Practical Book

7/25/2019 Practical Book

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UMM AL-QURA

UNIVERSITY FACULTY OF

MEDICINE DEPARTMENT OF

BASIC MEDICALBIOCHEMISTRY

Laboratory Manual

ABDULLATIF . T . BABAKR

(BSc., MSc., Biochemistry)

14351436


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BASIC MEDICAL BIOCHEMISTRY-Practical Notes

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Table of contents:

Week Practical Page

1Introduction to Biochemistry Lab., Evaluation methods; Labconduct and safety precautions

03

2Introduction to the commonly used instruments in the laboratory.Containers: glassware, Plastics, metals, Disposables

12

3 Methods expressing concentration 19

4Tutorial and E-Learning session

5Mechanistic Principles of Qualitative Identification ofCarbohydrateI 25

6 Mechanistic Principles of Qualitative Identification of

CarbohydrateII 267 Quantitative Analysis of Carbohydrates 27

8 Tutorial and E-Learning

9 Qualitative analysis of lipids-1 31

10 Qualitative analysis of lipids-2 34


12 Ionic properties of amino acids 36

13 Protein solubility and separation : Saltingin & salting out 4114 Protein size and separation (Gel Chromatography) 44


16 Revision


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I- INTRODUCTION:

Biochemistry is one of the modern , most important , basic medical sciences. The

term define itself, however, a short rich definition is that : Biochemistry is the

study of life at the molecular level.

Biochemistry: From Atoms to Molecules to Cells (fig. 1):

Biochemical studies lead to a more fundamental understanding of life & impacts

the treatment of disease & solves environmental problems.

The Roots of Biochemistry:

All Living Matter Contains C, H, O, N, P & S:

Essential elements that make up the bulk of mass of any living organism. All

biological molecules (amino acids, proteins, glucose, polysaccharides, lipids,

nucleotides, DNA, RNA, etc.) are constructed from these elements.

Biological Macromolecules (Bio-molecules):

major classes are proteins, polysaccharides (carbohydrates), nucleic acids (DNA,

RNA); (lipids are a major class of biomolecules, but not polymeric).

Organelles, Cells & Organisms:

self-assembly of macromolecules into higher levels of order cell -cell structure &

organelles for prokaryotes & eukaryotes.

Biochemistry seeks to describe the structure, organization, and function of living

organisms in molecular terms. To understand life on the molecular level, you

must:

-know the chemical structures & function of biological molecules, to:

-understand the molecular processes in the expression of genetic information.

-understand bioenergetics (the study of energy flow in cells).


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So, there are Three Areas of Study:

1. Conformational:

structure and 3D arrangements of biomolecules.

2. Metabolism:

energy production and utilization.

3. Informational:

language for communication inside and between cells.

DNA RNA Protein Cellular Processes.

FIG. -1- : BIOCHEMISTRY, FROM ATOMS TO ORGANISM


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Practical Biochemistry:

Biochemical studies are conducted in order to obtain information on chemical and

physico-chemical processes that take place in the cells and tissues of living

organisms in norm and in pathology.

Depending on the scope and target of intended investigation, special procedures

are applied to biological materials and adequate physico-chemical methods are

used that must be learned for conducting experiments in biochemistry.

Prior to getting down to practical work, it appears expedient to take a brief survey

of general principles and techniques employed in practical biochemistry.

Code of practice:

This code is a listing of the most essential laboratory procedures that are basic to

safe laboratory practice. In many laboratories, such a code may be given the status

of RULES for laboratory operation.

It is emphasized that good laboratory practice is fundamental to laboratory safety

and can't be replaced by specialized equipment which can only support it.

The most important rules are listed below, not necessarily in order to importance:

1- Mouth pipetting should be prohibited.

2- Eating, Drinking, Smoking and applying cosmetics should not be permittedin the laboratory work area.

3- The laboratory should be kept neat, clean and free of materials not pertinentto the work.

4- Work surface should be decontaminated at least once a day, and after anyspill of potentially dangerous material.

5- Cleaning the glassware after use.


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HAZZARDOUS PROPERTIES OF LABORATORY CHEMICALS

Incompatible Chemicals:

Many chemicals commonly found in the laboratory undergo dangerous reactionsif allowed to come into contact during storage or else where, some of which are

listed below:

Chemical subs. Reacts with

Acetic acid With chromic acid, hydroxy-contaning compounds,

ethylene plycol, perchloric acid peroxides and

permanganates.

Acetylene With copper (tubing). Fluoring, bromine, chloride,iodine, silver, mercury, carbon

Alkali metal Such as calcium potassium and sodium-with water,

carbon dioxide, carbon tertrachloride and other

chlorinated hydrocarbons.

Ammonium

anhydrous

With mercury, halogens, calcium hypochlorite

hydrogen fluoride.

Ammonium

nitrate

With acids, metal powders, Flammable liquids,

chlorates nitrates, sulfur and finely divided organics

or combustible.

Aniline With nitric acid, hydrogen peroxide.

Bromine With ammonia, acetylene, butadiene, butane,

hydrogen, sodium carbide.Turpentine and finely

divided metals.

Carbon acetate With calcium hypochloratewith all oxidizingagents.

Chlorates With ammonium salts, acids, metal powders. Sulfur

finely divided organics or combustibles carbon.

