Practical
BIOCHEMISTRY
Dr. AHMED KHAMIS MOHAMED SALAMA
Medical Laboratories Dept., Colleges compound at Zulfi
February 2013
Principles of Biochemistry - Practical part
Dr. Ahmed Khamis Salama
PRACTICAL BIOCHEMISTRY
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. The extent of the normal range depends on the constituent
being measured. Human serum chloride has a relatively narrow normal range
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of values (95 – 107 m equiv / liter), while fasting blood glucose varies more
widely (60–100mg/100 ml)
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.
Significant figures
There is a big difference in the precision when someone says that a mass of
substance is 4 kg and other one says it is 4.000 kg.
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In the first case, we can assume only that the mass of substance is nearer 4
kg than it is to 3 kg or 5 kg. This means that the mass of substance is
between 3.5 and 4.5 kg.
In the second case, if we say that a substance has a mass of 4.000 kg, this
means that its mass is between 3.9995 and 4.0005 kg. The rule followed is
that the last figure in any measurement is only approximate.
So a mass written as 4 kg is said to have only one significant figure, while
4.000 kg has four significant figures. The precision with which a scientific
measurement was made is always indicated by the number of significant
figures quoted.
The final result of any estimation indicates the accuracy of the measurement.
Thus, if the result of say a serum calcium is given as 11.2 mg/100 ml then
this means that the serum calcium is less than 11.3 but more than 11.1;
while 11.21 means that the serum calcium lies between 11.20 and 11.22.
The final result should include all the significant figures; that is , all the
certain digits and the first uncertain (doubtful) digit of that number. As a
general rule when rounding off numbers, add 1 if the last figure dropped is 5
or more. Thus, the figure obtained by calculation may be 11.18 mg/ 100 ml,
but this is expressed as 11.2 mg/ 100 ml to three significant figures.
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.
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Dr. Ahmed Khamis Salama
Primary standards for the calibration of a pH meter
pH
25°C
37°C
0.05 M Potassium hydrogen phthalate
4.01
4.02
0.025 M Potassium dihydrogen phosphate
6.86
6.84
0.01 M Sodium tetra borate
9.18
9.06
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.
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Dr. Ahmed Khamis Salama
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 is a common test for all carbohydrates larger than tetroses. The test
is on the basis that pentoses and hexoses are dehydrated by conc.
Sulphuric acid to form furfural or hydroxymethylfurfural, respectively.
These products condense with α-naphthol to form purple condensation
product.
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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 35 g of CuSO4.7H2O in water and make up to 500 ml.
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.
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Preparation of sodium potassium tartarate and Fehling B:
We need 173 g of Na, K-tartarate to be dissolved with 120 g KOH in 500 ml distilled water.
Molecular weight of Na, K-tartarate
Molecular weight of tartarate ion
210 g
148 g
173 g of Na, K-tartarate
? g of tartarate ion
Number of grams of tartarate ion = 121 g = 123 g of acid
Tartaric acid
23 g Na
150 g
23 g Na
123 g
? g Na
Amount of Na = 19 g NaOH
Tartaric acid
39 g K
150 g
39 g K
123 g
?gK
Amount of K = 32 g KOH
Method:
1. Dissolve 123 g of tartaric acid in a little amount of distilled water
and stir. Solution 1.
2. Dissolve 19 g NaOH + 32 g of KOH in a little amount of distilled
water. Solution 2.
3. Mix solution 1 and 2 and stir very well to give Na, K-tartarate
solution.
4. Dissolve 120 g KOH in little amount of distilled water to prepare
KOH solution.
5. Mix KOH solution and Na, K-tartarate solution and complete the
total volume of solution to 500 ml with distilled water to get
Fehling B solution.
Method:
5 g of glucose, fructose and sucrose, respectively, are dissolved in 100 ml
of distilled water. Three 500 ml conical measures are each filled with the
sugar solutions. Each solution is made up to the 500 ml mark with
distilled water warmed up to 60 °C. Afterwards 8 ml of Fehling reagent
are poured into each of the sugar solutions while stirring.
