51284 Protein

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Proteins

Introduction

Proteins are very large molecules with molecular weights that range from 6000 to Proteins are very large molecules with molecular weights that range from 6000 to millions of grams per mole. While proteins can be used as an energy source by the body, this is millions of grams per mole. While proteins can be used as an energy source by the body, this is not their primary role.

not their primary role. Proteins are essential for the catalysisProteins are essential for the catalysis of most of the body's chemical of most of the body's chemical reactions, for structural support and for the transport/storage of vital nutrients. As proteins and reactions, for structural support and for the transport/storage of vital nutrients. As proteins and amino acids (the components of proteins) are not stored in the body, some protein intake is amino acids (the components of proteins) are not stored in the body, some protein intake is required each day.

required each day.

I- General properties of amino acids

Amino acids are the building blocks of proteins. In humans, the amino acids used are Amino acids are the building blocks of proteins. In humans, the amino acids used are --amino acids

amino acids, which means the, which means the carboxylic acid group and the amino group are located on thecarboxylic acid group and the amino group are located on the same carbon.

same carbon. TheThe αα-amino and-amino and αα-carboxyl groups on amino acids act as acid–base groups,-carboxyl groups on amino acids act as acid–base groups,

donating or accepting a proton as the pH is altered. At low pH, both groups are fully protonated, donating or accepting a proton as the pH is altered. At low pH, both groups are fully protonated, but as the pH is increased first the carboxyl group and then

but as the pH is increased first the carboxyl group and then the amino group loses a hydrogen ion.the amino group loses a hydrogen ion. For the standard 20 amino acids, the p

For the standard 20 amino acids, the p K K is in the range 1.8–2.9 for theis in the range 1.8–2.9 for the αα-carboxyl group and-carboxyl group and

8.8–10.8 for the

8.8–10.8 for the αα-amino group. Those amino acids with an ionizable side-chain have an-amino group. Those amino acids with an ionizable side-chain have an

additional acid–base group with a distinctive p additional acid–base group with a distinctive p K K ..

Isoelectric Point (pI

Isoelectric Point (pI): is the pH at which the amino acid has no net charge. The): is the pH at which the amino acid has no net charge. The isoelectric point of an amino acid is the average of the p

isoelectric point of an amino acid is the average of the p Ka Ka values of the protonation transitionsvalues of the protonation transitions on

on either sideeither side of the isoelectric species. For example, of the isoelectric species. For example, aspartic acid isoelectric species exists inaspartic acid isoelectric species exists in the pH domain between p

the pH domain between p Ka Ka1 (2.1) and p1 (2.1) and p Ka Ka2 (3.9). Thus, the 2 (3.9). Thus, the pI of aspartic acid is 3.0.pI of aspartic acid is 3.0. Reactive groups in

Reactive groups in amino acids include -NHamino acids include -NH22 and -COOH groups and -COOH groups and and groups presentgroups present

on side R chains

on side R chains. In peptides and proteins only the side chain is available for reactions besides. In peptides and proteins only the side chain is available for reactions besides amino and carboxylic groups at the terminal ends. When amino acids are linked together,

amino and carboxylic groups at the terminal ends. When amino acids are linked together, amideamide or peptide bonds

or peptide bonds are formed. The formation of an amide group is shown in the reaction below, are formed. The formation of an amide group is shown in the reaction below, in which two amino acids react to form a dipeptide.

in which two amino acids react to form a dipeptide.

Reactions of this type can continue to link many amino acids together to form Reactions of this type can continue to link many amino acids together to form polypeptides. When the number of amino acids in the molecule reaches about

polypeptides. When the number of amino acids in the molecule reaches about5050, it is considered, it is considered a protein. Proteins can

a protein. Proteins can be classified as simple or complex. A simple protein is composed be classified as simple or complex. A simple protein is composed only ofonly of amino acids. Complex proteins, which are far more common, incorporate other non-amino acid amino acids. Complex proteins, which are far more common, incorporate other non-amino acid groups in their structure.

groups in their structure.

- H - H22OO

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Exp. # 1

Exp. # 1 The solubility of amino acids test

The solubility of amino acids test

Principle: Principle:

In general amino acids are

In general amino acids are soluble in aqueous solution and the soluble in aqueous solution and the lowest solubility aroundlowest solubility around their

theirisoelectric point (pI),isoelectric point (pI), but slightly soluble or insoluble in organic but slightly soluble or insoluble in organic solvents. Nearly allsolvents. Nearly all aliphatic amino acids exist as

aliphatic amino acids exist as zwitterionszwitterions (The doubly charged situation of the amino acid). The (The doubly charged situation of the amino acid). The strong positive charge on the NH3+ group induces a tendency for COOH group to loose a proton, strong positive charge on the NH3+ group induces a tendency for COOH group to loose a proton, so that amino acids are stronger acids than many of the weak acids. For example glycine has a so that amino acids are stronger acids than many of the weak acids. For example glycine has a pKa of 2.4 in comparison to Acetic acid 4.8.

pKa of 2.4 in comparison to Acetic acid 4.8.

Materials and reagents: Materials and reagents: 1.

1. Hydrochloric acid (0.1mol/l)Hydrochloric acid (0.1mol/l) 2. Sodium hydroxide (0.1mol/l) 2. Sodium hydroxide (0.1mol/l) 3. Ethanol

3. Ethanol 4. Chloroform 4. Chloroform 5. Amino acids

5. Amino acids (glycine, glutamic aci(glycine, glutamic acid, lysine, and ald, lysine, and alanine)anine)

Method: Method:

Examine the solubility of the

Examine the solubility of the above amino acids in water, above amino acids in water, dilute acid, and dilute alkali,dilute acid, and dilute alkali, ethanol, and chloroform.

ethanol, and chloroform. 1)

1) Add 1 – Add 1 – 2 ml of water, dilute acid, and 2 ml of water, dilute acid, and dilute alkali, ethanol, and chloroform solution todilute alkali, ethanol, and chloroform solution to different labeled tubes.

different labeled tubes. 2)

2) Add a very Add a very small amount of the solid amino acids.small amount of the solid amino acids. 3)

3) Examine the solubility of the above amino acids.Examine the solubility of the above amino acids. *** How do

*** How do you interpret the results?you interpret the results?

Exp.