Chromic acid With acetic acid, naphthalene caniper, alcohol,

glycerol turpentine and other flammable liquids.

Chlorine dioxide With ammonia, methan, phosphate, hydrogen sulfide.


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Chemical subs. Reacts with

Chlorine With ammonia, acetylene, butadiene, benzene and

other petroleum fractions, hydrogen, sodium carbide,

turpentine and finely divided powdered metals.Copper With acetylene, hydrogen peroxide.

Cyanides With acids and alkali.

Liquids With ammonium nitrate, chromic acid, hydrogen

peroxide, nitric acid, sodium peroxide and halogens.

Hydrogen

peroxide

With copper, chromium, iron, most metal or their

respective salts, flammable fluids, and other

combustible materials, aniline and nitromethane.

Hydrogen sulfide With fuming nitric acid, oxidizing gases.Hydrocarbons

general

With fluorine, chlorine, formine, chromic acid and

sodium peroxide.Iodine With acetylene, ammonia.

Mercury With acetylene, fulminic acid hydrogen.

Nitric acid With acetic, chromic and hydrocyanic acids, aniline,

carbon, hydrogen sulfide, fluids or gases and

substances that are readily nitrated.

Oxygen With oils, grease, hydrogen, flammable liquids, solids

and gases.

Perchloric acid With acetic anhydride, bismuth and its alloys, alcohol

paper, wood and other organic materials.

Phosphorus

pentoxide

With water.

Potassium

permanganate

With glycerol, ethylene, glycol, benzaldehyde,

sulfuric acid.

Silver With tartaric acid, ammonium compounds.

Sodium peroxide With any substance, for instance, methanol, aceticacid, acetic anhydride, benzaldehyde, carbon

disulfide, glycerol, ethylene, glycol, ethyl acetate,

furfural.

Sodium With carbon tetrachloride, carbon dioxide, water.

Sulfuric acid With chlorates, perchlorates, permanganates and

water.


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Other chemicals have serious acute and chronic effects, some of which are listed

below:

Chemical Reported effects

Acute Chronic

Trichloroethylene

(ethylene trichloride)

Narcotic effects Liver damage, nonspecific

neurological impairment.

m-Xylene (1,3-

dimethylbenzene)

Narcotic effects,

headache, dizziness,

fatigue, nausea

Nonspecific neurological

impairment.

o-Xylene (1,2-dimethylbenzene)

Narcotic effects,headache, dizziness,

fatigue, nausea

Nonspecific neurologicalimpairment.

p-Xylene (1,4-

dimethylbenzene)

Narcotic effects,

headache, dizziness,

fatigue, nausea

Nonspecific neurological

impairment.


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CHECKOUT PROCEDURE:

Checking out of the laboratory may count as one experiment. You will be graded

on how thoroughly you complete the following items.

1- clean your portion of the desktop, scrubbing it with soap and rinsing it ifnecessary. Clean through your desk and the sink at the end.2- Clean and dry all apparatus, leave no marks or labels on glassware other

than unavoidable scratches and etches.3- Oil the screws on the lamp and iron ring.4- Empty the desk completely and line the drawer with a paper towel.

5- Arrange the apparatus to be checked in on top of your desk inapproximately the order in which they appear on the check-in sheet.

6- Place all chipped or cracked items to one side. Discard (or otherwisesegregate) non-returnable.

7- Make a list of all missing returnable equipment, after checking withneighbors first to see if they have extras. Then obtain missing item.

8- Return safety glasses, if you were issued them.9- Return paper items to desk drawer as you check in.10- Lock desk.11- Additional cleaner assignment.


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How to protect yourself in the laboratorySAFETY RULES:

Every experiment is designed to minimize hazards, but the following rules are a

necessary adjust to that design.

1- Wear safety glasses all times when you are in the laboratory. Those whowear prescription glasses have considerable protection already. At the very

least, others should wear inexpensive plastic nonprescription glasses. By

"safety glasses" we mean industrial quality eye protective devices meetingthe standards of the American standard safety code for head, eye, and

respiratory protection. Those who were contact. Lenses are warned of a

special problem. if a chemical splashes onto the eyes, it may seep under theedge of the contact lenses. If you wash the eye with the lens in place you

will not the certain of flushing away all of the material, The lens must be

removed as soon as practicable, so that both lens and eye can be thoroughlywashed.

2- Learn the exact locations of eyewash fountains, fire extinguishers, fire

alarms, fire blankets, and other safety features in your laboratory, as will ashow to use these devices. sketch the laboratory and indicate their location.

3- Work only during the scheduled laboratory periods and perform only

authorized experiments. Your instructor will advise you about localregulations. An important safety rule, however is never work alone inlaboratory. If an accident occurs, the other person may able to aid you.

4- If you feel faint, sit down right away.5- If you burned and require the attention of a doctor, has someone accompany

you to the doctor's office. Do not apply salves or ointments on the burned

areas, let the doctor decide the treatment. Prompt cooling of a burned areawith cold water markedly reduces subsequent pain and facilitates heating ofthe area.

6- Some accidents happen when lapels are not read carefully. Get in the habitof reading out loud (but softly) the label of bottle you intend to use.

7- To avoid contamination;

(a)Discard unused chemicals : do not return them to reagent bottles ;claen

up anything you spilled..

(b)Never put a medicine cropper or a pipette from your desk into a regent

bottle but, instead pour a very small amount of the regent quantities.