Simplified 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.
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Results:
Glucose solution
Fructose solution
Sucrose solution
Orange-brown color is appeared.
Orange-brown color is appeared.
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.
R-COH + CuO + 2 OH-
R-COOH + Cu2O + H2O
Fehling
solution
brown
red ppt
alkali
media
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 orangebrown Cu2O.
Fig. 1: Redox reaction
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
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Dr. Ahmed Khamis Salama
anomeric carbon of glucose is involved in the glucose- fructose bond and
hence is not free to form the aldehyde in solution.
Fig. 2: Sucrose
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 ketoenol tautomerism.
Fig. 3: Conversion of fructose to glucose and mannose
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Dr. Ahmed Khamis Salama
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.
Fig. 4: Decomposition of fructose
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
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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.
Method:
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.
Compare the sensitivity of Benedict's and Fehling's test, using increasing
dilutions of 1% glucose.
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.
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
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Dr. Ahmed Khamis Salama
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. Formation of mucic acid:
Concentrated nitric acid oxidizes carbohydrates to the corresponding
saccharic acid. Galactose forms mucic acid with nitric acid which has
characteristic gritty crystals that separate out in dilute HNO3.
Method:
 Add 1g of solid sugar (Galactose, lactose or glucose) to 12
ml on conc. HNO3 in a porcelain basin.
 Evaporate on a boiling water bath until about 4 ml is left.
Mucic acid crystals should then separate out.
CHO
COOH
6. 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.
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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.
7. 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.
8. Bial’s Test :
Bial’s test is used to distinguish between pentoses and hexoses. They
react with bial’s reagent and is converted to furfural. Orcinol and furfural
condense in the presence of ferric ion to form a colored product.
Appearance of green colour or precipitate indicates the presence of
pentoses and formation of muddy brown precipitate shows the
presence of hexoses.
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Dr. Ahmed Khamis Salama
9. Osazone Test:
The ketoses and aldoses react with phenylhydrazine to produce a
phenylhydrazone which further reacts with another two molecules of
phenylhydrazine to yield the osazone. Needle-shaped yellow osazone
crystals are produced by Glucose, fructose and mannose whereas
lactosazone produces mushroom shaped crystals. Crystals of different
shapes will be shown by different osazones. Flower-shaped crystals are
produced by maltose.
Results
Test
Monosaccharide
glucose
fructose
ribose
Disaccharide
Sucrose
maltose
Polysaccharide
Starch
1. Molisch
2. Fehling
3. Benedict
4. Barfoed
5.mucic acid
6. Iodine
7.Seliwanoff
8.Bial
9.Osazone
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Dr. Ahmed Khamis Salama
Quantitative determinations of carbohydrates
Standardization of Fehling solution:
1. Prepare 5.0 g/L of glucose solution as standard by dissolving 0.5 g/100ml
distilled water.
2. Put 5.0 ml of Fehling A + 5 ml of Fehling B + 20 ml distilled water in a conical
flask and boil.
3. Titrate directly and slowly against standard glucose solution before the
solution be cooled.
4. Continue the titration process until orange-brown color appeared.
5. Record the consumed volume of the standard solution of glucose (V glucose).
6. Calculate the concentration of Fehling mixture in g/L.
Calculations:
Conc glucose x V glucose = Conc Fehling x V Fehling
5.0 g/L x V glucose = Conc Fehling x 30 ml
5.0 g/L x V glucose
Conc Fehling = ----------------------30 ml
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Principles of Biochemistry - Practical part
Dr. Ahmed Khamis Salama
Determination of the disaccharide, 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.
Method:
1. Take 10 ml from milk in a conical flask and then add 3 ml of Acetic anhydride.
