Protein Definition

7/25/2019 Protein Definition

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Protein Definition

Proteins are large biological molecules consisting of one or more chains of amino acids. Proteins perform a v

array of functions within living organisms, including catalyzing metabolic reactions, replication DNA, responding

stimuli, and transporting molecules from one location to another. Proteins differ from one another primarily in th

sequence of amino acids, which is dictated by the nucleotide sequence of their genes, and which usually resu

in folding of the protein into a specific three-dimensional structure that determines its activity.

A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds betwe

the carboyl and amino groups of ad!acent amino acid residues. "he sequence of amino acids in a protein is defin

by the sequence of a gene, which is encoded in the genetic code. #n general, the genetic code specifies $% standa

amino acids& however, in certain organisms the genetic code can include selenocysteine and in certainarch

pyrrolysine. 'hortly after or even during synthesis, the residues in a protein are often chemically modif

by posttranslational modification, which alters the physical and chemical properties, folding, stability, activity, a

ultimately, the function of the proteins. 'ometimes proteins have non-peptide groups attached, which can

called prosthetic groups or cofactors. Proteins can also wor( together to achieve a particular function, and they oft

associate to form stable protein complees.

)i(e other biological macromolecules such as polysaccharides and nucleic acids, proteins are essential parts

organisms and participate in virtually every process within cells. *any proteare enzymes that catalyze biochemical reactions and are vital to metabolism. Proteins also have structural

mechanical functions, such as actin and myosin in muscle and the proteins in the cytos(eleton, which form a syste

of scaffolding that maintains cell shape. +ther proteins are important in cell signaling, immune responses, c

adhesion, and the cell cycle. Proteins are also necessary in animals diets, since animals cannot synthesize all t

amino acids they need and must obtain essential amino acis from food. "hrough the process of digestion, anima

brea( down ingested protein into free amino acids that are then used in metabolism.

Proteins may be purified from other cellular components using a variety of techniques su

as ultracentrifugation, precipitation, electrophoresis, and chromatography& the advent of genetic engineering h

made possible a number of methods to facilitate purification. *ethods commonly used to study protein structure a

function include immunehistochemistry, site-directed mutagenesis, nuclear magnetic resonance and m

spectrometry

protein occurrence

e have used polyclonal antibodies against fusion proteins produced from cDNA fragments of a meio

chromosome core protein, or/, and a protein present only in the synapsed portions of the cores, 'yn/, to detect t

occurrence and the locations of these proteins in rodent meiotic prophase chromosomes. "he $01 amino acid o

protein is present in early unpaired cores, in the lateral domains of the synaptonemal comple and in tchromosome cores when they separate at diplotene. A novel observation showed the presence of or/ aial to t

metaphase # chromosomes and substantial amounts of or/ in association with pairs of sister centromeres. "

centromere-associated or/ protein becomes dissociated from the centromeres at anaphase ## and it is not found

mitotic metaphase centromeres. "he etended presence of or/ suggests that it may have a role in chromosom

dis!unction by fastening chiasmata at metaphase # and by !oining sister (inetochores, which ensures co-segregation

anaphase #. "wo-colour immunofluorescence of or/ and 'yn/ demonstrates that synapsis between homologo

cores is initiated at few sites but advances rapidly relative to the establishment of new initiation sites. #f the rap

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advance of synapsis deters additional initiation sites between pairs of homologues, it may provide a mechanism f

positive recombination interference. #mmunogold epitope mapping of antibodies to four 'yn/ fusion proteins plac

the amino terminus of 'yn/ towards the centre of the synaptonemal comple while the carboyl terminus eten

well into the lateral domain of the synaptonemal comple. "he 'yn/ fusion proteins have a non-specific DN

binding capacity. #mmunogold labelling of or/ antigens indicates that the lateral domain of the synaptonem

comple is about twice as wide as the apparent width of lateral elements when stained with electron-dense me

ions. 2lectron microscopy of shadow-cast surface-spread 's confirms the greater width of the lateral domain. "

implication of these dimensions is that the proteins that comprise the synaptic domain overlap with the prote

constituents of the lateral domains of the synaptonemal comple more than was apparent from earlier observation"his arrangement suggests that direct interactions might be epected between some of the synaptonemal compl

proteins.

Classification of Proteins and Their Functioons

No lass of

Protein

3unction in the body 2amples

/ 'tructural Provide structura

components.

Collagen is in tendons and

cartilage.

.Keratin is in hair, s(in, wool,and nails.

$ ontractile *ove muscles. Myosin and Actin contract

muscle fibers.

0 "ransport arry essential substances

throughout the body.

Hemoglobin transports oygen.

Lipoproteins transport lipids.

1 'torage 'tore nutrients. Casein stores protein in mil(.

Ferritin stores iron in the spleen

and liver

4 5ormone 6egulate body

metabolism and nervoussystem.

nsulin regulates blood glucose

level.Growth hormone regulates body

growth.

7 2nzyme atalyze biochemical

reactions in the cells.

Sucrase catalyses the hydrolysis

of sucrose. Trypsin catalyses the

hydrolysis of proteins.

8 Protection 6ecognize and destroy

foreign substances.

Immunoglobulins stimulate

immune

responses.