(c)Try to keep inner walls of bottle stoppers or corks from touching tops of

desks or shelves where they might pick up dust or other chemicals.


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8-Discard all waste solids-water-insoluble chemicals, litmus paper, used matches,

broken glass, paper towels-into crocks at the end of your laboratory bench.

When sinks are used as wastebaskets they may over flow.

9-Your shoes should cover your feet to protect them from spilled chemicals or

dropped objects.

10-If your hair is long, fluffed with chemicals, it is quite flammable. At least pin

or tie it back in some way while you work around benzene burner flames.

11-Neither food not beverage is allowed in the laboratory.

12-Every time you select a flask, cylinder, or test tube for some experiment,

examine it for cracks and broken edges. Some times a broken edge canbe tolerated, but under no circumstances use a cracked container.

13-Never taste chemical. Check odors only very cautiously.

14-Always pour concentrated acid into water (never water into acid).


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3- Other types of pipettes:These include the micropipette, which are used to measure smallquantities

that the regular pipettes cannot measure, i.e. micro litter amounts

(ul.). these types of pipettes are usually used in techniques that

require high accuracy such as in working with enzymes and

hormonal assays.

Automatic pipettes are two types:

1. Fixed volume pipette:This delivers only the exact volume which is

written on the top of the pipette (e.g. 20 ul,

100 ul, 1000 ul, etc.).2. Adjustable or variable volume pipette:This can be calibrated to deliver any amount

of solution which is written on the side of the pipette, (e.g. 20 ul.Pipette can be used to deliver volumes from 1 to 20 ul, and 100 ul.

Pipette can be used to deliver volumes from

10 to 100 ul.).

Use of pipettes:

1. Never use mouth, use pipette bulbs or pipette filters inaspirating solutions.

2. Pipettes must be clean from any dirt or grease that might be in oroutside the pipettes.

3. After aspirating the required volume, wipe off the surface of the

pipette with tissue paper.

4. Always read the bottom of the meniscus for clear solutions, andtop for

colored or viscous ones.

5. The graduation point and your eyes should be in a horizontalposition.

6. When delivering solutions, let the tip of the pipette touch the

inner surface of the container, and let the solution flow by capillaryaction.

7. Always choose the proper type of pipette in measuring therequired volume, i.e. use smaller pipette for small volumes and vice

verse.


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Other types of glassware in the laboratory include, but not limited to:

Beakers:Available in

different volumes.

Conical flasks:Used in titration

procedures and

other different uses,

also available indifferent volumes.

Graduated

cylinders:For measuring thevolume of solutions

and dilution

procedures.

Burette:For addition of

volumes in

titration

procedures.


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2- pH meterThe pH meter is an instrument that is used to measure the pH(Acidity or alkalinity) of any solution. It is composed of the

following:

1- Glass-bulb electrode.

2- Reference electrode.

3- Sensitive meter or measuring device.

pH measurement procedure:

1. Leave the pH meter in STD BY mode to eliminate warm up and

increase component life. Allow one half hour warm up if meter hasbeen disconnected.

2. Securely connect pH and reference electrodes, or combination pH

electrode into INPUT (pH) on the rear panel.3. Verify that reference filling solution level in the electrode is

adequate. The level of filling solution should be higher than the

sample level.4. Stir buffer and sample during analysis with a magnetic stirrer, if

possible.

5. Rinse electrodes with distilled water between measurements.


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3-Spectrophotometer

Spectrophotometer is one of the most commonly used instruments in

the biochemistry laboratory. Its main function is to measure the

absorbance or concentration of any substance (Carbohydrates,

proteins, etc.) in a solution.

Beer-Lambert Law:

Consider a ray of light of initial intensity (I0) passing through a

solution in a transparent vessel. Some of the light is absorbed, so that

the intensity of the transmitted light (I) is less than (I0). The ratio of

(I) to (I0) is known as the transmittance (T) and depends upon the

path length of the light through solution.

So, transmittance is a measure of the amount of light that is allowedto pass through a solution. The Lamberts law states that:

When a ray of monochromatic light (single wavelength) passesthrough an absorbing medium, its intensity decreases as the

length of the medium

increases.Similarly, Beers Law states that:

When a ray of monochromatic light passes through an absorbing

medium, the intensity decreases as the concentration of the absorbingmedium increases.


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These two laws are combined in the form of Beer-LambertLaw and expressed as: A = abc. Where:

A = Absorbance.

a = Extension coefficient (constant).

b = Length of light path. = 1cm.

c = Concentration of the substance in the solution.

It is clear from this law, that the absorbance of any colored solution

is directly proportional to the concentration of the substance

producing the color.

3- components of the spectrophotometer:Spectrophotometer is generally composed of the following major

parts:

i) Light source:Emits visible or UV light depending on the source itself.

ii) Monochromator:Filter which functions in isolating only one single light. i.e. one

wavelength.iii) Slit:This part functions in passing a very fine beam of isolatedwavelength.

iv) Cuvette holder:This is where the sample of the colored solution is inserted. When

measuring absorbance in the visible region the cuvette is usuallymade of glass, while in measuring absorbance at the low UV region

cuvettes are made of quartz.

v) Photo cell:This part converts light energy to electric energy so it can be

measured by the measured by the measuring device.

vi ) Galvanometer:This is where the electric pulses are received and converted on a

scale either to absorbance or to transmittance units.