2. Make stirring very well and the add 70 ml of distilled water and stir for 3-4
minutes.
3. Filter the solution and then neutralize it by using sodium carbonate solution
(Because Fehling A+B react with sugar in alkali or neutral media only ).
4. Sodium carbonate should be added drop by drop until the end of
effervescence process.
5. Complete the solution to 250 ml in volumetric flask and titrate against
Fehling (A+B).
6. Continue the titration process until orange-brown color appeared.
7. Record the consumed volume of Fehling mixture V Fehling.
8. Calculate the concentration of lactose?
Calculations:
Conc sugar x V sugar = Conc Fehling x V Fehling
Conc sugar x 10 ml sugar = Conc Fehling as determined above x VFehling
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Principles of Biochemistry - Practical part
Conc sugar (glucose + galactose) =
Dr. Ahmed Khamis Salama
Conc Fehling x V Fehling
-----------------------------10 ml
Molecular weight of lactose, C12H22O11 = 342
Molecular weight of glucose, C6H12O6 = 180
Molecular weight of galactose, C6H12O6 = 180
Molecular weight of glucose + galactose = 360
360 g of glucose + galactose
342 g lactose
Y g of glucose + galactose
? g lactose
Lactose concentration = Yg x 342 / 360
Lactose + acetic anhydride
glucopyranose + galactopyranose
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Principles of Biochemistry - Practical part
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Determination of Sucrose
α-D-glucopyranosyl-(1↔2)-β-D-fructofuranoside
Sucrose (common name: table sugar, also called saccharose) is a disaccharide
(glucose + fructose) with the molecular formula C12H22O11. Its systematic name is αD-glucopyranosyl-(1↔2)-β-D-fructofuranoside (ending in "oside", because it's not a
reducing sugar). It is best known for its role in human nutrition and is formed by
plants but not by other organisms such as animals.
sucrose + Hydrochloric acid
glucopyranose + fructofuranose
Method:
1. Take 50 ml of sucrose sample in a conical flask.
2. Add 5 ml of HCl + 50 ml of distilled water to the conical flask.
3. Boil the mixture for 30 minutes.
4. Let the solution to cool.
5. Neutralize the solution by using sodium carbonate and complete the volume
to 250 ml.
6. Take 20 ml of the final solution and transfer it into flask.
7. Titrate against Fehling A + B
7. Continue the titration process until orange-brown color appeared.
9. Record the consumed volume of Fehling mixture V Fehling.
10. Calculate the concentration of sucrose sample?
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Dr. Ahmed Khamis Salama
Calculations:
Conc sugar x V sugar = Conc Fehling x V Fehling
Conc sugar x 10 ml sugar = Conc Fehling as determined above x VFehling
Conc sugar (glucose + fructose) =
Conc Fehling x V Fehling
------------------------------ = Y g
10 ml
Molecular weight of sucrose, C12H22O11 = 342
Molecular weight of glucose, C6H12O6 = 180
Molecular weight of fructose, C6H12O6 = 180
Molecular weight of glucose + fructose = 360
360 g of glucose + fructose
342 g sucrose
Y g of glucose + fructose
? g sucrose
sucrose concentration = Yg x 342 / 360
Conc of sucrose = Conc of (G+ F) x 342/ 360
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Principles of Biochemistry - Practical part
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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.
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Principles of Biochemistry - Practical part
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Tests for fatty acids
Saponification:
When fats and oils are heated with alkali, free fatty acids and glycerol are
liberated and this process is known as saponification.
∆
Oil or fat + alkali media
glycerol + fatty acid
Saponification number:
It is the number of mg of NaOH or KOH which needed to saponificated 1 g fat or
oil.
According the previous equation:
1 mole fat or oil = 3 mole NaOH or KOH
M.wt fat or oil
= 3 M.wt NaOH or KOH
M.wt fat or oil
= 3 x 40 g NaOH or 3 x 56 g KOH
M.wt fat or oil = 3 x 40 x 1000 mg NaOH or 3 x 56 x 1000 mg KOH
1 g fat or oil
M.wt fat or oil
1g fat or oil
=
=
=
X mg NaOH or KOH (saponification number)
3 M.wt NaOH
saponification number
Saponification number(theoretical) = 3 x 40 x 1000 / M.wt of fat or oil
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Determination of the saponification number of
some fatty acids
Butyric acid (Mw, 88) CH3(CH2)2COOH milk fat
Stearic acid (Mw, 284) CH3(CH2)16 COOH animal & plant fat
Oleic acid (Mw, 282) CH3(CH2)7 CH=CH (CH2)7 COOH animal & plant fat