Exp. #2

#2 The

The ninhydrin

ninhydrin test

test

Ninhydrin (triketohydrindene hydrate) is a powerful oxidating agent which leads to the Ninhydrin (triketohydrindene hydrate) is a powerful oxidating agent which leads to the oxidative deamination of

oxidative deamination ofalpha-amino (NHalpha-amino (NH22) groups between pH 4 and pH 8) groups between pH 4 and pH 8. It is very. It is very

important for the detection and

important for the detection and the quantitative analysis of amino acids. Ninhydrin also reactsthe quantitative analysis of amino acids. Ninhydrin also reacts with primary amines and ammonia however

with primary amines and ammonia however the formation ofthe formation ofcarbon dioxide is quite diagnosticcarbon dioxide is quite diagnostic for amino acids

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Imino acids (Proline and hydroxyproline) yield a

Imino acids (Proline and hydroxyproline) yield a yellowyellow product (absorption maximum product (absorption maximum

440 nm

440 nm). Ninhydrin reaction is very sensitive and is ideal for the ). Ninhydrin reaction is very sensitive and is ideal for the detection of amino acids ondetection of amino acids on chromatograms and their quantitative determination.

chromatograms and their quantitative determination.

Ninhydrin reaction is very sensitive and is ideal for the detection of amino acids on Ninhydrin reaction is very sensitive and is ideal for the detection of amino acids on chromatograms and their quantitative determination in column fr

chromatograms and their quantitative determination in column fr actions.actions.

1. Amino acids(1g/lglycine, tyrosine, and tryptophan)Amino acids(1g/lglycine, tyrosine, and tryptophan)

2.

2. Ninhydrin (2g/l prepare fresh) Ninhydrin (2g/l prepare fresh)

Method: Method:

1-1- Place 1 ml of the amino acid solution in a test tube.Place 1 ml of the amino acid solution in a test tube.

2-2- Adjust the pH to about neutrality.Adjust the pH to about neutrality.

3-3- Add five drops of Ninhydrin solution.Add five drops of Ninhydrin solution.

4-4- Boil for 2min.Boil for 2min.

Optional:

Optional: Determine the limits of sensitivity of the reaction by Determine the limits of sensitivity of the reaction by carrying out the test on serialcarrying out the test on serial dilutions of glycine until a negative

dilutions of glycine until a negative result is obtained.result is obtained.

Exp. #3

Exp. #3 The The xanthoproteic reaction.xanthoproteic reaction. Principle:

Principle:

Xanthoproteic comes from the Greek word

Xanthoproteic comes from the Greek wordxanthosxanthos which means yellow. Boiling which means yellow. Boiling

concentrated nitric acid reacts with tyrosine, tryptophan and phenylalanine (phenylalanine give concentrated nitric acid reacts with tyrosine, tryptophan and phenylalanine (phenylalanine give weakly positive reaction as it contain an inactive benzene ring) to yield

weakly positive reaction as it contain an inactive benzene ring) to yieldyellowyellow products. The products. The intensity of yellow color deepens upon the addition of

intensity of yellow color deepens upon the addition ofalkalinealkaline solution that form solution that form orangeorange colored

colored salt in the basic medium salt in the basic medium..

Materials and reagents: Materials and reagents:

1-1- Amino acids (1g/l glycine, tyrosine, tryptophan and phenylalanine).Amino acids (1g/l glycine, tyrosine, tryptophan and phenylalanine).

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3-3- Nitric acid (conc.) Nitric acid (conc.)

4-4- Sodium hydroxide (10 mol/l)Sodium hydroxide (10 mol/l)

Method: Method:

1-1- Add an equal volume of conc. Nitric acid to about 0.5 ml of the amino acid solution.Add an equal volume of conc. Nitric acid to about 0.5 ml of the amino acid solution.

2-2- Cool, and observe the color change.Cool, and observe the color change.

3-3- Add sufficient NaOH to make Add sufficient NaOH to make the solution strongly alkaline.the solution strongly alkaline.

^-^-^-^-^-^- YellowYellow color in acid solution which turnscolor in acid solution which turns deeper yellowdeeper yellow then thenbright orangebright orange with alkali with alkali constitutes a positive result.

constitutes a positive result.

4-4- Repeat the test with the phenol solution. Phenylalanine gives a negative or weakly positiveRepeat the test with the phenol solution. Phenylalanine gives a negative or weakly positive solution.

solution.

Exp. # 4 Millon’s reaction

Compounds containing the

Compounds containing thehydroxybenzenehydroxybenzene radical react with millon’s reagent to form radical react with millon’s reagent to form

red

red complexes. The only phenolic amino acids are complexes. The only phenolic amino acids aretyrosinetyrosine and its derivatives and on and its derivatives and only thesely these amino acids give a

amino acids give a positive reaction. The original Millon’s reagent was a solution of mercuricpositive reaction. The original Millon’s reagent was a solution of mercuric

nitrate

nitrate in 50% v/v in 50% v/vnitric acidnitric acid, but modifications are now , but modifications are now used that are less liable to interferenceused that are less liable to interference from organic salts.

from organic salts.

1- Amino acids

1- Amino acids (1 g/l glycine, (1 g/l glycine, tyrosine, and tyrosine, and phenylalanine).phenylalanine). 2- Millon’s reagent (150 g/l solution of

2- Millon’s reagent (150 g/l solution of mercuricmercuricsulphatesulphate in 15% v/v in 15% v/vsulphuric acidsulphuric acid).). 3- Phenol (1 g/l).

3- Phenol (1 g/l). 4- Sodium nitrate (

4- Sodium nitrate (10 g/l10 g/l).). 5- Boiling water bath. 5- Boiling water bath.

Method: Method:

1-1- Add five drops of Add five drops of Millon’s reagent to 1 ml of the test solution.Millon’s reagent to 1 ml of the test solution.

2-2- Heat in a boiling water bath for 10 min. ThenHeat in a boiling water bath for 10 min. Thencoolcoolto room temperature.to room temperature.

3-3- Add five drops of Add five drops of sodium nitrate solution; asodium nitrate solution; apositive resultpositive result is indicated by a is indicated by a brick redbrick red color. color.

Exp.

Exp. #

# 5

5 Glyoxylic

Glyoxylic reaction

reaction for t

for tryptophan (Hopkins-Cole

ryptophan (Hopkins-Cole test)

test)

The

The indole group of tryptophanindole group of tryptophan reacts with reacts with glyoxylic acidglyoxylic acid in the presence of conc. in the presence of conc. Sulphuric acid to give a

Sulphuric acid to give apurplepurple color. color. Glacial acetic acid that has been exposed to the lightGlacial acetic acid that has been exposed to the light contains glyoxilic acid

contains glyoxilic acid. Also protein can be detected by this test as concentrated H. Also protein can be detected by this test as concentrated H22SOSO44 at the at the

solution interface hydrolyses protein to form free tryptophan,

solution interface hydrolyses protein to form free tryptophan, which reacts with glyoxylic acid towhich reacts with glyoxylic acid to form

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1.

1.Amino acid (1g/l glycine, tyrosine, and tryptophan).Amino acid (1g/l glycine, tyrosine, and tryptophan). 2.

