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BIOCHEMISTRY

PROTEINS
Peptides and proteins, while similar in many regards, have several key differences that are important to nderstand. To tully
apprecate the difterences between proteins and peptides, it is important to understand amino acids, the building bBocksof both, and
how allthree (amino acids, peptides, and proteins) relate to one another.

amino acids peptide protein

1.AMINO ACIDS
Proteins are poBynmers of amino acids, with each amino acid residue joined to its neighbor by a
peptide bond. specific type of covalent bond, a
1.1COMMON AMINO ACIDS
They include the 22proteinogenie (protein-building) amino acids, which combine into peptide chains
building-biocks of avast array of proteins. ("polypeptides") to form the
Twenty of the proteinogenic amino acids are encoded directly by triplet codons in thegenetic code and are
amino acids. The other two known as "standard"
coded directly by DNA), and (non-standard)
are selenocysteine (present in nmauy noneukaryotes as well as most
pyrrolysine (found only in some arehea and one eukaryotes, but not
bacterium).
1.2 STRUCTURAL FEATURES
selenocrsteini Aa
All amino acids are a-amino acids with

a carboxyl (-C0OH) group,

an amino (-NH,) group and 22.
Pyvvolgin
-H along with a side chain (R group) specific to each amino acid bonded to the a carbon.
The name "amino acid" is derived froni the amino group and
carboxyl-acid-group in their basie structure.
At neutral pH 7.0, the carboxyl group exists as -C00 and the aninagroup as-NH^.
The carboxyl group isproton donor’ weak acid and amino group is proton acceptor ’ weak base.
The aminogroup is primaryamine (arise when one of three hydrogenatoms in ammonia is
involed in amid bond formation. replaced by an alkyl or aromatic group)

The carboxyl group is involed involved in ester, ether and amide bonds formation,
L.3ISOMERISM
In protein molecules, all the amino acid residues are
-stereoisomers. The alpha carbon is a chial carbon atom, with the
exceptionof glycine. Therefore, all apha aminoacids but glycine can exist in either of two enantiomers, called L or Damino acids
which are mirror images of each other.

Aithough, D-Amino acid residues known to found only in bacterial cell walls and certain peptide antibioties.
Cells are able to speeifically synthesize the Lisomers of amino acids
because the active sites of enzyes are asymnetric, ea using the
reactions they catalyze to be stereospecific.
The Land D convention for amino acid eonfiguration refers not ta the
optical activity of the amino acid but rather hased on tbe
absolute configuration of the three-carbon sugar glyceraldehyde, aconvention proposed by Emil Fischer in 801.
Amina Acid 3 Ska chn
Letter ette

Nonpolar, Aliphatic Rgroups

Gheine Gk IhOnpolar neutral
Ala 5.97
Aianune onpolar neutrat 6.01
Proline Pro P ROnpolar hetral 648
Valine Val honpoBar neukral
597

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Leucine Leu nonpolar neutral 5.98
Isoleucine nonpolar neutral 6.02
Methionine
Aromatic Rgroups
Met M nonpolar neutral 5.74

Phenylalanine Phe nonpolar neutral 5.48 257, 2o6, 188

Tyrosine Tyr polar neutral 5.66 274, 222, 193
Tryptophan Trp W nonpolar neutral 5.89 280, 219
Polar, uncharged Rgroups
Serine Ser S polar neutral 5.68
Threonine Thr T polar neutral 5.87
Cysteine Cys C nonpolar neutral 5.07 250
Asparagine Asn polar neutral 5.41
Glutamine Gln Q polar neutral 5.65
Positively charged Rgroups
Lysine Lys K polar positive 9.74
Histidine His H polar Positive (10%) Neutral 7-59 211
(90%)
Arginine Arg R polar positive 10.76
Negatively charged Rgroups
Glutamic acid Glu polar negative 3.22
Aspartic acid Asp D polar negative 2.77