Use excess amount of NaOH for saponification process.

Determine the amount of NaOH which remained after saponification
using oxalic acid.

Calculate the reacted amount of NaOH.
Procedure:
1. Weigh 1 g of fat or oil, oleic acid (Mw = 282) and transfer it to a conical flask.
2. Add 25 ml of NaOH 0.5N to the fat amount in the conical flask.
3. Boil the solution for 10 minutes and then add 20 ml of distilled water.
4. Titrate the excess of NaOH with Oxalic acid (0.5 N) in the presence of ph.ph
indicator.
C2O4H2
+ 2 NaOH
C2O4Na2 + 2 H2O
Calculations:
To calculate the volume of NaOH reacted with fat (V2) , you have to calculate the
volume of nonreacted NaOH (V1) and subtract it from the total amount of NaOH
(Indirect method).
N x V oxalic acid = N x V1
sodium hydroxide
1. Added amount of NaOH to the fat = 25 ml
2. Nonreacted amount of NaOH wth fat
= reacted amount with oxalic acid
= V1
3. Reacted amount of NaOH with fat
= (25 ml – V1) x 0.5 N
4. Saponification number
= (25 ml – V1) x 0.5 N x eq. wt of NaOH (40) / weight of fat
= (25 ml – V1) x 0.5 N x 40 x 1000 / 282
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Principles of Biochemistry - Practical part
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Determination of the Acid value of some fats
During storage, fats may become rancid due to peroxide formation at the double
bonds by atmospheric oxygen and hydrolysis by microorganisms with the liberation
of free acid. The amount of free acid present therefore gives an indication of the age
and quality of the fat.
The acid value is the number of milligram of KOH required to neutralize the free acid
present in 1 g of fat.
Method:
1. Accurately weigh out 10 g of the test compound and suspend the melted fat
in about 50 ml of the fat solvent (equal volumes of 95% alcohol and ether
neutralized to phph).
2. Add 1 ml of phph solution (1% in alcohol) and mix thoroughly.
3. Titrate with 0.1 N KOH until the faint pink color persists for 20 to 30 sec.
4. Note the number of milliliters of standard alkali required.
5. Calculate the acid value of the fat.
Note: 0.1 N KOH contains 5.6 g/L or 5.6 mg/ml
Acid value = V1 x 0.1 N x eq. wt of KOH (56) / weight of fat (10 g)
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Principles of Biochemistry - Practical part
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Determination of Ascorbic Acid in Vitamin C Tablets
In the experiment, a Vitamin C tablet was extracted into dilute H 2SO4 and an aliquot
of standard KIO3 solution was added. This was followed by addition of an excess of
KI. The liberated I2 reacted with the ascorbic acid, and the amount of unreacted I 2
was determined by a back titration with the previously standardized Na2S2O3
solution.
By knowing the amount of I2 originally liberated and determining the amount of I2
that did not react with ascorbic acid, it is possible to determine the amount of
ascorbic acid in the tablet.
This determination can be very tricky because the starch endpoint is not as clear as
in the standardization of Na2S2O3 solution. Because this is a back titration,
overshooting the endpoint gives a low value for ascorbic acid.
Iodimetric determination:
The amount of unreacted I2 was determined by a back titration with the previously
standardized Na2S2O3 solution as the following:
1. Transfer 10 ml of sample which contains I2 to a conical flask>
2. Titrate using sodium thiosulate solution (0.1 M) until the color of solution be
faded.
3. Add 4 drops of starch indicator (Blue color appeared).
4. Complete the titration process using sodium thiosulate solution (0.1 M) until
the blue color disappeared.
5. Record the consumed volume of sodium thiosulfate.
6. Calculate the amount of Iodine.
Generation of I2:
Acidic media (6H+)
KIO3
+
5 KI
Pot. Iodate
excess
3I2 + 3 H2O
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Reaction of generated I2 with vitamin C:
Determination of unreacted I2 with excess I2 :
Starch indicator
2 Na2S2O3 + I2
2 NaI + Na2S4O6
Sodium tetrathionate
I2 + 2e-
2I2 S2O3
S4O6--
+ 2 e-
Calculations:
C x V iodine = C x V thiosulfate
Concentration of I2 = C x V thiosulfate / V iodine
Concentration of I2 = 0.1 M x V thiosulfate / 10 ml
Gram Iodine / liter = Conc of I2 (M) x M.wt of I2
Gram Iodine / liter = Conc of I2 (M) x 126.9
g/L iodine = N x 126.9
Total amount of I2 = 30 g/L
Total amount of I2 - nonreacted amount of I2 = reacted amount of I2
Ascorbic acid
= Iodine
C6H6O6
=
176 g
X g ascorbic acid
I2
= 126.9 g iodine
= gram iodine reacted
Ascorbic acid (g/L) = 176 x g iodine / 126.9
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Dr. Ahmed Khamis Salama
Proteins
Proteins, also known as polypeptides, are organic compounds made of amino acids
arranged in a linear chain and folded into a globular form. The amino acids in a
polymer chain are joined together by the peptide bonds between the carboxyl and
amino groups of adjacent amino acid residues.
The sequence of amino acids in a protein is defined by the sequence of a gene, which
is encoded in the genetic code. In general, the genetic code specifies 20 standard
amino acids; however, in certain organisms the genetic code can include
selenocysteine — and in certain archaea — pyrrolysine.
Shortly after or even during synthesis, the residues in a protein are often chemically
modified by post-translational modification, which alters the physical and chemical
properties, folding, stability, activity, and ultimately, the function of the proteins.
Proteins can also work together to achieve a particular function, and they often
associate to form stable complexes.
Like other biological macromolecules such as polysaccharides and nucleic acids,
proteins are essential parts of organisms and participate in virtually every process
within cells. Many proteins are enzymes that catalyze biochemical reactions and are
vital to metabolism. Proteins also have structural or mechanical functions, such as
actin and myosin in muscle and the proteins in the cytoskeleton, which form a
system of scaffolding that maintains cell shape. Other proteins are important in cell
signaling, immune responses, cell adhesion, and the cell cycle. Proteins are also
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Dr. Ahmed Khamis Salama
necessary in animals' diets, since animals cannot synthesize all the amino acids they
need and must obtain essential amino acids from food. Through the process of
digestion, animals break down ingested protein into free amino acids that are then
used in metabolism.
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Determination of total protein
Biuret assay
Reagent:

Stock biuret reagent: Dissolve 45 g of sodium potassium tartarate in about
400 ml of 200 mmol/l sodium hydroxide and add 15 g of copper sulphate
(CuSO4.5H2O), stirring continuously until solution is complete.
Add 5 g
potassium iodide and make up to 1 liter with 200 mmol/l sodium hydroxide.

Working biuret reagent: Dilute 200 ml of stock reagent to 1 liter with 200
mmol/L sodium hydroxide containing 5 g potassium iodide/L

Tartarate-iodide solution: Dissolve 9 g sodium potassium tartarate in 1 liter
of 200 mmol/l sodium hydroxide containing 5 g potassium iodide/L.

Bovine or human albumin standard, 80 g/l.
Method:
(a) Test. Add 0.1 ml serum to 5 ml working biuret solution.
(b) Serum blank. Add 0.1 ml serum to 5 ml tartarate-iodide solution.
(c) Standard. Add 0.1 ml standard to 5 ml working biuret solution.
(d) Standard blank. Add 0.1 ml standard to 5 ml tartarate-iodid solution.
(e) Reagent blank. Add 0.1 ml water to 5 ml working biuret solution.
Incubate all tubes at 37 C for 10 min. After cooling to room temp measure the
absorbances at 555 nm using the reagent blank to set the zero.
Calculation:
Serum total protein (g/l) =
[Reading of (a) – Reading of (b) / Reading of (c) – Reading of (d)] X Standard concs.
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The Folin-Lowery method of protein assay
Principle:
Protein reacts with the Folin-Ciocalteau reagent to give a coloured complex. The
colour so formed is due to the reaction of the alkaline copper with the protein as in
the biuret test and the reduction of phosphomolybdate by tyrosine and tryptophan
present in the protein.
Materials:

Alkaline sodium carbonate solution (2% Na2CO3 in 0.1 N NaOH).

Copper sulphate-sodium potassium tartarate solution (0.5% CuSO4 in 1% Na,
K tartarate). Prepare fresh by mixing stock solutions.

Alkaline solution: Prepare on day of use by mixing 50 ml of (1) and 1 ml of (2).

Folin-Ciocalteau reagent: Dilute the commercial reagent with an equal
volume of water on the day of use. This is a solution of sodium tungstate and
sodium molybdate in phosphoric and hydrochloric acid).

Standard protein (albumin solution 0.2 mg/ml).
Method:

Add 5 ml of the alkaline solution to 1 ml of the test solution.