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Methods Expressing ConcentrationObjectives:By the end of this topic the students should be able to:

Identify the molarity, normality and molality.

Ideal preparation of different types of solutions (Standard,Saturated, Unsaturated and Percent solutions).

Definitions:Normality (g eq/L): The concentration of a solution in equivalents per

liter.

Molarity (W/V): The concentration of a solution in units of moles of

solute per liter of solution.

Molality (W/W): Mole (mol.): A mass of a pure substance equal to its

formula weight in grams (g).

In case of solids:

1 molar solution = M.W in gms. x 100/ purity% g/l.Example :

1 molar solution of NaOH = 40 x 100/96.0 g/l.

In case of liquids:

1 molar solution = M.W x 100/purity x Sp.Gr. g/l.

Example:

1 molar solution of acetic acid = 60.05 x 100/99.5 x 1.049 g/l.

Experiment 1: Assessment of Normality and MolarityRead the given details of the reagent from its bottle:

M.W.

Sp. Gr. / or wt/v at 20 c.

Purity %Prepare the given table:

Table: Estimates of Normality and Molarity of analytical reagents:

Compound M.W Sp. Gr. Purity % Amount /L

1 N 1 M

CH3COOH

HCl

H2SO4

H3PO4

NH3

NaOH

KOH

NaCl

KH2PO4

K3HPO4

Liquids in ml. and solids in grams.


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III- CARBOHYDRATES

Introduction:

Sugars can be defined as polyhydroxy aldehydes or ketones. Hence the

simplest sugars contain at least three carbons. The most common are the aldo-and keto-trioses, tetroses, pentoses, and hexoses. The simplest 3C sugars are

glyceraldehye and dihydroxyacetone

All carbohydrates can be classified as either monosaccharides ,

oligosaccharides or polysaccharides. Anywhere from two to ten

monosaccharide units, linked by glycosidic bonds, make up an oligosaccharide.

Polysaccharides are much larger, containing hundreds of monosaccharide units.

The presence of the hydroxyl groups allows carbohydrates to interact with the

aqueous environment and to participate in hydrogen bonding, both within and

between chains. Derivatives of the carbohydrates may contain nitrogens,

phosphates and sulfur compounds. Carbohydrates also can combine with lipid

to formglycolipids or with protein to formglycoproteins.

Monosaccharides:

The monosaccharides commonly found in humans are classified according to

the number of carbons they contain in their backbone structures. The major

monosaccharides contain four to six carbon atoms.

Cyclic Fischer Projection of

-D-Glucose

Haworth Projection of

-D-Glucose
http://web.indstate.edu/thcme/mwking/lipid-synthesis.html#sphingolipidshttp://web.indstate.edu/thcme/mwking/protein-modifications.html#glycoprothttp://web.indstate.edu/thcme/mwking/protein-modifications.html#glycoprothttp://web.indstate.edu/thcme/mwking/protein-modifications.html#glycoprothttp://web.indstate.edu/thcme/mwking/lipid-synthesis.html#sphingolipidshttp://web.indstate.edu/thcme/mwking/lipid-synthesis.html#sphingolipids


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Disaccharides:

Covalent bonds between the anomeric hydroxyl of a cyclic sugar and the

hydroxyl of a second sugar are termed glycosidic bonds, and the resultant

molecules are glycosides. The linkage of two monosaccharides to form

disaccharides involves a glycosidic bond. Physiogically important

disaccharides are sucrose, lactose and maltose.

Sucrose: prevalent in sugar cane and sugar beets, is composed of glucose

- - g l y c o s i d i c b o n d .

Sucrose

Lactose: is found exclusively in the milk of mammals and consists of

galactose -(1,4) glycosidic bond.

Lactose

Maltose: the major degradation product of starch, is composed of 2 glucose-(1,4) glycosidic bond.

Maltose


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Polysaccharides:

Most of the carbohydrates found in nature occur in the form of high molecular

weight polymers called polysaccharides. The monomeric building blocks used

to generate polysaccharides can be varied; in all cases, however, the

predominant monosaccharide found in polysaccharides is D-glucose. When

polysaccharides are composed of a single monosaccharide building block, they

are termed homopolysaccharides. While the Polysaccharides composed of

more than one type of monosaccharide are termed heteropolysaccharides.

Preparation of reagents

1. Molischs reagent

5% naphthal in alcohol, i.e., 5g of naphthal dissolved in 100ml ofethanol.

2. Iodine solution0.005% in 3% KI, i.e., 3g of KI dissolved in 100ml water and then 5mg of

iodine is dissolved.

3. Benedicts solution

17.3g of sodium citrate and 10g of sodium carbonate are dissolved in 75ml

of water. 1.73g of CuSO4.5H2O is dissolved in 20ml of water. Mix the

CuSO4 solution with alkaline citrate with constant stirring, finally the whole

volume is made up to 100ml with water.

4. Barfoeds reagent

13.3g of copper acetate in 200ml of water and add 2ml of glacial acetic acid.5. Bials reagent

Dissolve 300mg of orcinol in 100ml of concentrated HCl.