2.GlyoxylicGlyoxylic acid; Glacial acetic acid which has been exposed to light. acid; Glacial acetic acid which has been exposed to light. 3.

3.Sulphuric acid (conc.).Sulphuric acid (conc.).

Method: Method:

1-1- Add 2ml of glacial Add 2ml of glacial acetic acid to 2ml of the acetic acid to 2ml of the test solution.test solution.

2-2- Pour about 2 ml of conc. HPour about 2 ml of conc. H22SOSO44carefully down the sides of a carefully down the sides of a sloping test tube so as to formsloping test tube so as to form

two layers. two layers.

3-3- Observe any color change at the liquid junction.Observe any color change at the liquid junction.

-- - - A positive result is shown by - - A positive result is shown by the formation of athe formation of apurple-violet ringpurple-violet ring at the junction of the two at the junction of the two liquids.

liquids.

Exp. # 6 Pauly’s reaction

The basic principle

The basic principle involved in pauly's test isinvolved in pauly's test isdiazotizationdiazotization. The sulphanilic acid gets. The sulphanilic acid gets diazotised in the presence of

diazotised in the presence of sodium nitrite and sodium carbonate with the sample. Diazotizedsodium nitrite and sodium carbonate with the sample. Diazotized sulphanilic acid reacts with the

sulphanilic acid reacts with thephenol group of tyrosinephenol group of tyrosine, and, and imidazole ring of histidineimidazole ring of histidine to to form highly-colored azo compounds. Th

form highly-colored azo compounds. This test reacts with histidine to give ais test reacts with histidine to give aredred color and with color and with tyrosine to give an

tyrosine to give an orangeorange color. Tryptophan givescolor. Tryptophan gives yellowyellow color. The diazonium compounds are color. The diazonium compounds are only formed in the

only formed in the coldcold, so all solutions are cooled in ice , so all solutions are cooled in ice before diazotization.before diazotization.

1.

1. Amino acids (1g/l glycine, tyrosine, histidine, and tryptophan).Amino acids (1g/l glycine, tyrosine, histidine, and tryptophan). 2.

2. Sulphanilic acid (10g/l solution in Sulphanilic acid (10g/l solution in 1mol/l Hydrochloric acid)1mol/l Hydrochloric acid) 3.

3. Sodium nitrate (Sodium nitrate (50g/l50g/l)) 4.

4. Sodium carbonate (10g/l)Sodium carbonate (10g/l)

Method: Method:

1-1- Add 2 ml of the Add 2 ml of the test solution to test tube,test solution to test tube,coolcool in ice. in ice.

2-2- Mix 1 ml of sulphanilic Mix 1 ml of sulphanilic acid with the test solution,acid with the test solution,keep on icekeep on ice..

3-3- Add 1ml of NaNOAdd 1ml of NaNO22 solution and solution andleaveleave in the cold for 3min. in the cold for 3min.

4-4- Make the solution alkaline by the addition of 2 ml of NaMake the solution alkaline by the addition of 2 ml of Na22COCO33 and note the colors formed. and note the colors formed.

Exp. #7

Ehrlich Ehrlich’’s reagents reagent Principle:

Principle:

Ehrlich's reacts with a number of

Ehrlich's reacts with a number of organic compounds such asorganic compounds such asindoles, aromatic amines,indoles, aromatic amines, and ureides

and ureides to give colored copmoundsto give colored copmounds..Ehrlich's reagent is well known strong electrophileEhrlich's reagent is well known strong electrophile which reacts with the electron-rich

which reacts with the electron-rich !!-carbon of-carbon of indoleindole rings to form a rings to form a blue-colouredblue-coloured compounds. compounds.

The reason it is used as the

The reason it is used as the confirmative test for tryptophan utilization by some bacterial speciesconfirmative test for tryptophan utilization by some bacterial species by Indole test. Also, biologically, Ehrlich's reagent used for detection and content determination by Indole test. Also, biologically, Ehrlich's reagent used for detection and content determination

of

of Urobilinogen in urine Urobilinogen in urine samples as samples as it giveit givedark pink or red colordark pink or red color. In. In industrial chemistryindustrial chemistry it it is used for

is used for hydrazinehydrazine (N (N22HH44) spectrophotometeric determination at 458 nm ) spectrophotometeric determination at 458 nm as Ehrlich's reagentas Ehrlich's reagent

form distinct

form distinct yellow coloryellow color with hydrazine. with hydrazine.

1. Ehrlich’s reagent (100g/lEhrlich’s reagent (100g/l p p-dimethylaminobenzaldehyde-dimethylaminobenzaldehyde in conc. Hydrochloric acid) in conc. Hydrochloric acid)

2.

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3.

3. Urea (1g/l).Urea (1g/l).

Method: Method:

1-1- Add 2 ml of EAdd 2 ml of Ehrlich’s reagent to 0.5 ml of the test solution.hrlich’s reagent to 0.5 ml of the test solution.

2-2- Observe the color.Observe the color.

Exp. # 8 The nitroprusside test

Thiol groups react with sodium nitroprusside (Na

Thiol groups react with sodium nitroprusside (Na22Fe(CN)Fe(CN)55 NO) NO) in the presence of excessin the presence of excess

ammonia to give

ammonia to givea reda red color. This test is specific for cysteine. color. This test is specific for cysteine.

1. Sulphur amino acids (1 g/l cysteine, cystine, and methionine).Sulphur amino acids (1 g/l cysteine, cystine, and methionine).

2.

2. Sodium nitroprusside (20 g/l prepare fresh).Sodium nitroprusside (20 g/l prepare fresh).

3.

3. Ammonium hydroxide.Ammonium hydroxide.

Method: Method:

1.

1. Mix 0.5 ml of a Mix 0.5 ml of a fresh solution of sodium nitroprusside with 2ml of the test solution.fresh solution of sodium nitroprusside with 2ml of the test solution. 2.

2. Add 0.5 ml of ammonium hydroxide.Add 0.5 ml of ammonium hydroxide. 3.

3. Observe the color.Observe the color.

Exp. # 9 The Sakaguchi reaction Exp. # 9 The Sakaguchi reaction Principle:

Principle:

The only amino acid containing the

The only amino acid containing theguanidine group is arginineguanidine group is arginine, and this reacts with, and this reacts with !! – –

naphthol and an oxidizing agent

naphthol and an oxidizing agentbromide waterbromide water to give a red color. to give a red color.

1-1- Amino acids (1 g/l glycine and arginine).Amino acids (1 g/l glycine and arginine).

2-2- Guanidine (1 g/l glycocyamine, methylguanidine and creatine).Guanidine (1 g/l glycocyamine, methylguanidine and creatine).

3-3- Urea (1 g/l).Urea (1 g/l).

4-4- Sodium hydroxide(10mol/l).Sodium hydroxide(10mol/l).

