Name: AQSA AROOJ

Practicals of biochemistry
Bs zoology
5th semester
21bsz004
To : Mam LAIBA
Submission date:30:11:2023
Day: Friday

1:Separation and identification of amino acids by paper

chromatography
The aim of this experiment is to examine amino acids by paper chromatography, by looking at the Rf
values and finding out the unknown amino acids.

PROCEDURE
 here we have use Whitman paper 1 and you can also use normal filter paper for this purpose.
 Filter paper has been cut into regular shapes where we have prepare 3 samples solution of
amino acids of 10 ml each.
 Solution A and solution B are the standard solutions of amino acids
 the third solution solution of sample mixture of a Amino acids A and B which we have prepared
for demonstration purpose
 separation paper chromatography by mixing small volume of sample A and B draw a starting line
and about 1.5 cm from the bottom of the filter by pencil.
 Draw another line the end up to which we will allow equal to move
 Mark the three points by pencil on the starting of equally spaced on which we use volume of
sample A press the capillary tube gently two paper to transfer the same volume of sample on
the mark 1 at one end of starting line
 Again apply the sport of standard solution B in the similar way on the mark point at starting
point using another capillary tube.
 Again apply third sport of sample C of the amino acid on 3 rd mark of starting line you can also
label each port below the starting for its identification we have stepped are thread on the top of
the filter paper to hold and support the filter paper in the upright of the type of development
while we have prepared 10 ml of an hydride solution by mixing and N butanol and glacial acetic
acid and water.
 In ratio of 4:1:5 and then allow the beaker to separate with the mobile face present in paper
immersed the filter paper vertically in the Centre of the beaker.
 Note that the level of the mobile phase is in the beaker and the sports should not be deemed
into the solvent comes the beaker with battery plate and then the mobile face to move over the
filter paper up to the end of the capillary action.
 Paper chromatography is based on the principle of precipitation here the corporate of the
mixture is partition of two liquid phase one is stationary and in which water held in the pour of
the filter paper.
 Second is mobile face the component in the mixture then separate by difference of component
toward the stationery phase and mobile sale when they solvent touches the end line which is at
the top of the filter paper take place at the paper.
 From the beaker and mark the the solvent front the pencil at the filter paper.
 Filter paper to draw at the room temperature and you can see the spot of amino acids are not
visible so after drying the filter paper at room temperature spray the filter paper with the
anhydride reagent.
 After spraying dry the filter paper in hot air oven 100 degree about 5 to 10 minutes ninhydrin
react with the amino acid to give bluish violet colour after drawing in the hot women you can
see the blue violet colour of amino acid
 After this outline the spot of the amino acid on the filter paper by the pencil and mark the centre
of each spot.
 After outlining each part of the amino acid and marking the Centre of each spot and record the
distance of each spot to the Centre of the spot.
 Also measure distance travel by the solvent from starting line and then calculate the FR value of
each spot.
 After calculating the RF value of each spot then identify the amino acid mixture by comparing it
with the RF of standard solution.

2:Practical to estimate free amino acids

Ninhydrin Reagent Preparation
A chemical test to detect the presence of

• Ammonia

• 1 and 2-degree amines

• Alpha-amino acid

• Proteins with free amino groups

 1. Extensively sensitive test for amino acids (can detect even microgram amounts)

2. Can be for both qualitative and quantitative assays.

3. Commonly used in the forensics to detect fingerprints

(terminal amines of lysine residues shed off from proteins present in finger-sweet secretions react with
ninhydrin)

About 100ml of any one of the following solvents

 Ethanol

 Acetones

 1:1 mixture of acetone and butanol


3:SDS polyacrylamide gel electrophoresis
Polyacrylamide gel electrophoresis (PAGE) is a technique widely used in biochemistry, forensic
chemistry, genetics, molecular biology and biotechnology to separate biological
macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility.
Mechanism of Polymerization

Polyacrylamide gels are formed by copolymerization of acrylamide and bis-acrylamide (“bis,”

N,N’-methylene-bis- acrylamide). The reaction is a vinyl addition polymerization initiated by a
free radical-generating system.
Procedure:
 The basic principle of PAGE is to separate analyses by passing them through the pores of a
polyacrylamide gel using an electric current.
 To achieve this, an acrylamide–bisacrylamide mix is polymerized (polyacrylamide) by the
addition of ammonium persulfate.
 Gel electrophoresis is a technique used to separate DNA fragments according to their size.
 DNA samples are loaded into wells (indentations) at one end of a gel, and an electric current is
applied to pull them through the gel.
 DNA fragments are negatively charged, so they move towards the positive electrode.
4: Estimation of DNA by diphenyl method:
To estimate the concentration of DNA by diphenylamine reaction.
Principle:
 This is a general reaction given by deoxygenates.
 The 2-deoxyribose of DNA, in the presence of acid, is converted to ω-hydroxilevulinic aldehyde,
which reacts with diphenylamine to form a blue colored complex, which can be read at 595 nm.
 The deoxyribose in DNA in the presence of acid forms β-hydroxylevulinaldehyde which reacts
with diphenylamine to give a blue color with a sharp absorption maximum .
  In DNA, only the deoxyribose of the purine nucleotides react, so that the value obtained
represents half of the total deoxyribose present.