6. Seliwanoffs reagent

Dissolve 50g of resorcinol in 100ml of con.HCl in the ratio of 1:2.7. Concentrated HCl

8. Concentrated H2SO49. Osazone ReagentPhenyl hydrazine hydrochlorideSodium acetateAcetic acid


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Mechanistic principles of qualitative identification of Carbohydrates

PRINCIPLE OF REACTIONS:

1. Molischs test:

Con. H2SO4 dehydrates carbohydrates to form furfural and its derivatives.This product combines with sulphonated naphthal to give purple colour.

2. Anthrone test:

The same principle outlined above, except that the furfural reacts with

anthrone (10-keto-9,10-dihydroanthracene) to give a blue-green complex.

3. Iodine test:

Iodine forms a coloured absorption complex with polysaccharides due tothe formation of micellae aggregate. Iodine will form a polysaccharideinclusion complex.

4. Benedicts test:

Carbohydrates with a potential aldehyde or ketone group have reducing

property when placed in an alkaline solution. Cupric ions present in thesolution will be reduced to cuprous ion. This will give a red coloured

precipitate. Moreover, this test is more specific for reducing sugars.

5. Barfoedtest:

Barfoeds reagent is weakly acidic and it is only reduced by

monosaccharides. Prolonged boiling may hydrolyze the disaccharide togive false positive test.

6. Bials test:

When pentose is heated with con.HCl, furfural, which condenses with

orcinol in the prescence of ferric ion to give a blue green colour.

7. Seliwanoffs test:

Ketoses are dehydrated more rapidly than aldose to give a furfuralderivatives, which then condenses with resorcinol to form a red colour

complex.


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GENERAL PROCEDURE FOR QUALITATIVE ANALYSIS OF CARBOHYDRATES

No. EXPERIMENT OBSERVATIONINFERENCE

1. Molischs testTo 1ml of test solution, add 2

drops of Molischs reagent.Then add con. H2SO4 carefully

along the sides of the test tube.

Violet coloured ring is

formed at the junction of

the 2 layers.

Presence of

carbohydrate.

2.Anthrone test

To 5 drops of sugar solution

add 2 ml. Anthrone reagent.

Blue green color complex

is formed

Presence of

carbohydrate.

3.Iodine test

To 1ml of the test solution, 2

drops of iodine is added and

observe the colour change.

(i) Deep blue colour is

obtained.

(ii)Dark brown colour is

obtained.

iii)No colour change.

Presence of

polysaccharide.

Presence of

polysaccharide.

(Glycogen)

Absence of

polysaccharide.4.

Benedicts test2ml of Benedicts reagent ismixed with 0.5ml of test

solution and the contents are

boiled for a few minutes.

(i)Orange red precipitate

is obtained.

(ii)No characteristic

colour change.

Presence of

reducing sugar.

Absence of

reducing sugar.


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5.Barfoeds testTo 2ml of test solution, 2ml of

Barfoeds reagent is added andboiled for 3 minutes and the

colour change is noted.

(i)Brick red precipitate is

obtained at the bottom of

test tube.(ii)No colour change.

Presence of

reducing

monosaccharideAbsence of

reducing

monosaccharide.

6.Bials testTo 1ml of the test solution,

5ml of Bials reagent is added.The contents are boiled and

cooled.

(i)Blue green colour

is obtained.

(ii)No colour change.

Presence of

pentose sugar.

Absence ofpentose sugar.

7.Seliwanoffs test

To 1ml of the test solution,

3ml of Seliwanoffs reagent isadded and the contents are

boiled

(i)Cherry red colour

is obtained.

(ii)No colour change.

Presence of

fructose.

Absence of

fructose.

8. FEARON'S TEST

To 1ml of the test solution,

2ml of Fearons reagent is

added and the content is

heated. Then NaOH was

added to the cold mixture.

i)Red coloration appear.

ii)no color change.

Presence of

reducing

disaccharide.

Absence ofreducing

disaccharide.


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B) Enzymatic methods:

1-Hexokinase method (The reference method).

Glucose +ATP +HKADP+G6P

G6P +NAD +G6PD6 P-gluconolactone +NADH+H (measured at 340)

2- Glucose Dehydrogenase Method:

Glucose +NADGDH

Gluconolactone +NADH+H (measured at 340)

3- Glucose oxidase method:

Glucose Oxidase for blood glucose estimation (Experiment #1)PRINCIPLE OF THE METHOD

Glucose oxidase (GOD) catalyses the oxidation of glucose to gluconic acid.

The formed hydrogen peroxide (H2O2), is detected by a chromogenic

oxygen acceptor, phenol-aminophenazone in the presence of peroxidase

(POD):

Principle: (Trinders method )

-D-glucoseMutarotase

-D-glucose

-D-glucose +H2O+O2Glucose oxidase D-gluconic acid+H2O2

H2O2+ 4-aminophenazone + phenolPeroxidase

Quinonemine +4 H2O

The intensity of the color formed is proportional to the glucose concentration

in the sample.

CLINICAL SIGNIFICANCEGlucose is a major source of energy for most cells of the body; insulinfacilitates glucose entry into the cells. Diabetes is a disease manifested by

hyperglycemia; patients with diabetes demonstrate an inability to produceinsulin. Clinical diagnosis should not be made on a single test result; it shouldintegrate clinical and other laboratory data.


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PREPARATIONWorking reagent (WR):Dissolve the contents of one vial R 2 Enzymes in one bottle of R 1 Buffer.

Cap and mix gently to dissolve contents.