5-5- !! –Naphthol(10g/l in alcohol). –Naphthol(10g/l in alcohol).

6-6- Bromine water (add a few drops of bromine to 100 ml of water and shake; do this in a fumeBromine water (add a few drops of bromine to 100 ml of water and shake; do this in a fume chamber).

chamber). Caution

Caution: Bromine gives very nasty burns if spilt on the : Bromine gives very nasty burns if spilt on the skin.skin.

Method: Method:

1-1- Mix 1 ml of Mix 1 ml of strong alkali with 3 ml of the amino acid solution.strong alkali with 3 ml of the amino acid solution.

2-2- Add two drops ofAdd two drops of !! –naphthol. –naphthol.

3-3- Mix thoroughly and add four or five drops of bromine water.Mix thoroughly and add four or five drops of bromine water.

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II- General reactions for proteins

Albumins Albumins

Albumin (Latin: albus, white) refers generally to any

Albumin (Latin: albus, white) refers generally to any protein readily soluble in water,protein readily soluble in water, dilute acids and alkalies which

dilute acids and alkalies which may be precipitated out from solution using high saltmay be precipitated out from solution using high salt concentration in a

concentration in a process calledprocess called'salting out''salting out'. Albumin experiences heat coagulation . Albumin experiences heat coagulation (protein(protein denaturation). Substances containing albumin, such as egg white, are called

denaturation). Substances containing albumin, such as egg white, are called albuminoidsalbuminoids.. Albumin is the main protein of plasma; it

Albumin is the main protein of plasma; it binds water, cations (such as Cabinds water, cations (such as Ca2+2+, Na, Na++ and K and K ++), fatty), fatty acids, hormones, bilirubin and drugs -

acids, hormones, bilirubin and drugs - its main function is to regulate the colloidal osmoticits main function is to regulate the colloidal osmotic pressure of blood.

pressure of blood.

Globulins Globulins

Globulins are insoluble or sparingly soluble in

Globulins are insoluble or sparingly soluble in water, but their solubility is greatlywater, but their solubility is greatly increased by the addition

increased by the addition of neutral salts such as sodium chloride in a of neutral salts such as sodium chloride in a process calledprocess called'salting in''salting in'.. These proteins are coagulated by heat. They are deficient in methionine. Example - Serum

These proteins are coagulated by heat. They are deficient in methionine. Example - Serum globulin, Fibrinogen, Myosin of muscle

globulin, Fibrinogen, Myosin of muscle

Casein Casein

Casein

Casein (from Latin "cheese") is the predominant phosphoprotein that accounts for nearly (from Latin "cheese") is the predominant phosphoprotein that accounts for nearly 20% of proteins in cow

20% of proteins in cow milk and cheese. Casein is not coagulated milk and cheese. Casein is not coagulated by heat. It is precipitated byby heat. It is precipitated by acids and by rennet enzymes.

acids and by rennet enzymes.

Casein consists of a fairly high number

Casein consists of a fairly high number of proline peptides, which do not interact. of proline peptides, which do not interact. ThereThere are also no disulfide bridges.

are also no disulfide bridges. As a result, it has relatively little tertiary structure. Because of this,As a result, it has relatively little tertiary structure. Because of this, it cannot denature. It is

it cannot denature. It is relatively hydrophobic, making it poorly soluble in relatively hydrophobic, making it poorly soluble in water. The purifiedwater. The purified protein is water insoluble. While it is also ins

protein is water insoluble. While it is also insoluble in neutral salt solutions, it is readilyoluble in neutral salt solutions, it is readily dispersible in dilute alkalis and in diluted salt solutions such as sodium ox

dispersible in dilute alkalis and in diluted salt solutions such as sodium ox alate and sodiumalate and sodium acetate.

acetate.

Gelatin Gelatin

Gelatin is produced by partial hydrolysis of collagen extracted from the boiled bones, Gelatin is produced by partial hydrolysis of collagen extracted from the boiled bones, connective tissues, organs and some intestines of

connective tissues, organs and some intestines of animals. The natural molecular bonds betweenanimals. The natural molecular bonds between individual collagen strands are broken

individual collagen strands are broken down into a form that rearranges more down into a form that rearranges more easily. Gelatineasily. Gelatin melts to a liquid when

melts to a liquid when heated and solidifies when cooled again. heated and solidifies when cooled again. Gelatin is slightly soluble in coldGelatin is slightly soluble in cold water that disperse more easily in the

water that disperse more easily in the acidic solution. Gelatin is also soluble in most polaracidic solution. Gelatin is also soluble in most polar solvents

solvents

Although gelatin is 98-99%

Although gelatin is 98-99% protein by dry weight, it has less nutritional value than protein by dry weight, it has less nutritional value than manymany other protein sources. Gelatin is unusually

other protein sources. Gelatin is unusually high in the non-essential amino acids glycine andhigh in the non-essential amino acids glycine and proline (i.e., those produced by the h

proline (i.e., those produced by the human body), while lacking certain essential amino acids (i.e.,uman body), while lacking certain essential amino acids (i.e., those not produced by the human body). It contains no tryptophan and is deficient in isoleucine, those not produced by the human body). It contains no tryptophan and is deficient in isoleucine, threonine and methionine.

threonine and methionine.

Peptones (Peptides) Peptones (Peptides)

Peptones (from the Greek

Peptones (from the Greek "to digest") are short polymers of amino acids. They have "to digest") are short polymers of amino acids. They have thethe same chemical structure as proteins, but are

same chemical structure as proteins, but are shorter in length. One convention is that peptideshorter in length. One convention is that peptide chains that are short enough to be made synthetically from the constituent amino acids are called chains that are short enough to be made synthetically from the constituent amino acids are called peptides rather than proteins.

peptides rather than proteins. Peptones are derived

Peptones are derived from animal milk or meat digested by proteolytic digestion. Infrom animal milk or meat digested by proteolytic digestion. In addition to containing small peptides,

addition to containing small peptides, the resulting spray-dried material includes fats, metals,the resulting spray-dried material includes fats, metals, salts, vitamins and many other biological compou

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growing bacteria and fungi. growing bacteria and fungi.

Glutathione Glutathione

Glutathione is a tripeptide thiol

Glutathione is a tripeptide thiol composed of glutamic acid, cysteine, and glycine. composed of glutamic acid, cysteine, and glycine. TheThe sulfhydryl (thiol) group (SH) of cysteine serves as a proton

sulfhydryl (thiol) group (SH) of cysteine serves as a proton donor and is responsible for thedonor and is responsible for the biological activity of glutathione as a scavenger for reactive oxygen species (ROS) such as free biological activity of glutathione as a scavenger for reactive oxygen species (ROS) such as free

radicals and peroxides. Glutathione has been called the "

radicals and peroxides. Glutathione has been called the "master antioxidantmaster antioxidant", moreover, it", moreover, it regulates the actions of lesser antioxidants such as vitamin

regulates the actions of lesser antioxidants such as vitamin C, and vitamin E within the bodC, and vitamin E within the body. Ay. A deficiency of glutathione can

deficiency of glutathione can cause hemolysis and oxidative stress.cause hemolysis and oxidative stress.