In terms of structure, proteins can also be classified as:

Simple Proteins9 #f they yield only amino acids when they are hydrolyzed.

Conugate! Proteins9 #f they yield amino acids and additional products when hydrolyzed.

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+n a nutritional basis, proteins are classified as:

Complete9 #f they supply all the essential amino acids.

Incomplete9 #f they are deficient in one or more essential amino acids.

Proteins show four structural levels namely, primary, secondary, tertiary and quaternary.

"he linear sequence of amino acids in a polypeptide chain represents the primary structure. "he enzym

ribonuclease and the protein myoglobin function only in their primary structure.

#f the polypeptide chain is coiled into a spiral or heli to have a three-dimensional structure, then it is call

secondary structure. e.g., (eratin of s(in.

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Levels of Structure in Proteins

#f the helical polypeptide molecule is folded on itself assuming a comple but specific structure such spherical or rod li(e, then it is called tertiary structure. e.g., globulins of blood.

'ome proteins have two or more polypeptides, each with primary, secondary and tertiary structures then it

called quaternary structure. e.g., #nsulin and hemoglobin.

protein isolation and purification

"he first step in protein purification involves a cell disruption step. "he method of choicedepends on the type of cell. #n general, animal cells are easier to disrupt than bacteria, yeast orplant cells. "he tabbelow summarizes some of the methods. "his list is by no meanscomplete, as there are as many methods

disruption as there are types of cell.

ell "ype *ethod omment

;acteria 3rench press 'hearing forces disrupt cell

wall as the cells are forced

through a small opening under

very high pressure. Not

practical for large volumes;acteria 'onication

;ead *ill

Disruption of cell walls by

shearing and

avitation

ell wall sheared through

abrasion with

glass beads

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Animal ells ;lender 5omogenization of tissue or

cells will

disrupt cell walls

Plant ells

;acteria

'pores

;lender

are used to

disrupt some bacteria, plant

cells and

bacterial and fungal spores

;acteria )ysis 'olubilization of cell

membranes by

treatment with lysozyme and

2D"A& .$. eigh out the required amount of ammonium sulfate for 4%@ saturation at % =see table>."he initial concentration of ammmonium sulfate is %@.

0. Add /0 of the ammonium sulfate to the 4% m) centrifuge tube, swirl the tube and allow to dissolve. 6epeat withe remaining portions of ammonium sulfate.

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1. Place the centrifuge tube on ice for /4 minutes.

4. ;alance your tube with another studentBs tube. entrifuge for /% minutes at /%,%%% gravity.

6emember the position your tube was placed in the rotor.

7. )abel a /4 m) tube C4%-8%. Decant the supernatant from the 4% m) centrifuge tube into the /4 m) tube mar(4%-8%. *easure the volume of the 4%-8% supernatant in the /4 m) tube and save for step .

8. "ransfer the supernatant from the /4 m) tube labeled 4%-8% into the 4% m) centrifuge tube .

E. eigh out the required amount of ammonium sulfate for 8%@ saturation at % 6emember the initial concentration of ammonium sulfate is 4%@.

F. Add /0 of the ammonium sulfate to the 4% m) centrifuge tube, swirl the tube and allow to dissolve. 6epeat wi

the remaining portions of ammonium sulfate./%. 6epeat steps 1 and 4.

//. Decant the supernatant from the 4% m) centrifuge tube. Discard the supernatant into the waste container.

"eneric outline for protein purification

#n general, protein purification entails essentially five types of steps: /> efficient etraction from biological materi$> separation from non-protein components =nucleic acids and lipids>& 0> precipitation steps, initially to recover t

bul( protein from a crude etract, followed by preliminary resolution into manageable fractions& 1> use of io

echange chromatographysize fractionation or hydrophobic chromatography columns to further separate the targ

protein-containing fraction from the bul( protein& 4> a more refined set of steps including an Caffinity matrienable recovery of the target protein in a highly purified state along with a high yield. A variety of agarose-bas

matrices with immobilized reactive dyes, covalently bound nucleotides, metals and numerous other ligands a

commercially available =supplied by 'igma, Amicon, etc.>.#n order to evaluate the progress of purification, a convenient assay procedureGbased on enzymatic activity or som

other easily monitored property specific to the proteinGshould be available. A spectrophotometric or colorimet

method for enzymatic activity measurement is most convenient and a progressive increase in specific activity =enzymes, activity in units mg protein> is an ecellent indicator of the efficacy of the purification step. 3or protei

lac(ing a readily measurable biological activity, it may be feasible to use an immunochemical procedure such

western blotting or 2)#'A =2nzyme-)in(ed-#mmuno-sorbent Assay>, provided suitable antibodies are available.this case, electrophoretic resolution of the protein population in samples at each stage of purification will

required.

