is a simple and rapid colorimetric method. A
polyhydroxy aldehyde like glucose undergoes dehydration in the
presence of concentrated mineral acid like H2SO4. This
dehydration results in formation of 5 hydroxymethyl furfural,
which reacts with phenol and forms bright coloured complex. The
intensity of colour is directly proportional to the concentration of
glucose. The optical density is measured at 470 nm.
Anthrone method:
This method can be used to identify saccharide moiety present
in any given sample. it is a simple and rapid colorimetric method
for identification of mono, di and polysaccharides. First the
polysaccharides are hydrolysed to their monomeric forms.
Dehydration of these monosaccharides forms hydroxymethyl
furfural, which reacts with the Anthrone which is a tricyclic
aromatic ketone, to give a bluish-green coloured complex. The
optical density is measured at 620nm.
Lowry’s method:
The principle behind this method lies in the reactivity of
nitrogen in peptide bonds to the copper sulphate in alkaline
condition and the subsequent reduction of Folin-Ciocalteay
phosphomolybdic or phosphotungstic acid to
heteropolymolybdenum blue by copper-catalysed oxidation of
aromatic amino acids or phenolic compound. The intensity of
colour is directly proportional to protein concentration. The
optical density is measured at 660nm. This method is sensitive
to pH changes hence the pH should be maintained between 10-
10.5.
Bradford assay:
This assay is based on the absorbance shift between three
forms of Coomassie blue G-250 dye. Under acidic condition it is
in double protonated form showing red colour, upon binding to
protein it is in more stable unprotonated form showing blue
colour. The two bond interactions taking place in this assay are
as follows:
1. Red form of the dye donates electrons to the ionisable group
of the protein which exposes its hydrophobic pockets.
2. These hydrophobic pockets on the protiens non-covalently
bind to the non-polar region of the dye via Van der Waal’s
forces.
When the dye and the protein bind in acidic conditons its
maximum absorbance shifts from 465 to 595.
Caesin-
Casein is a conjugate protein- phosphoprotein majorly found in
milk, the salt of casein that can be encountered in milk is
calcium caseinate. In pure form it is amorphous, white, solid and
odourless. It exists in 4 subtypes as alpha 1 and 2, beta and k
form. It is insoluble in water. The isoelectric point of this protein
is 4.5 and the pH of milk is 6.6, casein possess negative charge
at this point and hence is solubilized as salt. The protein begins
to aggregate and precipitates at its isoelectric point.
Vit. C by DCPIP:
It is the titrometric assay of vit.C or ascorbic acid, based on
simple oxidation-reduction reaction. Vit.C is an important
dietary factor that is not synthesized by the body. In this the
ascorbic acid reduces 2,6 dicholorophenol indophenol dye to
colourless leuco base, it itself gets converted to anhydrous Vit. C
or dehydro ascorbic acid. The dye is pink in oxidised form which
indicates the end point.
SEM - II
Alpha amylase:
Alpha amylase is a glycosidic hydrolase, majorly produced by the
salivary glands and pancreas. it catalyses the cleveag of of
a 1 -> 4 glycosidic bonds of starch which is a
homopolysaccharide and gives maltose, maltotriose , glucose
and limit dextrins. The activity of alpha amylase is measured by
colorimetric method using 3,5 dinitro salicylic acid reagent. The
amount of starch hydrolysed is directly proportional to the
enzyme activity.
Temperature:
as with many chemical reactions, the rate of enzyme catalysed
reaction increases with increase in temperature. However, at
higher temperatures (which for most of the enzymes is 50*C)
the rate decreases due to denaturation of the enzymes as they
are proteins in nature, this is caused by breakage of hydrogen
bonds and change in arrangement of the active site and they
lose their function . The temperature at which maximum
velocity is reached is called the optimum temperature. The
velocity increases with increase in temperature and it begins to
drop drastically at temperature above its optimum value and
bell shaped curve is observed.
Metal ion:
Metal ions usually act as cofactors to certain enzymes, when
present in trace amounts these metal ions have shown to assist
in the enzyme activity. They usually work as the prosthetic
group. But, when present in larger amounts they may interfere
with the catalytic efficiency of the enzyme by reducing the rate
or all together halting the reaction. Hence when the
concentration of metal ions increases the catalytic activity is
decreased. These bind to the site other than the active site and
inhibit the enzyme from functioning.
Amyloglucosidase:
Amyloglucosidase is an enzyme which acts primarily on starch
and glycogen to split them in to their monomeric forms that is
glucose. This enzyme is more efficient that the salivary amylase
as it can cleave both a 1 -> 4 and a 1 -> 6 linkages. The most
common source for amyloglucosidase is A. niger. The activity
can be detected by DNSA method. The action of enzyme is
directly proportional to the activity of enzyme and the optical
density is measured at 530 nm.
Invertase:
Invertase also known as B- fructofuranosidase is named as such
because, it irreversibly hydrolyses sucrose into fructose and
glucose, it shows high activity over wide range of pH. It is
mostly found in yeast species. Its activity is given as ug of
reducing sugar/ ml/ min. The enzyme activity is determined by
DNSA method. The intensity of coloured is directly proportional
to the concentration of reducing sugar. The optical density is
measeured at 530 nm.
Chromatography:
Paper chromatography is a planar partition chromatography
technique used to separate compounds based on the
differential solublity in the stationary phase and mobile phase.
Stationary phase usually is a water ( atmospheric moisture) this
is held within the fibers of the filter paper mobile phase is
usually some sort of volatile liquid. All of the proteins in nature
comprise of chain sequence of made of 20 different amino
acids, all these are similar but differ in their R group. These can
be sperated based on that factor. The mixture separated
mighrates at different points on the paper which can be
identified using the RF value.
Saccharomyeces cerevisiae:
In order to study the activity of enzyme invertase produced by
yease called saccharomyces cerevisiae, it is necessary to first
immobilize the enzyme. It can be done by performing various
methods but the one used here is carried out by matrix
entrapment method using sodium alginate that reacts with
CaCl2 to form the beads which are further used for studying the
activity by using sucrose. The activity Is determined by DNSA
method.