Exp. # 10 The Biuret test for peptide bonds

The name of the test comes from the compound biuret, which gives a typically positive The name of the test comes from the compound biuret, which gives a typically positive reaction. Biuret is obtained by heating urea to about 180 C˚. In a kind of chelation reaction a blue reaction. Biuret is obtained by heating urea to about 180 C˚. In a kind of chelation reaction a blue color is formed with Cu

color is formed with Cu2+2+..

In a similar chelation reaction Cu

In a similar chelation reaction Cu2+2+ reacts with protein to form reacts with protein to form Cu Cu2+2+-protein complex.-protein complex.

In this experiment, alkaline copper sulphate reacts with compounds containing two or In this experiment, alkaline copper sulphate reacts with compounds containing two or more peptide bonds to give the violet-colored complex.

more peptide bonds to give the violet-colored complex.The depth of the color obtained is aThe depth of the color obtained is a measure of the number of peptide bonds present in the protein

measure of the number of peptide bonds present in the protein..

The reaction is not absolutely specific for peptide bonds, since the compounds containing The reaction is not absolutely specific for peptide bonds, since the compounds containing two carbonyl groups linked through hydrogen or carbon atom will give a positive result.

two carbonyl groups linked through hydrogen or carbon atom will give a positive result.

1.

1. Copper sulphate (10g/l of CuSOCopper sulphate (10g/l of CuSO44.5H.5H22O). O). 250 250 mlml

2.

2. Sodium Sodium hydroxide hydroxide (10mol/l). (10mol/l). 2 2 ll 3.

3. Proteins (5g/l albumin, casein, gelatin, and Peptone:Proteins (5g/l albumin, casein, gelatin, and Peptone:casein is dissolved a little dilutecasein is dissolved a little dilute NaOH

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4.

4. Glutathione Glutathione (5g/l). (5g/l). 500 500 mlml

Method: Method:

1-1- Add five drops of Add five drops of copper sulphate solution to 2 ml of the copper sulphate solution to 2 ml of the test solution.test solution.

2-2- Add 2 ml of NaOH;Add 2 ml of NaOH;mix thoroughlymix thoroughly..

3-3- Note the produced colors. Note the produced colors.

Exp. # 11 Denaturation by heat and extreme pH.

1.

1. Proteins as in exp.10Proteins as in exp.10 2.

2. Hydrochloric acid (1mol/l)Hydrochloric acid (1mol/l) 3.

3. Sodium hydroxide (1mol/l)Sodium hydroxide (1mol/l) 4.

4. Nitric acid (conc.) Nitric acid (conc.) 5.

5. Boiling water bathBoiling water bath

Method: Method:

1-1- Place 5 ml ofPlace 5 ml of eacheach protein in protein in threethree test tubes. test tubes.

2-2- Add 0.5 ml of HCL, 0.5ml of NaOH, and 0.5ml of water to these 3 tubes of each protein.Add 0.5 ml of HCL, 0.5ml of NaOH, and 0.5ml of water to these 3 tubes of each protein.

3-3- Place the tubes in Place the tubes in a boiling water bath for 10 min then cooa boiling water bath for 10 min then cool to room temperature.l to room temperature.

4-4- Adjust the acid and alkaline tubes to neutrality.Adjust the acid and alkaline tubes to neutrality. *** Comment on your observations.

*** Comment on your observations.

5-5- Slowly pour 2 ml of conc. HNOSlowly pour 2 ml of conc. HNO33 down the sides of test tubes containing down the sides of test tubes containing 2 ml of protein2 ml of protein

solution so as to form two layers. solution so as to form two layers.

6-6- Carefully mix the two liquids.Carefully mix the two liquids. *** Record your observations. *** Record your observations.

Exp. # 12 Precipitation by heavy metals.

At

At pH7 and abovepH7 and above, proteins are usually, proteins are usually negatively chargednegatively charged so that addition of a so that addition of a positively charged metal ion neutralizes this charge and the proteins come out of solution. positively charged metal ion neutralizes this charge and the proteins come out of solution.

Precipitation by heavy metals is therefore, most effective at neutral to slightly alkaline pH values, Precipitation by heavy metals is therefore, most effective at neutral to slightly alkaline pH values, although the

although the solution must not be too alkalinesolution must not be too alkaline otherwise there is a risk of metal hydroxides otherwise there is a risk of metal hydroxides being precipitated.

being precipitated. The formed precipitate is frequently soluble in excess of The formed precipitate is frequently soluble in excess of the heavy metal ionthe heavy metal ion solution since the excess ions confer a

solution since the excess ions confer a stabilizing positive charge on the pstabilizing positive charge on the particles.articles.

1.

1. Proteins as in exp.10 .Proteins as in exp.10 . 2.

2. Heavy metals (0.1 mol/l copper sulphate, lead acetate and mercuric nitrate)Heavy metals (0.1 mol/l copper sulphate, lead acetate and mercuric nitrate)

Method: Method:

1-1- Add a few Add a few drops of the heavy metal solution to 2 ml drops of the heavy metal solution to 2 ml of the protein solution.of the protein solution.

2-2- Record your observations.Record your observations.

*** What happens when excess reagent is added? *** What happens when excess reagent is added?

Exp. # 13 Precipitation by acidic reagent.

These compounds are acids and carry a

These compounds are acids and carry alarge negative chargelarge negative charge which neutralizes a which neutralizes a positively charged protein to form an insoluble salt.

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effective at acidic pH values where

effective at acidic pH values where proteins are positively charged.proteins are positively charged.

5-5- Proteins as in exp.10 .Proteins as in exp.10 .

6-6- Acidic reagents (20% w/v sulphosalicylic acid, Acidic reagents (20% w/v sulphosalicylic acid, saturated picric acid, 10% w/v tannic acid,saturated picric acid, 10% w/v tannic acid, and 20% w/v trichloracetic acid).

and 20% w/v trichloracetic acid).

Method: Method:

1-1- Add five drops of the Add five drops of the acidic reagent to 1-2 ml of the protein acidic reagent to 1-2 ml of the protein solution.solution. *** What is the effect of adding an excess of the reagent?

*** What is the effect of adding an excess of the reagent?

2-2- Slowly add dilute NaOH.Slowly add dilute NaOH.

3-3- Observe the result as the pH Observe the result as the pH decreases.decreases.