Purification of native proteinshile purification of the native proteins is a challenging eercise, several reliable approaches have stood the test

time. ompared to soluble proteins, membrane-bound.proteins are more difficult to purify. 'olubilization

membrane proteins can be achieved by the use of detergents but removal of the detergent is necessary for subsequeanalytical manipulations. A detailed treatment of the properties of various detergents and their applications

available in reference /. #n the following a representative procedure for purification of soluble Neurospora proteins

outlined./. Preparation of crude etracts: 2fficient etraction of the total protein from the starting material is vital for succe

of any purification procedure. omplete disruption of cells and release of contents from cellular debris is the m

important step in the process. 3or purification of Neurospora proteins in the native state, the first step involves t

etraction of bul( protein fraction from mycelial cells. All steps in the procedure are carried out at 1H to minimiprotein degradation. *ycelial cultures are grown for /E to $% h in a medium conducive to optimal production of t

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target protein, harvested, lyophilized and stored at I8%o. "en to $% g of lyophilized mycelial powder is suspend

in /% volumes of an etraction buffer =4% m* "ris-5l, p5 8.4, %./ m* 2D"A, / m* J-mercaptoethanol

dithiothreitol> and the miture is stirred for 14 min in the cold room. "he presence of 2D"A serves to inhibit prote

action and J-mercaptoethanol =or D""> is necessary for maintenance of a reducing environment. "his slurryhomogenized using a glass homogenizer and the homogenate is centrifuged at /$ %%% g for $% min =to remo

cellular debris> in a refrigerated entrifuge. "he pellet is discarded and the supernatant is used in subsequent step

At this stage it may prove helpful to add a miture of protease inhibitors =omplete coc(tail: 6oche or 'igma> if ttarget protein is suspected to be unstable. KNote: Nucleic acids can be removed from the etract by addition

protamine sulfate to a final concentration of %.$@, while stirring. "he precipitated nucleic acids are removed

centrifugation. 3or most purposes, nucleic acid removal is not necessary& the precipitate may also bind the proteininterestL.

$. Precipitation of proteins: 'everal methods are available for precipitation of proteins utilizing changes in p5 a

temperature, or addition of salts and organic solvents. Ammonium sulfate is the most commonly used precipitant f

salting out of proteins. At saturation =0.F * at %o and 1.%1 * at $%o> it precipitates most proteins and proteproteins in solution from denaturation and bacterial growth. "o the supernatant from step

/, sufficient solid =N51>$'+1 =Mltrapure reagent or 2nzyme grade> is added to achieve 1%@ saturation K'ee 6e

for "able showing relationship between =N51>$'+1 concentration and @ saturationL. "o avoid surface denaturatiothe solution should not be stirred vigorously and =N51>$'+1 should be added gradually, in small amounts, allowi

each successive batch to dissolve completely before addition of the net. "he precipitated protein is removed

centrifugation at /$ %%% g for /% min and to the supernatant more =N51>$'+1 is added to yield E%@ saturatio

"he fraction of precipitated proteins between 1% and E%@ saturation is recovered by centrifugation, resuspendgently in 4 to /% ml of a suitable buffer =e.g. $% m* "ris-5l, p5 8.4, $% m* Nal, /% m* *gl$> and dialyzed

the cold room against several, 1-) changes of the same buffer over a /7-h period to remove residual =N51>$'+"he dialyzed suspension is then centrifuged at /$ %%% g for /% min to remove insoluble particulate matter and t

supernatant is tested for the presence of the target protein =p>.

0. #on-echange chromatography: "he dialyzed fraction is applied to a /7 mm 0% cm column pac(ed with

anion-echanger, D2A2-cellulose ='igma 3ast 3low 3ibrous D2A2 ellulose> or D2A2-'epharose, previouequilibrated against the above-mentioned dialysis buffer. "he column is connected to a Pharmacia P-/ pump and

3rac-/%% fraction collector and is washed with O7%-/%% ml of buffer to remove unbound proteins. "he prote

fraction bound to the matri =including the target protein> is eluted with /4% ml of a linear % to /.84 * Nal or gradient, prepared in the same buffer, generated by a Pharmacia $'+1Gcan be applied to a column containing a hydrophobic matri, such as Phenyl- or +ctyl 'epharose, pr

equilibrated with $% m* "ris-5l =p5 8.4> containing 0%@ saturated =N51>$'+1. "he column is washsuccessively with buffer containing $4@, $%@, /4@, /%@ and 4@ saturated =N51>$'+1 . 3inally the protein

eluted with the original buffer. "he fractions containing p are combined and concentrated using entricon fil

concentrators =/% or 0% (Da cutoff>.4. Affinity chromatography: An eample of affinity chromatography for separation of NAD=P>-binding proteins

the use of agarose or sepharose-bound reactive dyes. "he protein sample is loaded onto a /% mm $% cm colum

pac(ed, for instance, with ibacron ;lue-agarose 0


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concentrated and electrophoresed in 'D'-polyacrylamide gels to determine the state of purity of the protein. +th

types of affinity matrices, such as ADP-, A"P-agarose or on A-'epharose =containing immobilized adenosi

nucleotides or oncanavalin A> can be employed to capture A"PADP-binding proteins or glycoprotei

respectively.KNote: 'election of the appropriate combination of purification steps will depend on the properties of the targ

protein. #f it is a (nown protein, such as an enzyme that has been isolated and characterized from another organism

good starting point will be to try out some of the steps in the published procedure.L"he state of purity of the sample is !udged by 'D'-PA at I$%o

I8%o, along with 0%@ glycerol for maimum stability. All of the above steps can be scaled-up.