Palar, wneharged R groups

(eat
hph
Nonpalar, aliphatic Rgroups H,N-C-H HN-C-H
(O0 coo COO

H,N-C-H HN-¢-H HN--H mos

HOH H-C-OH,
CH,
H H, H Serise Thrsenin Cystrine
H,ccu, CH, CH,
Giyeine Alanine Proline Valine
Aromatíe Hgroupa
A
coo Coo coo co0 fooiel
H,G-C-H H,N-CH H,N H H,N-C-H HN-C-H
H-C-CH, CH BCH CH
H CH HCH
N.
CH, CH, Ch

sojeueine Mothionine Phenytalasoe Tyrose Poine

Trypohan
Asparagine Gltamie
Positively charged Rgroups
COO CO0
HN CH H,N- H
CHE CH, Negatively charged R groups
CH -NH ÇO0 COO

CIE CH H,N-C-H H,N--C-H

CH NH CH, CH
C-NH COQ CH
Ly
NH,
ArgiineR Hintidine
H oo
Aspartats Glutamate

Prr DEPgafHR
.4 AMINO ACIDS CLASSIFICATION Aad bogayis
Oeuned intofive main classes based on the properties of their R groups, in particular, their polarity, or tendency to interact with
water at biological pH (see table).
a. Nonpolar or Hydrophobic (Aliphatic RGroups) Amino Acids
Ghcine, alanine, valine, leucine, isoleucine, proline and methionine,
Clveine has the small R-H) and no chiral center Absence of small zide chain in glyeine give more conformational flexibilite
presents in turn or bends in proteis moBecule.
aline,leucine are isoleucine are present more in proteins due to to their emal and
tnin and involved hydrophobic interactions hydrophobie, presents inside of
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Iiydrophobicity [increasing order] : Alanine >valine >leucine >isoleucine
Methionine, one of the two sulfur-containing amino acids, has a nonpolar thioether group in its side chain.
Proline has an aliphatie side chain with adistinctive cyclic structure. The amino group of proline is asecondary amine (imino)
group is held in a cyclic ring that decreases the structural flexibility of polypeptide regions. Presence of proline in proteins creates
bend or turns.

b. Aromatie AminoAcids
Phenylalanine, tyrosine and tryptophan.
Tryptophan and tyrosine absorbs ultraviolet light at a wavelength of 28o nm, a property used for the estimation of protein
concentration.

Tyrosine and tryptophan are significantly more polar than phenylalanine, because of the tyrosine hydroxyl group and the nitrogen of
the tryptophan indole ring.
The number aromaticamino acids present in proteins are less due to their large size, they interfere
inprotein folding.

0
eiesih (nn)
c. Polar (uncharged) Amino Acids
Serine, threonine, cysteine, asparagine and glutamine.
The Rgroups are more soluble in water [hydrophilic], because the contain functional groups that form hydrogen bonds with
water. These amino actds present outside of the protein.
The serine and threonine polaritycontributed by their hydroxylgroups [-0H] and of eysteine by its thiol or
sulfhydryl group (-SH)
Two close by -SH groups of cysteines readily oxidized to form a covalently linked
dimeric amino acid called eystine, in which two
cysteine nolecules or residues are joined by a disulfide bond [-s-s-).

The disufide bond is covalent and strongly hydrophobie, a special role in

the structures of many proteins by forning covalent links
hetween parts of a polypeptide nolecule or between tyo different polypeptide chains.
B-Mercaptoethanol is sed to reduce disulide bonds and can act s a biological antioxidant. Glutathione inplieated in the
formation of native disulphide bonds.
AsDaragine and glutamine are the amides of two other amine acids also
which asparagine and ghutamine are easily hydrolyzed by acid or base. tound n protetns, aspartate and glutanate, respectively, to
d. Basie (positively charged) Amino Acids
Histidine, arginine and lysine.
Basic amino acids are polar and positivly charged at neutral pH.
The ysine, which hus a second prinary amino group at the poton on h
aiphatie chain; arinine, which has ositivey
haceed guanidinium group Isecond amino group at pasthon; and bistdine. which las an aromatie innldao
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C NH,
NH