III- Colorimetric Methods for Quantitative

Determination of Proteins.

In general, there

In general, there is no completely satisfactory or best way to determine the is no completely satisfactory or best way to determine the concentrationconcentration of protein in a given sample.

of protein in a given sample. The choice of the method depends on:The choice of the method depends on: 1)

1) The nature of the protein under test, The nature of the protein under test,2)2) The nature of the components in the protein sample (i.e. The nature of the components in the protein sample (i.e. contaminants),

contaminants), 3)3) The desired speed, The desired speed,4)4) Accuracy, and Accuracy, and5)5) Sensitivity of assay. Moreover, such Sensitivity of assay. Moreover, such assays needed to be

assays needed to be6)6) Reproducible, Reproducible,7)7) Simple and quick to perform, and Simple and quick to perform, and8)8) Inexpensive. Inexpensive.

There are two general

There are two general classes for the most commonly used colorimetric assay methods toclasses for the most commonly used colorimetric assay methods to determine protein concentration;

determine protein concentration; B)

B) Cu redoxCu redox based chemistry as in 1) based chemistry as in 1)Biuret,Biuret, 2) 2) LowryLowry and 3) and 3)BCABCA assay methods. assay methods. C)

C) Coomassie blueCoomassie blue binding as in binding as inBradfordBradford assay method. assay method.

-- Protein denaturation in these methods is required in order to get maximal color change.Protein denaturation in these methods is required in order to get maximal color change. Denaturation in Biuret, Lowry, and BCA due to

Denaturation in Biuret, Lowry, and BCA due tohigh NaOHhigh NaOH concentration. Denaturation in concentration. Denaturation in Bradford due to

Bradford due to high Hhigh H33POPO44 concentration. concentration.

In addition to the

In addition to the common ways to express concentration bycommon ways to express concentration by MolarityMolarity: mol/l,: mol/l, %%

solution: 1%

solution: 1% solution = (1 g/100 mlsolution = (1 g/100 ml); the above mentioned ass); the above mentioned assay methods usually expressed inay methods usually expressed in

amount/vol

amount/vol:: mg/mlmg/ml and sometimes in mg/dl that express the and sometimes in mg/dl that express the total proteintotal protein content. content. Moreover, Moreover,

proteins can be expressed by another unit;

proteins can be expressed by another unit;Enzyme activityEnzyme activity::μμmol product/(min ml E).mol product/(min ml E). EnzymeEnzyme

activity

activity means the means the amount amount of of enzyme enzyme needed needed to to convert convert 1 1 mol mol substrate substrate to to product product in in 11 min

min = = 1 EU or U (Enzyme Unit).1 EU or U (Enzyme Unit).

General Guidelines for all Colorimetric Methods: General Guidelines for all Colorimetric Methods:

1. Preparation of range of

1. Preparation of range of protein standard protein standard concentrations (usually BSA). concentrations (usually BSA). 2.

2. Addition of fixedAddition of fixed volume of standard to volume of standard to fixedfixed volume of dye solution. volume of dye solution. 3. Incubation for

3. Incubation for specific timespecific time and specific and specific temperaturetemperature.. 4.

4. Measure of absorbanceMeasure of absorbance at single wavelength specific to at single wavelength specific to method (i.e. specific to dye color).method (i.e. specific to dye color). 5.

5. Plotting absorbancePlotting absorbance vs. vs.BSA standardBSA standard concentration ( concentration (mg/ml)mg/ml) to generate the standardto generate the standard curve.

curve.

6. Repeat step 2 - 4 for the unknown protein. 6. Repeat step 2 - 4 for the unknown protein.

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7. Application of unknown absorbance reading to the standard curve to find the working 7. Application of unknown absorbance reading to the standard curve to find the working

unknown concentration. Finally, multiply the working unknown concentration with dilution unknown concentration. Finally, multiply the working unknown concentration with dilution factor to find the original unknown concentration.

factor to find the original unknown concentration.

A-A- Biuret Method (540 nm)

Biuret Method (540 nm)

Introduction:

• The “

• The “GrandfatherGrandfather” of all colorimetric methods.” of all colorimetric methods. •• Principle:Principle: Two reactions are involved: Two reactions are involved:

(1)

(1) Chelation reaction in basic mediumChelation reaction in basic medium::

(2)

(2) Redox reaction:Redox reaction:

Cu

Cu2+2+ + (Tyr, Trp, polar AA’s) + (Tyr, Trp, polar AA’s)redred !! Cu Cu++ + (AA’s) + (AA’s)oxox

• Is the

• Is the basis for the Lowry and BCAbasis for the Lowry and BCA methods. methods.

•• Least sensitiveLeast sensitive (100 – (100 – 1000 fold less sensitive than Lowry, BCA, or Bradford).1000 fold less sensitive than Lowry, BCA, or Bradford). •• Disadvantages:Disadvantages:

1) Requires relatively large amounts of pro

1) Requires relatively large amounts of protein (1-20 mg).tein (1-20 mg). 2) Not suitable in the

2) Not suitable in the presence of ammonium salts.presence of ammonium salts.

•• Lipoid materialsLipoid materials may yield a cloudy reaction mixture which can be cleared by shaking with may yield a cloudy reaction mixture which can be cleared by shaking with 1.5 ml of diethyl or petroleum ether and then centrifuge and read the aqueous phase. Bile 1.5 ml of diethyl or petroleum ether and then centrifuge and read the aqueous phase. Bile pigments absorb light very weakly in the region of 540 – 560 nm.

pigments absorb light very weakly in the region of 540 – 560 nm.

Materials and reagents:

A

A. Biuret reagent:. Biuret reagent: 1.

1. 1.5 g CuSO1.5 g CuSO44.5H.5H22OO

2.

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- Dissolve each compound

- Dissolve each compound separately in sufficient distilled water, add (1) to (2).separately in sufficient distilled water, add (1) to (2). 3.

3. 300 ml 10 % NaOH300 ml 10 % NaOH

-Add solution from (3) with constant swirling to the solution of (1+2) -Add solution from (3) with constant swirling to the solution of (1+2) 4.

4. 1 g KI.1 g KI. 5.

5. Dilute to 1 liter with distilled water. Store in Dilute to 1 liter with distilled water. Store in paraffin-lined bottle.paraffin-lined bottle.

Note:

Note: This reagent can This reagent can be kept indefinitely but must be discarded if, as be kept indefinitely but must be discarded if, as a result ofa result of contamination or faulty preparation, it

contamination or faulty preparation, it show signs of depositing any black or reddishshow signs of depositing any black or reddish precipitate. Biuret reagent may be prepared without KI, however, the addition of 0.1 % precipitate. Biuret reagent may be prepared without KI, however, the addition of 0.1 % KI may prevent excessive reduction and has not detectable effect on the rate, degree, or KI may prevent excessive reduction and has not detectable effect on the rate, degree, or quality of biuret color.

quality of biuret color.