6epresentative purification procedure:

#solation of the protein, resulting from fusion of the coding sequence with the metal-binding tag sequence, involv

the following: =/> growth and induction of transformed bacterial cultures& =$> lysis of cells in a suitable buffcontaining a detergent and lysozyme& =0> DNase and 6Nase treatment for removal of the nucleic acid fraction& =

passage of the etract through an affinity resin =NiR-N"A agarose for 5is-tagged proteins> and =4> elution of bouprotein. *any of the

so-called Cone-step purification systems, offered by commercial suppliers, frequently require multiple steps

recovery of a homogenous product.

#xpression and purification of the fusion protein:

"he initial inoculum is prepared in '+;-amp medium =$% g tryptone, 4 g yeast etract, %.4 g Nal, %./E7 g

%.%/* *gl$

per litre containing 4% Sgml ampicillin> by addition of 0-4 Sl of the stoc( culture and incubation

04o for 1-7 h while sha(ing. "wo-ml aliquots of the culture are used to inoculate fresh 4%-ml '+;-amp mediu

which is incubated at 0%o while sha(ing for /$ h. 3or epression, $% to 0% mllitre of this culture is used

inoculate '+;-amp medium, yielding a starting +D7%%nm

of %.%8 to %.//. #ncubation is resumed in the sha(er

0%o for 0.4 to 1.4 h, until +D

7%%of %.04 to %.14 is attained. Addition of / 9 $ m* #P"< during the last hour

growth can, often though not invariably, enhance the yield of the fusion protein. "he culture is harvested

centrifugation and the pellet frozen at I$%o, thawed on ice, resuspended in cold 2traction ;uffer =4% mphosphate buffer, 0%% m* Nal, p5 E.%, containing $% m* imidazole and /% m* J-mercaptoethanol> and the ce

are homogenized using a glass homogenizer. A detergent such as "riton -/%% is added to a final concentration%.%/@ followed by freshly prepared lysozyme =/%% Sgml> and the miture sha(en at room temperature for $% min

Net, DNase =4 Sgml> and 6Nase =7.$4 Sgml> are added and sha(ing continued for an additional 0% m

and the crude cell etract is centrifuged at /$ %%% g for $% min. "he resulting supernatant is clarified by r

centrifugation, mied with / to /.4 ml of the affinity resin =NiR

-N"A agarose& Qiagen>, pre-equilibrated with $% mimidazole in phosphate buffer =4% m* Na5

$P+1, 0%% m* Nal, p5 E.%>. "he miture is sha(en for F% min at 1

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to allow binding of the 5is-tagged protein. "he resin is poured into a column and washed in two steps: first with t

above phosphate buffer containing $% m* imidazole, followed by two washes with 1% m* imidazole to remove t

non-specifically bound protein fraction.

2lution is conducted in a batchwise manner with $ ml of /4% m*, 0 ml of $%% m* and 0 ml of $4% m* imidazo

Alternatively, a linear gradient of /%% m* to 1%% m* imidazole can be employed if larger volumes of the affin

resin and the supernatant are to be used. "he eluted fractions are monitored for the presence of the fusion protein 'D'-PA


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Protein sequencing is a technique to determine the amino acid sequence of a protein, as well as which conformati

the protein adopts and the etent to which it is compleed with any non-peptide molecules. Discovering t

structures and functions of proteins in living organisms is an important tool for understanding cellular processes, a

allows drugs that target specific metabolic pathways to be invented more easily.

"he two ma!or direct methods of protein sequencing are mass spectrometry and the 2dman degradation reaction. #t

also possible to generate an amino acid sequence from the DNA or m6NA sequence encoding the protein, if this

(nown. 5owever, there are a number of other reactions which can be used to gain more limited information abo

protein sequences and can be used as preliminaries to the aforementioned methods of sequencing or to overcom

specific inadequacies within them.

Determinin' amino acid composition

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#t is often desirable to (now the unordered amino acid composition of a protein prior to attempting to find t

ordered sequence, as this (nowledge can be used to facilitate the discovery of errors in the sequencing process or

distinguish between ambiguous results. nowledge of the frequency of certain amino acids may also be used

choose which protease to use for digestion of the protein. A generalized method often referred to as amino ac

analysisfor determining amino acid frequency is as follows:

/. 5ydrolyse a (nown quantity of protein into its constituent amino acids.

$. 'eparate the amino acids in some way.

#-terminal amino acid analysis

'angers method of peptide end-group analysis: A derivatization of#-terminal end with 'ange

reagent =DN3;>, ; total acid hydrolysis of the dinitrophenyl peptide

Determining which amino acid forms the#-terminus of a peptide chain is useful for two reasons: to aid the orderi

of individual peptide fragments sequences into a whole chain, and because the first round of 2dman degradation

often contaminated by impurities and therefore does not give an accurate determination of the#-terminal am

acid. A generalised method for#-terminal amino acid analysis follows:

/. 6eact the peptide with a reagent which will selectively label the terminal amino acid.

$. 5ydrolyse the protein.