Mix thoroughly and allow to stand at room temperature for 10 min or longer.

Add 0.5 ml of diluted Folin-Ciocalteau reagent rapidly with immediate mixing.

After 30 min read the extinction against the appropriate blank at 750 nm.

Estimate the protein concentration of an unknown solution after preparing
standard curve.
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Enzyme assay
Enzyme assays are laboratory methods for measuring enzymatic activity. They are
vital for the study of enzyme kinetics and enzyme inhibition.
UV/VIS Spectrophotometer.
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.
A more practical and commonly-used value is 1 enzyme unit (EU) = 1 μmol min-1 (μ =
micro, x 10-6).
One unit of enzyme (1 EU) = 1 μmol min-1 = 16.6 nano katal
1 U corresponds to 16.67 nanokatals.
1 katal = 1 mol/sec.
The specific activity of an enzyme is another common unit. This is the activity of an
enzyme per milligram of total protein (expressed in μmol min-1mg-1).
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Specific activity gives a measurement of the purity of the enzyme.
Activity = μmol min-1
Specific Activity = μmol. min-1. mg-1
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 steadystate 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.
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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.
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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.
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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 nonfluorescent. 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.
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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.
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Chromatographic assay:
Chromatographic assays measure product formation by separating the reaction
mixture into its components by chromatography. This is usually done by highperformance 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
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Dr. Ahmed Khamis Salama
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 pH 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.
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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).
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Determination of total protein
(Lowry method)
Reagents:

Solution (A): Dissolve 4 gram NaOH in 1 liter distilled water (D.W) to give 0.1
N and then add 20 gram Na2CO3 and 0.5 g Na, K-tartarate.

Solution (B): Dissolve 1 gram CuSO4 in 1 liter D.W

Solution (C) Mix 50 ml of reagent (A) with 1 ml of reagent (B)

1N Folin phenol reagent: Dilute 2N Folin reagent 1:1 with D.W
Procedure:

Prepare standard curve of bovine serum albumin (BSA).
Stock solution 20 mg BSA/ml DW = 20 μg BSA/μl = 1000 μg /50 μl
Working standard solution:
1000 μg /50 μl X V =
20 μg /50 μl X 2000 μl
1000 μg /50 μl X V =
40 μg /50 μl X 2000 μl
1000 μg /50 μl X V =
60 μg /50 μl X 2000 μl
1000 μg /50 μl X V =
80 μg /50 μl X 2000 μl
1000 μg /50 μl X V = 100 μg /50 μl X 2000 μl
Table for preparation of Standard concentrations of BSA):
BSA concentration
(μg /50 μl)
20
40
60
80
100
Stock solution
40 μl
80 μl
120 μl
160 μl
200 μl
Distilled water
(DW)
1.96 ml
1.92 ml
1.88 ml
1.84 ml
1.80 ml
Total volume
2 ml
2 ml
2 ml
2 ml
2 ml
Add 50 μl of plasma (dilution necessary for accurate measurement) or 10 μl of 10%
(w/v) brain homogenate to the tubes.
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Dr. Ahmed Khamis Salama
The addition table:
Addition
Blank (B)
Replicates (R1, R2, R3)
3 ml
3 ml
Plasma sample
-
50 μl
Standard BSA
-
50 μl of each concentration (20, 40, 60,
Reagent [C]
80, 100 μg/ 50 μl )
Distilled water
50 μl
-
Incubate at room temperature for 10 minute
Folin
0.3 ml
0.3 ml
Mix and stand at room temp for 30 min
Read the OD at 750 nm.
Calculations:
1. OD readings were used to calculate K value.
2. Plot the protein concs versus OD and draw a straight line.
3. Calculate the slope
y2-y1 / x2-x1
4. The OD values of the test sample are divided by K value to calculate the
protein concentrations ( x μg/50 μl) for sample.
Concentration of protein (μg/50 μl) = OD / K
Concentration of protein (mg/50 ml) = OD / K
Concentration of protein (mg)
= OD / K
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Determination of Adenosine Triphosphatase
(ATPase)
The significant role of ATPase enzyme is to hydrolyze ATP to release energy required
for all metabolic and biological processes.
So, if ATPase is inhibited, the adenosine triphosphate (ATP) will be accumulated. The
temperature of the body will be elevated and there is no energy released. Finally,
the metabolic and biological processes will be altered.
Reagents:
100 mM NaCl: Dissolve 4.97 g / 100 ml D.W
20 mM KCl:
Dissolve 1.26 g / 100 ml D.W
5 mM MgCl2: Dissolve 0.86 g / 100 ml D.W
30% TCA:
Dissolve 30 g / 100 ml D.W
10 mM Tris-0.32 M sucrose-0.001 M EDTA buffer pH 7.5:
Dissolve 1.21 g tris + 109.54 g sucrose + 0.37 g EDTA in D.W and adjust pH to 7.5 and
complete the volume to 1 liter.
10 mM Tris-0.001 M EDTA buffer pH 7.5:
Dissolve 1.21 g tris + 0.37 g EDTA in D.W and adjust pH to 7.5 and complete the
volume to 1 liter.
5 mM ATP: Dissolve 0.047 g in 2 ml D.W
Color reagent:
Prepare 10 N H2SO4 by dissolving 27.7 ml of sulfuric acid (36 N) in 100 .DW
Dissolve 10 g of ammonium molybedate in the above acid solution.
Solution A: 10 ml from ammonium molybdate solution + 20 ml D.W.
Solution B: Dissolve 5 g FeSO4 in 50 ml D.W
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Dr. Ahmed Khamis Salama
Then the color reagent is prepared by adding 30 ml of solution A to 50 ml of
solution B and then complete the volume to 100 ml.
In case of Na2HPO4. 12 H2O: dissolve 0.017 g / 100 ml DW
Dilute the stock solution according to the following table:
Blank
20 μM
40 μM
60 μM
80 μM
100 μM
Color reagent
Stock (ml)
0.2
0.4
0.6
0.8
1.0
4.0
Measure the OD at 740 nm
D.W (ml)
1.0 ml
4.8
4.6
4.4
4.2
4.0
4.0
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Procedure:
Make addition according to the following table:
Calculations:
Calculate K value from the calibration curve of inorganic phosphate.
Read the absorbance in case of enzyme assay method.
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Acetyl Cholinesterase Enzyme (AChE)
The significant role of AChE enzyme is to hydrolyze the neurotransmitter
Acetylcholine (ACh) to regulate the cholinergic cycle and to stop the release of
sodium pump.
So, if AChE is inhibited, the neurotransmitter, acetylcholine (ACh) will be
accumulated. Thus, sodium channels will opened and the release of sodium will be
continued leading to convulsions, paralysis and finally death.
Determination of AChE activity According to Ellman method
The rate of hydrolysis of acetylcholine by a red cell suspension at pH 7.2 is measured
at 412 nm by the reaction of thiocholine base with DTNB to give 5-thio-2nitrobenzoate anion.
Reagents:
Phosphate buffer pH, 7.2:
50 mmol in 9.0 g/L sodium chloride
Substrate solution (Acetyl thiocholine iodide):
Prepare 31 mM solution of substrate>
Color reagent (DTNB):
Dissolve 250 mol/L in phosphate buffer, pH 7.2
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Procedure:
1. To 1 ml cell volume suspension add 4 ml buffer and remix>
2. Into two silica cuvettes , pipette 2.0 ml (for test) or 2.1 m; (for blank)