NH

e. Acidie (negatively charged) Amino Acids

Aspartic acid and glutamic acid.
1he two amino acids having R groups with a net negative charge at pH 7.0 are aspartate and glutamate, each of which has a second
carboxyl group at ß- and y-position, respectively.
lonic bonds result from electrostatic attractions between positively and negatively charged side chains of an aspartic acid earboxylate
ion and a lysine ammonium ion helps to maintain a particular folded area of a protein.
a. Nonpolar or Small, hydrophic and most commonly present inside of the protein
Hydrophobic (Aliphatic R
Groups) Amino Acids Large,less commonly present and helps in identification and quantification of the protein
b. AromaticAmino Acids
c. Polar (uncharged) Amino Mostly present outside andinvolved in solubility of the protein.
Acids
d. Basic (positively Gives positive charge for the proteins.
charged) Amino Acids
e. Acidic (negatively charged) Gives negative charge for the proteins.
Amino Acids

1.5 UNCOMMON AMINO ACIDS

These are created by modification of normal amino acids. 4-hydroxyproline and 5-hydroxylysine are found in collagen, a fibrous
protein of connective tissues. 6-N Methyllysine is aconstituent of myosin,a contractile protein of muscle.
-carboxyglutamate, found in the blood clotting protein prothrombin and in certain other proteins that bind Ca2+ as part of their
biological function.
Desmosine, a derivative of four Lys residues, whiclh is found in the fibrous protein, elastin.
Selenocysteine is introduced during protein synthesis rather than created through a postsynthetic modification. It contains
selenium rather than the sulfur of cysteine.
1.6 ACID-BASE PROPERTIES
a. An amino acid has both a basicamine group (-NH) and an acidic carboxylic acid group (-COOH).
NH NH + H0
NH* NH3t
NH3t A.CHCO RCH-COO ACHCO0 + H(eq RCHCOOH
B-CH-COo Addacid

Nofos fst tiss IwE0al ve k

azwitterion

There is an internal transfer of ahydrogen ion from the -COOHgroup to the -NHa group to leave an ion with both a negative charge
anda positive charge. This is called azwitterion (charge Zero).
hIfvou inerease the pH of asolutionof an amino acid by adding NaOH, the His removed from the -NH, group. Now amino acid
exists as anegative ion moves towarts the anode tthe positive electrode) during electrophoresis.
If vou deerease the pH by adding an acid to asoution of an amino acid, the -C0O part of the zwitterion picks up a hvdrogen ion,
becomes positive ion and inove towards the cathode(the negative electrode) during electropboresis
Substances having this dual nature are amphoteric and are often called ampholytes. Asimple monoamino monocarboxylic o
aino acid, such as alanine, is a diprotic acid when fully protonated-it has two groups, the -COOH
group and the-NH group, that
can yield 2 protons:

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H H

Ne NH -NH, NH,
charge +1

e. Amino Acid Titration Curves

Procedure Figure shows the titration curve of the diprotic form of glycine [1M] titrated with NaOH [2M)
NH, NH. NH
CH
CH, CH,
COOH CO0 oo
13
Glyeine
pk,960