Instrumentation:

-Photometer adjusted on wavelength 540 nm

-Cuvette (light path) 1 cm

-Water bath at 37 C

-Automatic pipettes, disposable test tubes , racks and disposable tips.PROCEDURE

1. Assay conditions:

Wavelength: . . . . . . . . . . . . . .. . 505 nm (490-550)Cuvette: . . . . . . . . . . . . . . . . . . . . .. 1 cm light pathTemperature. . . . . . . . . . . . . . . . . . . 37C / 15-25C2. Adjust the instrument to zero with distilled water.3. Pipette into a cuvette:

Sample Standard Blank

1.0 1.0 1.0 WR (mL)

-- 10 -- Standard (L)10 -- -- Sample (L)

4. Mix and incubate for 10 min at 37C or 15-20 min at room temperature (15-

25C).

5. Read the absorbance (A) of the samples and standard, against the Blank.

The colour is stable for at least 30 minutes.

CALCULATIONS(A) Sample x 100 (Standard conc.) = mg/dL glucose in the sample(A) Standard


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Discussion:

*Physiological & Biochemical Background:

Glucose metabolism, Insulin action and other hormonal effects on glucose in thehuman body.

*Pathological & Disease Correlation:

Diabetes Mellitus, Cushing syndrome ,Hyperthyroidism ..etc

Questions:

1- What are the basis of reduction methods for glucose

estimation. ?2- Give short notes on Trinders method for glucose estimion.


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Introduction :

QUALITATIVE ANALYSIS OF LIPIDS

Lipids are naturally occurring compounds that are esters of long chain fatty acids.

They are insoluble in water but soluble in fat solvents such as acetone, alcohol,chloroform or benzene.

Alkaline hydrolysis (known assaponification) gives rise to the alcohol and the Na

or K salts of the constituent fatty acids.

Chemically, lipids can be divided into two main groups:

1- Simple lipids.

2- Compound lipids.

Steroids and the fat soluble vitamins are also considered as lipids because of their

similar solubility characteristics and are known as derived lipids.

Major Roles of Biological Lipids:

Lipids of physiological importance for humans have four major functions:

1. They serve as structural components of biological membranes.

2. They provide energy reserves, predominantly in the form of

triacylglycerols.

3. Both lipids and lipid derivatives serve as vitamins and hormones.

4. Lipophilic bile acids aid in lipid solubilization.

1- Simple lipids:

Esters of glycerol and fatty acids are known as acylglycerols orglycerides.

The acylglycerols are known asneutral lipids, they are called fats or

oils, depending on whether they are

solid or liquid at room temperature.

Shown to the right is the structure of

Triglycerides.


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PRINCIPLES:

The main groups of lipids have different solubility characteristics. When fats and

oils are heated with alkali, free fatty acids and glycerol are liberated and this

process is known as saponification. The excess alkali present reacts with the

liberated fatty acids to form the Na or K salts which give the solution a

characteristic soap appearance.

The fatty acids in animal fats are usually fully saturated whereas those found in

vegetable oils contain one or more double bonds. Hydrogenation of the double

bonds converts the oils into solid fats and this is carried out commercially for the

production of margarine.

Halogens also readily add across the double bonds and the decolorize

tion of a solution of bromine or iodine by a lipid indicates the presence of double

bonds.

MATERIALS:

Reagents

Alco. KOH (100 g/l in ethanol). Ethanol (absolute).

HCl. Conc. Diethyl ether.

NaOH (1N). Petroleum ether.

NaCl (solid).

Chloroform. KI (10% w/v. Benzene.

Copper acetate (1% w/v).

Standard liquids

Phospholipids: Lecithin.

Sterols: cholesterol (solid and

0.5% in ethanol).

1- SOLUBILITY:

Note the physical state of:

Fatty acids: palmitic acid,

Stearic acid.

Fats: Butter.

Oils: Olive oil.

f)

g)

Water.

Ethanol.

a)

b)

Palmetic acid.

Stearic acid.

h)

i)

j)

Diethyl ether.

Petroleum ether.

Chloroform.

c)

d)

e)

Oleic acid.

Olive oil.

Butter.

Carefully observe the differences between the above groups of lipids, write your

remarks.


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2- GREASE STAIN TEST:

Place one drop of the above lipids on a filter and leave to dry, observe the

formation of a clear grease spot and give your remarks.

3- COPPER ACETATE TEST:

Dissolve a few drops of the oil in 3 ml. of petroleum ether and add equal volumes

of copper acetate (1%), mix once by inversion (Dont shake). Leave the tube until

the emulsion will separate into two layers.

A. Saturated fatty acid: upper layer is clear and precipitate in the lower layer.

B. Unsaturated FA: upper layer is greenish-Blue color and lower layer is

colorless.

C. If the two layers are clear then the test.4- TEST FOR UNSATURATION:

Add one drop of Olive oil, one spatula-point of Butter and one spatula-point of

Lecithin to separate dry test tubes, and dissolve the lipids in about 1 ml. of

chloroform.

Add 1 ml. of chloroform to other test tube to act as Blank.

By means of Pasteur pipette add drop wise a solution of bromine in chloroform

until define yellow color is produced, Note the number of drops in each case, andcomment on the results.