0. Determine the amino acid by chromatography and comparison with standards.

"here are many different reagents which can be used to label terminal amino acids. "hey all react with amine grou

and will therefore also bind to amine groups in the side chains of amino acids such as lysine - for this reason it

necessary to be careful in interpreting chromatograms to ensure that the right spot is chosen. "wo of the mo

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common reagents are 'angers reagent =/-fluoro-$,1-dinitrobenzene> and dansyl derivatives such as dan

chloride. Phenylisothiocyanate, the reagent for the 2dman degradation, can also be used. "he same questions app

here as in the determination of amino acid composition, with the eception that no stain is needed, as the reagen

produce coloured derivatives and only qualitative analysis is required, so the amino acid does not have to be elu

from the chromatography column, !ust compared with a standard. Another consideration to ta(e into account is th

since any amine groups will have reacted with the labelling reagent, ion echange chromatography cannot be use

and thin layer chromatography or high pressure liquid chromatography should be used instead.

C(terminal amino acid anal%sis

"he number of methods available for -terminal amino acid analysis is much smaller than the number of availab

methods of N-terminal analysis. "he most common method is to addcarboypeptidases to a solution of the prote

ta(e samples at regular intervals, and determine the terminal amino acid by analysing a plot of amino a

concentrations against time.

Predictin' protein se)uence from D*+-*+ se)uences

#n organisms that do not have introns =e.g. pro(aryotes> the amino acid sequence of a protein can also be determin

indirectly from the m6NA or the DNA that codes for the protein. #f the sequence of the gene is already (nown, th

this is all very easy. 5owever, it is rare that the DNA sequence of a newly isolated protein will be (nown, and so

this method is to be used, it has to be found in some way. +ne way that this can be done is to sequence a shosection, perhaps /4 amino acids long, of the protein by one of the above methods, and then use this sequence

generate a complementary mar(er for the proteins 6NA. "his can then be used to isolate the m6NA coding for t

protein, which can then be replicated in a polymerase chain reaction to yield a significant amount of DNA, wh

can then be sequenced relatively easily. "he amino acid sequence of the protein can then be deduced from th

5owever, it is necessary to ta(e into account the possibility of amino acids being removed after the m6NA h

been translated.

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-eactions

As amino acids have both a primary amine group and a primary carboyl group, these chemicals can undergo mo

of the reactions associated with these functional groups. "hese includenucleophilic addition, amide bond formati

and imine formation for the amine group and esterification, amide bond formation and decarboylation for

carboylic acid group."he combination of these functional groups allow amino acids to be effective polydentaligands for metal-amino acid chelates "he multiple side-chains of amino acids can also undergo chemi

reactions."he types of these reactions are determined by the groups on these side-chains and are, therefore, differe

between the various types of amino acid.

"he 'trec(er amino acid synthesis

Chemical s%nthesis

'everal methods eist to synthesize amino acids. +ne of the oldest methods begins with the bromination at the

carbon of a carboylic acid. Nucleophilic substitution with ammonia then converts the al(yl bromide to the ami

acid #n alternative fashion, the 'trec(er amino acid synthesis involves the treatment of an aldehyde with potassiu

cyanide and ammonia, this produces an U-amino nitrile as an intermediate. 5ydrolysis of the nitrile in acid th

yields a U-amino acid.Msing ammonia or ammonium salts in this reaction gives unsubstituted amino acids, wh

substituting primary and secondary amines will yield substituted amino acids.)i(ewise, using (etones, instead

aldehydes, gives U,U-disubstituted amino acids."he classical synthesis gives racemic mitures of U-amino acids

products, but several alternative procedures using asymmetric auiliaries or asymmetric catalysts have be

developed.

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At the current time, the most-adopted method is an automated synthesis on a solid support =e.g., polystyrene bead

using protecting groups =e.g., 3moc and t-;oc> and activating groups =e.g.,D and D#>.

Peptide bond formation

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The condensation of t.o amino acids to form a dipeptidethrou'h apeptide bond

As both the amine and carboylic acid groups of amino acids can react to form amide bonds, one amino ac

molecule can react with another and become !oined through an amide lin(age. "his polymerization of amino acids

what creates proteins. "his condensation reaction yields the newly formed peptide bond and a molecule of water.

cells, this reaction does not occur directly& instead the amino acid is first activated by attachment to a trans

6NA molecule through anesterbond. "his aminoacyl-t6NA is produced in an A"P dependent reaction carried out

an aminoacyl t6NA synthetase."his aminoacyl-t6NA is then a substrate for the ribosome, which catalyzes the atta

of the amino group of the elongating protein chain on the ester bond.As a result of this mechanism, all proteins maby ribosomes are synthesized starting at their N-terminus and moving towards their -terminus.

5owever, not all peptide bonds are formed in this way. #n a few cases, peptides are synthesized by specific enzyme

3or eample, the tripeptide glutathione is an essential part of the defenses of cells against oidative stress. "h

peptide is synthesized in two steps from free amino acids.#n the first step gamma-glutamylcyste

synthetase condensescysteine and glutamic acid through a peptide bond formed between the side-chain carboyl

the glutamate =the gamma carbon of this side-chain> and the amino group of the cysteine. "his dipeptide is th

condensed with glycine byglutathione synthetase to form glutathione.