B

B.. Standard protein solutionStandard protein solution. Bovine serum albumin (BSA): 10mg/ml in . Bovine serum albumin (BSA): 10mg/ml in distilled water.distilled water.

C

C.. Unknown protein sampleUnknown protein sample. 4ml, it will be given by . 4ml, it will be given by your assistant.your assistant.

Procedure:

1.

1. Prepare several dilutions ofPrepare several dilutions of standard (BSAstandard (BSA) solution with water to a final volume of) solution with water to a final volume of 1.0 ml1.0 ml

as shown in table (

as shown in table (1) together with a1) together with ablank blank tube containing all reagents except the protein. tube containing all reagents except the protein. 2.

2. Dilute theDilute the unknownunknown protein solution to protein solution to 1 ml1 ml as in table 1. as in table 1. MixMixsolutions thoroughly.solutions thoroughly. 3.

3. AddAdd 4 ml4 ml of Biuret reagent, of Biuret reagent,mixmix the solutions thoroughly.the solutions thoroughly. 4.

4. AA

llow the samples to stand for 30 minutes at room temperature

OR OR

place all the

test tubes, and contents into a water bath at 50˚

CC

for 10 minutes

for 10 minutes (The color is

(The color is

stab

stable for about 60 min

le for about 60 min

at room temperature) at room temperature)

..

5.

5. MeasureMeasure the intensity of the color in each tube at 540 nm against the intensity of the color in each tube at 540 nm againstblank blank which is set at zero which is set at zero absorption.

absorption.

6.

Prepare thePrepare thestandard protein calibration curvestandard protein calibration curve (OD 540 versus mg/ml protein) from these (OD 540 versus mg/ml protein) from these data and

data andreportreport the protein concentration of the unknown sample. A typical Biuret the protein concentration of the unknown sample. A typical Biuret calibration curve is shown in next figure.

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Table 1:

Table 1: Preparation of Blank, Standard and Unknown Preparation of Blank, Standard and Unknown samples for thesamples for the

Biuret

assayassay

..

Tube Tube No. No. Protein Protein solution (ml) solution (ml) H H22O O (ml) (ml) BiuretBiuret reagent (ml) reagent (ml) O.D O.D 540 540 Blank 1 Blank 1 0.0 0.0 1.0 1.0 4.04.0 Blank Blank Blank 2 Blank 2 0.0 0.0 1.0 1.0 4.04.0 Std. 3 Std. 3 0.1 0.1 0.9 0.9 4.04.0 Std. 4 Std. 4 0.2 0.2 0.8 0.8 4.04.0 Std. 5 Std. 5 0.5 0.5 0.5 0.5 4.04.0 Std. 6 Std. 6 0.8 0.8 0.2 0.2 4.04.0 Standard Standard protein protein BSA BSA (10 mg/ml) (10 mg/ml) Std. 7 Std. 7 1.0 1.0 0.0 0.0 4.04.0 Ukn. 8 Ukn. 8 0.1 0.1 0.9 0.9 4.04.0 Ukn. 9 Ukn. 9 0.5 0.5 0.5 0.5 4.04.0 Unknown Unknown protein protein Ukn. 10 Ukn. 10 1.0 1.0 0.0 0.0 4.04.0 Note:

Note: Duplicates can be used to reduce any error that may be generated during preparation. Duplicates can be used to reduce any error that may be generated during preparation.

B-B-Lowry Method: (660 nm)

Lowry Method: (660 nm)

Introduction:

•• PrinciplePrinciple:: The final

The final deep blue colordeep blue color formed by the F formed by the Follin- Lowry reagent is due to:ollin- Lowry reagent is due to:

1)

1) Reaction of proteins with cupric ionReaction of proteins with cupric ions in alkaline solution as in Biuret assay.s in alkaline solution as in Biuret assay.

2)

2) Redox reaction of Biuret (Generation of CuRedox reaction of Biuret (Generation of Cu++) involved in a) involved in a second redox reactionsecond redox reaction which is responsible for the enhanced color change:

which is responsible for the enhanced color change: (i)

(i) Cu Cu2+2+ + (Tyr,Trp, polar AA’s) + (Tyr,Trp, polar AA’s)redred !! Cu Cu++ + (AA’s) + (AA’s)oxox

(ii)

(ii) Cu Cu++ + Folin’s + Folin’soxox!! Cu Cu2+2+ + Folin’s + Folin’sredred (Dark blue) due to(Dark blue) due to

Folin’s reagent contain

Folin’s reagent contain phosphomolybdic phosphotungticphosphomolybdic phosphotungtic acid. acid. Therefore, the intensity of the color dep

Therefore, the intensity of the color depends mainly on the amount oends mainly on the amount offTyr and TrpTyr and Trp amino amino acids present in the protein.

acids present in the protein.

•• More sensitive (About 100 times)More sensitive (About 100 times) than Biuret assay in which detection may reach 5 than Biuret assay in which detection may reach 5µµg/ml.g/ml.

•• Disadvantages:Disadvantages:

- Sensitive to variety of

- Sensitive to variety ofcontaminantscontaminants..

-- Non-linearNon-linear concentration dependence. Therefore, standard curve must be generated each concentration dependence. Therefore, standard curve must be generated each time.

time.

-- Correct mixingCorrect mixing (there are two reagents) and (there are two reagents) and timingtiming are critical. are critical.

• Lowry method is very good for proteins containing chromophores such as hemes and flavins. • Lowry method is very good for proteins containing chromophores such as hemes and flavins.

Materials and reagents:

A.

A. Alkaline copper (Reagent B) reagent Alkaline copper (Reagent B) reagent :: Reagent I : 2 g CuSO

Reagent I : 2 g CuSO44.5H.5H22O.O.

Reagent II: 2 g

Reagent II: 2 g sodium potassium tartrate or sodium or potassium tartrate.sodium potassium tartrate or sodium or potassium tartrate. Reagent A: 2 g Na

Reagent A: 2 g Na22COCO33in 0.1ml NaOH.in 0.1ml NaOH.

Reagent B

Reagent B: Mix in order; 0.1 ml reagent : Mix in order; 0.1 ml reagent I, 0.1ml reagent II, 10ml reagent A. I, 0.1ml reagent II, 10ml reagent A. PreparePrepare

fresh

fresh before use. before use.

B.

B. Follin-Phenol reagentFollin-Phenol reagent(2 N solution):(2 N solution):

Dilute Follin-Phenol reagent 1:1 with distilled water,

Dilute Follin-Phenol reagent 1:1 with distilled water, make just before usemake just before use. Keep in dark.. Keep in dark.