CH3 (CH2)7 CH=CH (CH2)7 COOH + Br2

Oleic acid Bromine

H H

CH3 (CH2)7 C C (CH2)7 COOH

Br Br

Dibromo-Stearic acid


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5- TEST FOR CHOLESTEROL (Liebermans test):

Add 10 drops of acetic anhydride and 2 drops of conc. H2SO4 to 2 ml of each of the

following:

a) Cholesterol solution in chloroform (0.5 %).

b) Egg yolk solution in chloroform (0.5%).

c) Butter solution in chloroform (0.5%).

d) Chloroform.

Give possible interpretation of the reaction in each case.


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e.g. : Glycine :

pKa= 2.34 9.60

H3N+---CH2---COOH H++H3N+---CH2---COO- H++H2N---CH2---COO-

Gly+ Glyo Gly-

Buffering:

According to Henderson-Hasselbalch equation:

pH = pKa + log[A-]/[HA]

At the point of the dissociation where the concentration of the conjugate base [A-] is

equal to that of the acid [HA]: pH = pKa + log[1]

The log of 1 = 0. Thus, at the mid-point of titration of a weak acid:

pH = pKa

At this point, when the pH = pKa, the slope of the curve (i.e. the change in

pH with addition of base or acid) is at a minimum, so the buffer solution best resists

addition of either acid or base, and hence has its greatest buffering ability. As a

general rule, buffer solution can be made for a weak acid/base in the range of 1 pH

unit from the pKa of the weak acids.

addition of strong base produces weak conjugate base:

CH3CO2H + OH-------------> CH3CO2

-+ H2O

addition of strong acid produces weak acid:

H3O+

+ CH3CO2------------> CH3CO2H + H2O

Blood Buffering:

(to understand the role of imidazole ring of Histidine in buffering capacity ofhaemoglobin)

The pH of blood is maintained in a narrow range around 7.4. Even

relatively small changes in this value of blood pH can lead to severe metabolic

consequences. Therefore, blood buffering is extremely important in order to

maintain homeostasis.The primary buffers in blood are hemoglobin in erythrocytes

and bicarbonate ion (HCO3-) in the plasma. Buffering by hemoglobin is

accomplished by ionization of the imidazole ring of histidines in the protein.


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EXPERIMENT-1

Preparation of Normal Titration Curve for Glutamic Acid

The dissociation of glutamic acid can be represented as:

We are going to use the pH meter to explore the acid-base behavior of Glutamic

acid .

Materials:- Burette

- Beaker.- pH standards.

- 0.05M NaOH.

- 0.05M HCl.

- 0.05M Glu.

- 0.05M Hi.

- Ph meter.

Procedure:

1- Titrate 10 ml. of 0.05M Glutamic acid against 0.05M NaOH. Repeat the

titration with 0.05M HCl.

2- Record your data in the given table.3- Sketch a curve from your data on a graph paper.

(Plot pH (Yaxis) versus volume of NaOH expended (Xaxis).

Volume (ml.)

NaOH 0.05M

pH Volume (ml.)HCl. 0.05M

pH

0.0 0.0

0.5 0.5

1.0 1.0

1.5 1.52.0 2.0

2.5 2.5

3.0 3.0

3.5 3.5

4.0 4.0

4.5 4.5

5.0 5.0


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EXPERIMENT-2

Preparation of Normal Titration Curve for Histidine

Repeat the above procedure using 0.05M Histidine.

The dissociation of Histidine can be represented as:

Here , is the ionizable groups of Histidine:


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Protein solubility and separation

PROTEINS:

Proteins are polymers of the bifunctional monomers, amino acids. The twenty

common naturally-occurring amino acids each contain an -carbon, an -aminogroup, an -carboxylic acid group, and an -side chain or side group. These sidechains (or R groups) may be either nonpolar, polar and uncharged, or charged,

depending on the pH and pKa of the ionizable group.

Amino acids form polymers, the amino group from one amino acid attached to

the carboxylic group of the adjacent amino acid, the resulting link between them

is an amide link which biochemists call a peptide bond. In this reaction, water is

released. In a reverse reaction, the peptide bond can be cleaved by water

(hydrolysis).

O O

H2N C ------ HN C OH

C C

R1 R2

Peptide bond

When two amino acids link together to form an amide link, the resulting structure

is called a dipeptide. Likewise, we can have tripeptides, tetrapeptides, and other

polypeptides. At some point, when the structure is long enough, it is called a

protein. There are many different ways to represent the structure of a polypeptide

or protein.


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Solubility of proteins:

Introduction:

The solubility of protein depends on, among other things, the salt concentration in

the solution.

At low concentrations, the presence of salt stabilizes the various charged groups

on a protein molecule, thus attracting protein into the solution and enhancing the

solubility of protein. This is commonly known as salting-in. However, as the salt

concentration is increased, a point of maximum protein solubility is usually

reached. Further increase in the salt concentration implies that there is less and

less water available to solubilize protein. Finally, protein starts to precipitate when

there are not sufficient water molecules to interact with protein molecules. This

phenomenon of protein precipitation in the presence of excess salt is known as

salting-out.

Precipitation of Proteins at isoelectric Point:

Protein solubi li ty:

The solubility of proteins in aqueous buffers depends on the distribution ofhydrophilic and hydrophobic amino acid residues on the proteins surface.

Proteins that have high hydrophobic amino acid content on the surface havelow solubility in an aqueous solvent.

Charged and polar surface residues interact with ionic groups in the solventand increase solubility.