#n chemistry, peptides are synthesized by a variety of reactions. +ne of the most-used in solid-phase pepti

synthesis uses the aromatic oime derivatives of amino acids as activated units. "hese are added in sequence onto t

growing peptide chain, which is attached to a solid resin support."he ability to easily synthesize vast numbers

different peptides by varying the types and order of amino acids =using combinatorial chemistry> has made pepti

synthesis particularly important in creating libraries of peptides for use in drug discovery through high-throughp

screening.

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http://en.wikipedia.org/wiki/Esterhttp://en.wikipedia.org/wiki/Ester


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Colour reaction of proteins

Terms Definitions

;iuret "est

=procedure>

Add 4 drops of /@ u'+1 to 1 ml of 4@ Na+5. Divide into two

portions. Add $ml of distilled water to one and $ml of protein solution tothe other.

;iuret "est

=principle>

#n al(aline medium copper from u'+1 will form a coordination

comple with the peptide bond. A minimum of 0 peptides are required to

answer this test. ;iuret test is not performed on urine because of presence

of peptide li(e lin(age.

Ninhydrin "est=procedure> ;oil /ml of protein solution with 4 drops of ninhydrin reagent.

Ninhydrin "est

=principle>

"his test is given by the free amino acids, small peptides and protein will

react to give purple color. Ninhydrin reacts with amino acids to form

hyrindantin and then it further forms 6uheumans purple by reacting with

amonia and another ninhydrin. #mino acids give yellow color owing to

absence of alpha amino acids.

anthoproteic

"est =procedure>

"o /ml of protein solution add /ml of concentrated 5N+0. ;oil for 0%

seconds. oo under tap water and add $-0 ml of 1%@ Na+5.

anthoproteic

"est =principle>

"his test is answered by aromatic amino acids li(e tyrosine and

tryptophan. Phenyl alanine gives a wea( positive reaction. hen a

preotein solution is heated with concentrated nitric acid, the benzene ring

under goes nitration to form yellow nitro derivatives. hen treated with

Na+5 the sodium salt formed is tense orange in color.

*illons "est

=procedure>

Add $-0 drops of mercuric sulphate in /%@ 5$'+1 to /ml of protein

solution. ;oil gently of flame for 0% seconds. Add $-0 drops of /@

sodium nitrate solution.

*illons "est 'pecific to phenolic group of thyrosine. "he hydroy benzene group of

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=principle> thyrosine reacts with *illons reagent to form red colored comple.

Aldehyde

6eaction

=procedure>

Add / drop of /%@ mercuric sulphate in /%@5$'+1 to / ml of protein

solution and / drop of formaline. *i well. Add $ ml of concentrated

5$'+1 along sides of test tube.

Aldehyde

6eaction

=principle>

"he test indicates presence of indole group containing amino acid. "he

oidizing agents are 5g'+1 and 5$'+1 which oidize indole nucleus of

tryptophan. "his oidized indole ring reacts with formaldehyde to give the

violet colored ring at the !unction of two liquids.

'a(aguchis "est

=procedure>

"o 0ml of protein solution and add $ drops of *olischs reagent. "hen add

7 drops of al(aline hypobromite.

'a(aguchis "est

=principle>

"est is answered by arganine. "he guanidine group reacts with alpha

napthol and sodium hypobromite to form deep red color.

Al(ali )abile

6eaction

=procedure>

"o 0ml of protein solution in a boiling test tube. Add equal volume of

1%@ Na+5. ;oil on direct flame for at least two minutes. "hen add 0-1

drops of lead acetate.

Al(ali )abile

6eaction

=principle>

hen a protein solution is boiled with an al(ali, the sulfur of cysteine and

cystine splits to form sodium sulfide. "his forms blac( lead sulfate on

acting with lead acetate.

"his test is not answered by methionine because the sulphur is involved in

a thioether lin(age. "his is not split by boiling with strong al(ali.

Paulys "est

=procedure>

"o $ml of protein solution, add diazotized sulphanilic acid followed by

/ml of $%@ Na$+0.

Paulys "est

=principle>

#midazole group reacts with diazotized aulphanilic acid to form highly

colored azocompouds. #n al(aline medium this is red in color. Diazotized

sulphanilic acid is also called Van Den ;ergs reaction.

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Colour reaction of amino acid

ommon amino acids have the general structure . "hey contain in common a central alpha carbon to which

carboylic acid group =-++5> and amino group =-N5$> and a hydrogen atom are covalently bonded. #n addit

the alpha carbon atom is bound to a specific chemical group, designated 6 and called the side chain, that uniqu

defines each of the $% common amino acids. #n solution at about p5 8,% the amino group is protonated and is in

ammonium ion form =-N50R

>, carboylic group is in its unprotonated or carboylate ion form = negatively charg-++->. +ptical activity. "he alpha carbon atoms of the amino acids, ecept glycine, are each lin(ed to four differ

chemical groups. "his is the characteristic of an asymmetric carbon atom =chiral>. "his amino acids eist

stereoisomeric pairs called enantiomers = D and ) >.5owever only ) isomers are found in proteins. Amphote

properties. Amino acids are amphoteric molecule =dipolar ions>, that is they have both basic and acidic groups.

/. *onoamino-monocaboylic amino acids eist in aequeous solution as dipolar molecules, which means thhave both positive and negative charges and the overall molecules is electrically neutral.