 The procedure entails inserting a catheter into the peritoneal cavity, initially to aspirate blood or
fluid, and subsequently to infuse fluid and lavage the cavity, if necessary.
 The current role for DPL/DPA in the trauma patient is reviewed

5: Effect of pH on enzyme activity:

 Enzymes are affected by changes in pH

 The most favorable pH value – the point where the enzyme is most active – is known as
the optimum pH.
 This is graphically illustrated .
 Extremely high or low pH values generally result in complete loss of activity for most
enzymes.
 Enzymes are also proteins, which are also affected by changes in pH.
 Very high or very low pH will lead to the complete loss of the activity of most enzymes.
 The pH value at which the enzyme is most active is called the optimal pH value.
 These are the ionic and hydrogen bonds.
 Extreme pH. can therefore cause these bonds to break.
  When the bonds holding the complementary active site of an enzyme break, it cannot
bind to its substrate.
 The enzyme is thus denatured, as no enzyme-substrate or enzyme-product complexes
can form.
 In general, an enzyme has an optimum pH.
 Although most enzymes remain high activity in the pH range between 6 and 8, some
specific enzymes work well only in extremely acidic (i.e. pH <5.0) or alkaline (i.e. pH
>9.0) conditions.

6: Effect of temperature on enzyme activity:

Temperature:
 Raising temperature generally speeds up a reaction, and lowering temperature slows
down a reaction.
 However, extreme high temperatures can cause an enzyme to lose its shape (denature)
and stop working. pH: Each enzyme has an optimum pH range. Changing the pH outside
of this range will slow enzyme activity.
 At low temperatures, an increase in temperature increases the rate of an enzyme-
catalyzed reaction.
 At higher temperatures, the protein is denatured, and the rate of the reaction
dramatically decreases.
 An enzyme has an optimum pH range in which it exhibits maximum activity
 As the temperature increases, molecular motion increases resulting in more molecular
collisions. If, however, the temperature rises above a certain point, the heat will
denature the enzyme, causing it to lose its three-dimensional functional shape by
denaturing its hydrogen bonds.
 The proteins in enzymes are usually globular.
 The intra- and intermolecular bonds that hold proteins in their secondary and tertiary
structures are disrupted by changes in temperature and pH.
 This affects shapes and so the catalytic activity of an enzyme is pH and temperature
sensitive.
7:RNA estimation by orcinol method :
Principle:
  This is a general reaction for pentose and depends on the formation of furfural when the
pentose is heated with concentrated hydrochloric acid.
 Orcinol reacts with the furfural in the presence of ferric chloride as a catalyst to give a green
color, which can be measured at 665 nm.
 HiPer® RNA Estimation Teaching Kit is designed for rapid and accurate determination of RNA by
orcinol reagent.
 This method depends on the conversion of pentose in the presence of hot acid to furfural which
then reacts with orcinol to yield a green color.

Method:

 Method used to detect the presence of pentose’s with a test reagent consisting of orcinol, HCl
and ferric chloride.
 This test is used to detect the presence of a pentose in urine. In the presence of pentose, the
test reagent dehydrates pentose to form furfural

 The traditional method for assessing RNA concentration and purity is UV spectroscopy.
 The absorbance of a diluted RNA sample is measured at 260 and 280 nm.
 The nucleic acid concentration is calculated using the Beer-Lambert law, which predicts a linear
change in absorbance with concentration.
 The orcinol–sulfuric acid method, in which the recommended reagent is a solution of orcinol
(0.1%, m/v) in diluted sulfuric acid (70%, v/v), was the first to be adapted for use in an
automated carbohydrate analyzer.
 The components include orcinol, hydrochloric acid, and ferric chloride.
 A pentose, if present, will be dehydrated to form furfural which then reacts with the orcinol to
generate a colored substance.
8: estimation of amylase activity from blood serum:
Amylase (U/dL) x 1000 Amylase/Creatinine (U/g) = Creatinine (mg/dL) Up to 400
mylase activity exceeds 400 U/dL, the sample must be diluted with 0.85% NaCl. Multiply the
result by the appropriate dilution factor.
Dilute the sample so that the obtained value is around 80 and 320 U/dL.

Amylase activity is determined using a coupled enzymatic assay, which results in a colorimetric
(405 nm) product, proportional to the amount of substrate, ethylidene-pNP-G7, cleaved by the
amylase.
Method
 Estimation of serum amylase by the saccharimetric method is based upon the digestion of a starch
solution with serum followed by the determined- nation of the reducing substances produced.
 To quantify the activity of amylase from saliva samples, we are going to measure the rate at
which substrate (starch) is reacted away. Iodine readily reacts with starch to produce a purple
color.
 We will use an spectrophotometer to quantitatively determine the intensity of the purple color.
 For an amylase blood test, a health care professional will take a blood sample from a vein in your
arm, using a small needle.
 After the needle is inserted, a small amount of blood will be collected into a test tube or vial. You
may feel a little sting when the needle goes in or out.
 Amylase test measures the amount of amylase in blood or urine (pee).
 Amylase is an enzyme made by your pancreas and salivary glands that helps your body break
down carbohydrates.
 If an amylase test finds too much amylase in your blood or urine, it may indicate a pancreas
disorder or other health condition.