I soelectric point (pI ):

Is the pH-value of a solution at which the total net charge of a protein equalszero.

At a solution pH that is above the pI the surface of the protein is predominantlynegatively charged and therefore like-charged molecules will exhibit repulsive

forces.

Likewise the surface of the protein is predominantly positively charged at asolution pH that is below the pI, and repulsion between proteins occurs.

However, at the pI the negative and positive charges are eliminated, repulsiveelectrostatic forces are reduced and the dispersive forces predominate. The dispersive forces will cause aggregation and precipitation. The pI of most proteins ranges between the pH 4 to 6. When microorganisms grow in milk, they often produce acids and lower the

pH of the milk. The phenomenon of precipitation or coagulation of milk protein (casein) at low

pH as milk becomes spoiled is one of the common examples of protein

isolation due to changes in the pH.


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Procedure:1. Into a 50 ml volumetric flask add 20 ml of water.2. Add 0.25 g of pure casein, followed by the addition of 5 ml of 1 N NaOH

solution.3. Once casein is dissolved, add 5 ml of 1 N acetic acid solution, then dilute with

H2O to 50 ml and mix well. The resulted solution is a 0.1 N casein acetatesodium.

4. Setup a series of 9 test tubes.5. In the first test tube put 3.2 ml1 N CH3COOH, and 6.8 ml H2O and mix

thoroughly.

6. In each of the other test tubes (2-9) put 5 ml H2Od.7. From the test tube 1 transfer 5 ml to the test tube 2, and mix thoroughly.

8. Repeat step 7 for the rest of test tubes (3 - 9).9.Now to each test tube (1 -9) add 1 ml of the casein acetate sodium solution, andshake the test tubes immediately.

10.Let the samples stand for 30 min, and note the turbidity in the 9 test tubes.11.Use)+(and )(signs to describe the turbidity in the different test tubes.12.You should observe the most precipitation in the test tube which has the pH

around 4.7 (close to the isoelectric point of casein).

Results:

987654321TUBE

0.0060.0120.0250.050.10.20.40.81.61N CH3COOH

5.95.65.35.04.74.44.13.83.5PH

TURBI-DITY


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PROTEIN SIZE AND SEPARATION

Separation of proteins

The molecular weights of proteins are very high. Due to their wide variety of

amino acid configurations, proteins behave very differently. These differencesconstitute the bases of the biochemical function of proteins. And these basic

differences are also the parameters which are used to separate proteins:

1- Physical Size:

Which reflects molecular weight of the protein, Gel filtration uses this

parameter, also referred to as Exclusion chromatography.

2- Electric Charge:

Some amino acid residues are positively charged while others are negatively

charged. Variations in the pH of an amino acid system cause variations in the

charge of amino acid residues. Consequently, the net surface charge of a

protein (comprised of amino acid residues) also varies with its environment.

It is these variations in the net charge of proteins which allow them to be

separated by such techniques as ion exchange chromatography or by

electrophoretic techniques.

3- Hydrophobic character:

Hydrophobic regions available to interact with a hydrophobic stationary

phase provide the important characteristic of proteins which is used in

adsorption chromatography.

4- 3-dimentional substructure:

Provide the basis for very specific separation methods. Bio-specific affinity

chromatography is used to separate proteins according to their biologicalfunctions. Proteins, such as enzymes, antibodies and glycoproteins are

particularly appropriate for separation by affinity chromatography.


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Exclusion Chromatography

Experiment:

Objectives:

a- To demonstrate the principle of molecular exclusion (gel permeation)chromatography using a bead of G-100 Sephadex gel to separate a mixtureof:Fluorescin : Yellow M.wt. = 332 (size of a dipeptide).

Hemoglobin : Red M.wt. = 68,000 (protein).

Blue Dextran : Blue M.wt. = 200,000 (large protein).

b- To show that the technique can be used to determine the Molecular Weightof a newly discovered protein.

Principle:

In gel filtration the gel acts as a molecular sieve separating molecules with

differences in molecular size and weight. The gel matrix contains numerous

porous beads (stationary phase) with (mobile phase) in between. If the ample of

mixture is applied at the top of the column, the large molecules in the sample will

not be able to enter the pores in the bead but will pass between them and so be

eluted first, smaller molecules that have access to the pores are retarded in the gel

to a certain extent and will therefore be eluted after the large molecules in order ofdecreasing M.wt. and size.

Materials:

Sephadex G-100: 0.5 g of (S.G-100) /100 ml. of 0.3% (w/v) NaCl; leave toswell for 1 hr. prior to experiment.Eluant: 0.3% (w/v) NaCl.Mixture of : Flurescin, hemoglobin and blue dextran (each 0.1 g/10 ml.

H2O). To prepare Hemoglobin dilute 1 ml. of blood to 10 ml. with H 2O.

Chromatography column (0.1 to 1.5 cm. diameters).Microperpox peristaltic pump: Flow rate = 0.5 ml/min.Disposable syringes (5ml, needle size 20 G).

Method:

1. Set up the column replacing the waist flask by a fraction collector.

2. Pour slurry of Sephadex G-100, into the column and allow the gel beads tosettle. Once about 10 cm. have settled, allow the solvent to run out of the

button of the column. Do not allow the liquid level to fall below the top ofthe Sephadex. Continue to add Sephadex until you have a column whose

settled height is at least 15 cm.


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