$. At low p5, the carboyl group accepts a proton and become uncharged, so that the overall charge on t

molecule is positive.

0. At high p5, the amino group loses its proton and becomes uncharged =neutral> , thus the overall charge on

molecule is negative.p5 at which an amino acid is electrically neutral is (nown as the isoelectric point for tmolecule and the symbol is p#.

#n protein! almost of the carboyl and amino groups are combined on peptide lin(age and are not available

chemical reactions. "hus it is nature of the side chains that ultimately dictates the role an amino acid plays

protein. #t is useful to classify amino acids according to the properties of their side chain 9 that is , whether

nonpolar or polar uncharged, acidic or basic.

"he chemical reactions of the amino acids are relatively numerous because of the different reactive groups t

are present in the same molecule. Aliphatic monoamino 9 monocarboylic acids give all reactons epected f

carboyl and amino groups. "he other amino acids give these reactions and in addition those reactio

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characteristic of additional groups that may be present. *any of these reactions are also given by protein becau

they contain these amino acids.

-eaction .ith *inh%drin/

Amino acids react with ninhydrin = tri(etohydrindene hydrate> to yield carbon dioide, ammonia and aldehy

containing one less carbon than the amino acid.

"he reaction also yields a blue or purple color , which is useful for the colorimetric quantitative estimation

amino acids . Proline, an imino acid, also react with ninhydrin, but gives a yellow color.

"he reaction of ninhydrin with alpha- amino acids is as follows:

0illon reaction/

A red color is obtained when phenole compounds are heated with 5g=N+0>$

#n nitric acid containing a trace of nitrous acid. Protein containing "yrosine give this reaction:

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1anthoprotein reaction/

Aromatic amino acids =Phenylaalanine, "yrosine, "ryptophan > reacts with nitric acid on heating to give a yello

and in al(aline solution orange compound.

Sa2a'uchi reaction/


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+dam2ie.ic3( $op2ins reaction/

ith glio(salic acid in sulfuric acid a red 9violet color is obtained with "ryptophan in proteins.

ystine and its reduction product, the sulfhydryl containing ysteine after heating in al(aline solution give th

brown precipitate = Pb'> with reagent which contain Pb$R.

Amino acids can be lin(ed together in amide bonds through the carboyl group of one and the amino group

another to form a substituted amide bond which is termed a peptide bond or lin(age.

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The biuret assa%/

hen substances containing two or more peptide bonds react with al(aline copper sulfate a violet comple

formed. "he colored product is the result of coordination of peptide nitrogen atoms with u$R."he amount

product formal depends on the concentration of peptides.

Importance of isoelectric point in proteins:

"he isoelectric point of a protein is the p5 where the +V26A)) charge on the protein is neutral. #n isoelectr

focusing gel electrophoresis allows for the separation of protein purely on the basis of their charge characteristics.

polyampholyte will migrate in an electric field li(e other proteins , if the protein have a net electric charge. #f the g

with a stable p5 gradient covering a wide p5 range, each polyampholyte molecule migrates to the position of

isoelectric point and accumulates there. ?ou can establish such a gradient by using a miture of low9molecu

ampholytes as the gel buffer. "his method produces distinct bands of accumulated polyampholyte and can reso

molecules with very small differences in isoelectric points.

+mino acids as amphol%tes :

Ampholytes are molecules containing both acidic and basic groups.

All of the common amino acids found in proteins =3igure 4.0> are ampholytes because they contain a carboyl gro

=-++5> that acts as an acid and an amino group =-N5$> that acts as a base.

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As free amino acids, each amino acid has at least two pa values =some have more because they have addition

acidic or basic groups>.

"he titration of an ampholyte generates a more comple plot of p5 versus moles of acid =or base> added than a

obtained for a simple buffer with only a single ionizing species because the ionization of each acidic and basic gro

of the ampholyte is represented by a step in the titration curve. #n 3igure $./E, for eample, there are two steps in t

titration curve of the ampholyte glycine, whereas in 3igure $./8the titration curves of N51Rand 5++5 have onone step each. 3igure $./Fshows the fraction of each molecular species of glycine present in the solution as

function of p5.

"he presence of both acidic and basic groups in a single molecule means the molecule may eist in several differe

charged states. 3or eample, glycine can have a charge of R/, %, or -/, depending on the p5 of the solution in whi

it is dissolved. "he state at a net charge of zero arises when the basic amine group is charged R/ and the acid

carboyl group is charged -/.