of the color reagent and add 1 ml cell suspension to each.
3. After 3 minute add 100 l substrate to the test.
4. Mix and determine the absorbance of the test against the blank at
412 nm.
5. Repeat the readings every minute for at least 6 minutes.
6. Calculate ∆ A412/min value averaging the two samples containing
substrate and subtracting the values for the blank.
7. Determine the number of mg protein as described before
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Determination of Tocopherols (Vitamin E)
Serum tocopherols can be measured by their reduction of ferric to ferrous ions
which then form a red complex with -- dipyridyl.
Tocopherols and carotenes are first expected into xylene and the absorbance is read
at 460 nm to measure the carotenes.
A correction for the carotenes is made after adding ferric chloride and reading at 520
nm.
Reagent:
1. Absolute alcohol, aldehyde-free
2. Xylene
3. -- dipyridyl, 1.2 g/L in n-propanol
4. Ferric chloride solution, 1.2 g in 1L of ethanol
5. Standard solution of -tocopherol, 10 mg/L in ethanol
Technique:
Sample
Add 1.5 ml serum
1.5 ml ethanol
1.5 ml xylene
Standard
Blank
Add 1.5 ml standard
Add 1.5 ml DW
1.5 ml ethanol
1.5 ml ethanol
1.5 ml xylene
1.5 ml xylene
Stopper, mix and centrifuge
Take 1 ml of xylene layer
Add 1ml dipyridyl reagent Add 1ml dipyridyl reagent Add 1ml dipyridyl reagent
Read the absorbance of sample and standard against the blank at 460 nm
(absorbance of carotenes only)
Add 0.33 ml of ferric chloride solution, mix and set wavelength at 520 nm
Read again the absorbance at 520 nm (absorbance of carotenes + tocopherols)
Calculations:
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Determination of serum phosphate
The body contains about 17 mole of phosphorus of which 87% is present in bones,
the remainder being found in cells and soft tissues i.e. more widely distributed than
is calcium.
Phosphorus is a constituent of many important biological compounds, e.g. some
proteins, some lipids, nucleic acids and some coenzymes.
It is also play a part in acid-base regulation, particularly by the kidneys.
Reagents:
1. Trichloroacetic acid (TCA) solution, 100 g/L DW.
2. Sulphuric acid, 5mM:
Add 450 ml H2SO4 conc. Slowly while cooling to 1.3 L water, dilute some
of this 1:10 and titrate with 1 M NaOH and make any necessary
adjustment to the original solution.
3. Ammonium molybdate solution:
Dissolve 7.5 g/200 ml DW, add 100 ml of 5M H2SO4 and make to 400 ml
with DW.
4. Metol (p-methyl amino phenol sulphate) solution:
1g/100 ml sodium bisulphate solution 30 g/L
5. Stock standard phosphate solution, 20 mM:
Dissolve 1.36 g KH2PO4 in 300 ml DW and make to 500 ml
6. Working standard phosphate solution (2mM):
Dilute the stock solution at 1:10 with water.
Technique:
1. Add 0.8 ml serum to 7.2 ml TCA, mix and filter or centrifuge.
2. Set up three tubes containing respectively, 5ml of the filtrate (= 0.5 ml
serum)( the sample) , 0.5 ml standard plus 4.5 ml TCA (the standard),
and 5ml TCA (the blank).
Follow the table below:
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Principles of Biochemistry - Practical part
The sample
5 ml serum filtrate
Dr. Ahmed Khamis Salama
The standard
0.5 ml standard
The blank
5ml TCA
+ 4.5 ml TCA
+ 1ml ammonium
+ 1ml ammonium
+ 1ml ammonium
molybdate solution
molybdate solution
molybdate solution
+ 1ml metol
+ 1ml metol
+ 1ml metol
Allow to stand 30 minutes and read at 680 nm against blank
Calculations:
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Determination of Acid Phosphatase (ACP)
Reagent:
Acid buffer-substrate solution (AcBS):
citrate buffer (0.05 M): 0.41 g citric acid + 1.125 g sodium citrate
Add 0.12 of p-nitrophenyl phosphate
NaOH (0.1 N): 4 g/1L DW
Standard curve for p- nitrophenol:
1. Dissolve 696 mg p-nitrophenol / 0.02 N NaOH and make up to 1L as
stock solution (5 x 10-3 M)
2. Take 10 ml of stock and dilute to 1 L with 0.02 N NaOH to prepare
working standard solution (5 x 10-5 M)
3. Dilute 1, 2, 3, 5, and 7 ml of the working standard solution with 0.02 N
NaOH to 11.1 ml and measure the absorbance against 0.02 N NaOH
4. The corresponding values for these dilutions are:
0.05, 0.10, 0.15, 0.25, and 0.35 mole p-nitrophenol
The reading multiplied by 5.2 and then divided by 22.2 to obtain the
unit of acid phosphatase.
Technique:
1. Add 1 ml AcBS in a test tube to 200 l serum.
2. Mix and incubate for 30 minutes at 37 C
3. Add 4 ml of 0.1 N NaOH
4. Measure the absorbance at 405 nm
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Calculation:
Kits for determination the followings:
1. Alkaline phosphatase
2. Triglycerides
3. Uric acid
51