C.

C. Bovine serum albumin (BSA) standards: Bovine serum albumin (BSA) standards:

1-1- 0.02 mg/ml.0.02 mg/ml. 2-2- 0.05 mg/ml.0.05 mg/ml.

3-3-

0.10 mg/ml.0.10 mg/ml.

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4-4- 0.15 mg/ml.0.15 mg/ml.

5-5- 0.20 mg/ml0.20 mg/ml

Standards are prepared in

Standards are prepared in distilled water and can be stored in refrigerator distilled water and can be stored in refrigerator but they shouldbut they should be discarded if bacterial growth is evident or if a change in a standard curve is not

be discarded if bacterial growth is evident or if a change in a standard curve is not attributable to reagent.

attributable to reagent.

D.

D. Unknown protein sample:Unknown protein sample:

Dilute the unknown

Dilute the unknown protein sample with distilled water as in Table 2. If the protein sample with distilled water as in Table 2. If the sample to besample to be analyzed contains ammonium sulfate, dilute with 10% Na

analyzed contains ammonium sulfate, dilute with 10% Na22COCO33. If ammonium sulfate. If ammonium sulfate

concentration is greater than 0.25% after all reagents have been added, readings will be concentration is greater than 0.25% after all reagents have been added, readings will be low.

low.

Procedure:

1-1-

Take 0.5 ml aliquots of BSA standard in small slender test tubes for a Take 0.5 ml aliquots of BSA standard in small slender test tubes for a standardstandard calibration curve and 0.

calibration curve and 0.5 ml distilled water for reagent blank as shown 5 ml distilled water for reagent blank as shown in Table 2.in Table 2.

2-2-

Take 0.5 ml, 0.25 ml and 0.1ml aliquots of unknown sample to test tubes. Make upTake 0.5 ml, 0.25 ml and 0.1ml aliquots of unknown sample to test tubes. Make up volume of 0.25 ml

volume of 0.25 ml aliquots and 0.1 ml aliquots to 0.5 aliquots and 0.1 ml aliquots to 0.5 ml with distilled water (Table 2)ml with distilled water (Table 2)

3-3-

Add 2.5 ml ofAdd 2.5 ml ofreagent B (alkaline copper reagent)reagent B (alkaline copper reagent) into the test tubes containing into the test tubes containing 0.50.5 ml of the sample or standard

ml of the sample or standard solutions.solutions.Mix by vortex and allow standingMix by vortex and allow standing undisturbed for 10 minutes.

undisturbed for 10 minutes.

4-4-

Add 0.25 ml inAdd 0.25 ml inFollin-phenol reagentFollin-phenol reagent rapidly. rapidly. Mix immediatelyMix immediately within 2 seconds by within 2 seconds by vortex before the addition of Follin-phenol reagent to the next tube.

vortex before the addition of Follin-phenol reagent to the next tube.

5-5-

Allow the samples to stand for 3Allow the samples to stand for 30 minutes at room temperature0 minutes at room temperatureOR OR place all the test place all the test tubes, and contents into

tubes, and contents into a water bath at 50˚C for 10 minutes (The a water bath at 50˚C for 10 minutes (The color is stable for atcolor is stable for at least two hours at room temperature).

least two hours at room temperature). CoolCool rapidly in a beaker of tab water. rapidly in a beaker of tab water.

6-6-

Measure the intensity of color in Measure the intensity of color in each tube at 660 nm each tube at 660 nm against Blank which is set atagainst Blank which is set at zero absorbance.

zero absorbance.

7-7-

Plot the standard calibration curve from the corresponding absorbance values of BSAPlot the standard calibration curve from the corresponding absorbance values of BSA standards (OD 660 versus mg

standards (OD 660 versus mg protein).protein).CalculateCalculate protein concentrations of the protein concentrations of the dilutedilute sample solution by using the

sample solution by using the standard curve.standard curve.MultiplyMultiply by the dilution factor for by the dilution factor for protein concentrations of the given sample.

protein concentrations of the given sample.

Table 2:

Table 2: Preparation of Blank, Standard Preparation of Blank, Standard and Unknown samples for and Unknown samples for

Lowry

assay

..

Tube Tube No. No. Protein Protein solution, ml solution, ml H H22O,O, ml ml Reagent Reagent B, ml B, ml Follin Follin Phenol, ml Phenol, ml O.D O.D 660 nm 660 nm Blank 1 Blank 1 0.0 0.0 0.5 0.5 2.5 2.5 0.250.25 Blank Blank Blank 2 Blank 2 0.0 0.0 0.5 0.5 2.5 2.5 0.250.25 0.05 mg/ml BSA std. 3 0.05 mg/ml BSA std. 3 0.5 0.5 - - 2.5 2.5 0.250.25 0.1 mg/ml BSA std. 4 0.1 mg/ml BSA std. 4 0.5 0.5 - - 2.5 2.5 0.250.25 0.15 mg/ml BSA std. 5 0.15 mg/ml BSA std. 5 0.5 0.5 - - 2.5 2.5 0.250.25 0.2 mg/ml BSA std. 6 0.2 mg/ml BSA std. 6 0.5 0.5 - - 2.5 2.5 0.250.25 Ukn. 8 Ukn. 8 0.1 0.1 0.4 0.4 2.5 2.5 0.250.25 Ukn. 9 Ukn. 9 0.25 0.25 0.25 0.25 2.5 2.5 0.250.25 Unknown Unknown protein protein Ukn. 10 Ukn. 10 0.5 0.5 - - 2.5 2.5 0.250.25 Note:

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C- BCA Method (560 nm):

•• BCABCA = = BBiiCCinchoninicinchoninicAAcid.cid. • Same redox

• Same redox reaction of Biuret that generates Cureaction of Biuret that generates Cu++.. • Cu

• Cu++ forms coordination complex with BCA reagent. forms coordination complex with BCA reagent. • Markedly influenced by

• Markedly influenced by protein-to-protein variation.protein-to-protein variation.

D- Bradford Method (595 nm):

•

• Recently this method is the Recently this method is the most commonly used protein colorimetmost commonly used protein colorimetric assay.ric assay. •• Principle:Principle:

-- Coomassie blueCoomassie blue + protein + protein !! Blue complex Blue complex •• Advantages:Advantages:

- Simple one-step method with 5 minute incubation time at Room Temp. - Simple one-step method with 5 minute incubation time at Room Temp. - Sensitive and accurate.

- Sensitive and accurate. •• Disadvantages:Disadvantages:

-- Non-linearNon-linear concentration dependence. concentration dependence.

- Stains glassware and glass cuvettes (Disposable plastics can solve this point). - Stains glassware and glass cuvettes (Disposable plastics can solve this point). - Interfered by detergents.