*olecules containing a miture of charges that result in the molecule having an overall charge of % are called

Wwitterions. "he zwitterion form of the amino acid, glycine,is as follows:

The use of carrier ampholyte-free IEF !"F-IEF# $ith IT% mo&ili'atio( a() co()ucti*ity )etectio( i( I

mo)e for preco(ce(tratio( a() a(alysis of ami(o aci)s is )emo(strate)+ The a(alytical proce)u

co(sists of three su&se,ue(t steps+ I( the rst step. ami(o aci)s are co(ti(uously )ose) from a

i((ite *olume reser*oir &y electromi/ratio( to the colum(. $here a sharp. statio(ary (eutrali'ati

reactio( &ou()ary # is create) i( &et$ee( aci)ic a() &asic primary electrolyte+ ere. ami

aci)s are selecti*ely focuse) trappe)#. if their pIfalls to the p )iere(ce o( &oth si)es of the

p /ap#+ "mi(o aci)s create sharp recta(/ular 'o(es. arra(/e) accor)i(/ to their p I*alues+ I( t

seco() step. focuse) 'o(es are mo&ili'e)+ "fter accumulatio( of the )etecta&le amou(t of ami

aci)s. )osi(/ electrolyte i( the i((ite *olume reser*oir is cha(/e) for the mo&ili'i(/ electrolyte+ T

mi/ratio( mo)e is cha(/e) from !"F-IEF to IT% a() su&sta(ces start to mi/rate to$ar) the a(alytic

capillary+ I( the thir) step. a(alytes are tra(sferre) i(to the a(alytical colum( e,uippe) $ith

co()ucti*ity )etector a() are )etecte) i( the (e$ lea)i(/ electrolyte i( a( IT% mi/ratio( mo)e+ T

prese(te) !"F-IEF-IT%-IT% $ith time-)epe()e(t accumulatio( of the lar/e-*olume sample e(a&les

achie*e i( a reaso(a&le time a 100 times lo$er c- here i( or)ers of (mol#. tha( ca( &e reach

&y co(*e(tio(al hyphe(ate) IT%-IT%+

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Levels of protein structure

"here are four distinct levels of protein structure.

Protein structure, from primary to quaternary structure.

Primar% structure

"he primary structure refers to amino acid linear sequence of the polypeptide chain. "he primary structure is h

together by covalent or peptide bonds, which are made during the process of protein biosynthesis or translation. "

two ends of the polypeptide chain are referred to as the carboyl terminus =-terminus> and the amino terminus =

terminus> based on the nature of the free group on each etremity. ounting of residues always starts at the N

terminal end =N5$-group>, which is the end where the amino group is not involved in a peptide bond. "he prima

structure of a protein is determined by the gene corresponding to the protein. A specific sequenof nucleotides in DNA is transcribed into m6NA, which is read by the ribosome in a process called translation. "

sequence of a protein is unique to that protein, and defines the structure and function of the protein. "he sequence

a protein can be determined by methods such as 2dman degradation or tandem mass spectrometry. +ften however

is read directly from the sequence of the gene using the genetic code. e (now that there are over /%,%%% proteins

our body which are composed of different arrangements of $% types of amino acid residues =it is stric

recommended to use the word Xamino acid residuesX as when peptide bond is formed a water molecule is lost s

protein is made up of amino acid residues>. Post-translational modifications such as disulfide formati

phosphorylations and glycosylations are usually also considered a part of the primary structure, and cannot be re

from the gene.

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+mino acid residues

2ach U-amino acid consists of a bac(bone part that is present in all the amino acid types, and a side chain that

unique to each type of residue. An eception from this rule is proline. ;ecause the carbon atom is bound to fo

different groups it is chiral, however only one of the isomers occur in biological proteins.


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An alpha-heli with hydrogen bonds =yellow dots>

'econdary structure refers to highly regular local sub-structures. "wo main types of secondary structure, the alp

heli and the beta strand or beta sheets, were suggested in /F4/ by )inus Pauling and cowor(ers "hese seconda

structures are defined by patterns of hydrogen bonds between the main-chain peptide groups. "hey have a regu

geometry, being constrained to specific values of the dihedral angles Y and Z on the 6amachandran plot. ;oth t

alpha heli and the beta-sheet represent a way of saturating all the hydrogen bond donors and acceptors in tpeptide bac(bone. 'ome parts of the protein are ordered but do not form any regular structures. "hey should not

confused with random coil, an unfolded polypeptide chain lac(ing any fied three-dimensional structure. 'eve

sequential secondary structures may form a Xsupersecondary unitX.

Tertiar% structure

"ertiary structure refers to three-dimensional structure of a single protein molecule. "he alpha-helices and be

sheets are folded into a compact globule. "he folding is driven by the non$speci%ichydrophobic interactions =

burial of hydrophobic residues from water>, but the structure is stable only when the parts of a protein domain

loc(ed into place byspeci%ictertiary interactions, such as salt bridges, hydrogen bonds, and the tight pac(ing of si

chains and disulfide bonds. "he disulfide bonds are etremely rare in cytosolic proteins, since the cytosol is genera

a reducing environment.

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&uaternar% structure

Quaternary structure is the three-dimensional structure of a multi-subunit protein and how the subunits fit togeth

#n this contet, the quaternary structure is stabilized by the same non-covalent interactions and disulfide bonds as t

tertiary structure. omplees of two or more polypeptides =i.e. multiple subunits> are called multimers. 'pecifica

it would be called a dimer if it contains two subunits, a trimer if it contains three subunits, and a tetramer if

contains four subunits. "he subunits are frequently related to one another by symmetry operations, such as a $-fo

ais in a dimer. *ultimers made up of identical subunits are referred to with a prefi of Xhomo-X =e.g

homotetramer> and those made up of different subunits are referred to with a prefi of Xhetero-X =e.g

heterotetramer, such as the two alpha and two beta chains of hemoglobin>.

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Protein Definition

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