pH 3pl 597

pA- 2.34

05

OH (equivalents
The plot has two distinct stages, corresponding to deprotonation of two diferent groups on glycine,
titration curve ofa monoprotic acid, such as acetic acid. resembles the shape of the
Stage 1: At very low pH, the predominant ionic species of glycine is the fully protonated form, *H,N-CH,-C0OH.
Stage 2: At the midpoint in the first stage of the titration, in wbich the -COOH group of glycine loses its
concentrations of the proton-donor (*H,N-CHa-C00H) and proton-acceptor proton, equimolar
(*H3N-CH-CO0) species are present.
Stage 1 2 3 4 5
Stage 3: Here the pH is equal to the pK, of the
NH, NH, NH, NH, NH, NH, NH, titrated. For glycine, the pH at the midpoint isprotonated group being
2.34. thus its -COOH
CH, CH, CH, CH, CH, = CH, CH, group has apKa of 2.34.
CoOH
COOH Coo C00 COO COo COO As the titration proceeds, another
important point is reached at plH
5.97 At this pH glycine is present largely as the dipolar ion HN
Charge + CHa-C00 [zwitter ion]
pK pKcooH pKNH3
Stage 4: The second stage of the
of a proton from the -NH, group of glycine. The pH at the uidpoint of this stage is 9.60, equal to titration corresponds to the
the pKa for the -NH group. removal
Stage 5: The titrationis essentially comnplete at apH of about 12, at which point the predominant form of glycine is HaN-CH-Co0.
Titration Curve of Glycine gives
1.aquantitative measure of the pKa of each of thetwo ionizing groups: 2.34 for the -COOH group (pKeooH) and 9.60 for the -NIL
group (pKNH).
2. Glycine has tuo regions of buffering power.1pH unit on either side of the first pkcooH of 2.34, indicating that
glvcine is a good
buffer near this pH. The other baffering zone is centered around pH 9.60.
3.The relationship between its net electric charge and the pH of the solution (see table).
4. At pH 5.97, glvcine is present predominantly as its dipolar form, fully ionized but with no net electric charge. The
characteristie plH
at which the net electric charge is zero is called the isoelectric point or isoelectricpH, or pl.
5 For glveine, which has no ionizable group in its side chain, the isoelectric point is simply the arithmetic mean of the
two pKa values:
pl= 2 (pKcoon+ pKNy")= V2 (2.34 +9.60) =5.97
6.Glycine bas a net negative charge at anypH above its pl and positive charge at any pH below its pl.
B.Acid-Base Properties of Acid/Basic Amino Acids
Out of 20 anino acids, 15 amino acids with asingle s-amino group, u-carboxyl group have titration curves reserabhgthat of aheine

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emaining 5 amino acids (basic and acidic) hve an ionizable R group (-COOH or NH,) have more complex titration curves, with
they have three pKa values (pKcooss pKNH pkCOOH) Or
orresponding to the three possible ionization steps; thus
pkas").
pl of Acidicamino acid Va (pKooon + pkcoon)
pl of Basic aminoacid (pKxt + p k )
ypical pK, values of these groups are shown in Table. The B-carboxyl group of aspartic acid and the y-carboxyl side chain of glutamic
typical aliphatic carboxyl groups on the other hand.
acid exhibit pA,values intermediate to the o-COO1H on one hand and
acids
pk, Valus of Acidic and Basic amino
Amino acid -C0OH pk,a-NH, pK, Rgroup pk. pl
9.60 3.65 27
Aspartic acid L.88 322
Glutamicacid 2.19 9.67 4.25
8.95 10.53 974
Lysine 2.18
9.04 12.48 10.76
Arginine 2.17 Z59
9.17 6.00
Histidine 1.82

exhibits a pK, that is higher than that of the a-amino

group but similar to that for a
In a simlar fashion, the -amino group of lysine
typical aliphatic amino group.
pH with apKa of
partially protonated with positive charge) at physiological
The imidazole side chain of histidine (which is
approximately 6.0.
values.
The imidazole/imidazolium ring of histidine is aromatic at all pH

Imsdazale many enzyme

as proton donors and acceptors in
Imidazolium
histidine side chains play important roles
With a pKa near neutrality,
reactions.
(-C-(NH)), making arginine
guanidino group of arginine is also protonated to give the guanidinium form
At physiological pH, the
a charged, aliphatic amino acid.
N HNe
HNcNH
HN
lon guanidnium
HN

2. PEPTIDES more amino

the tern peptide" generally refers to a compound made up of two or
amino acids and
a. Peptides are short chains ofclassified as oligopeptides and polypeptides.
acids, peptides can be further numbers of amino acids, generally less than ten.
are made up of relatively small
b. Meaning "few," "oligo" denotes that oligopeptides
theother hand, are cormposed of nore than ten amino acids.
Polypeptides, on bond, to yield a
joined through a substituted amide linkage, termed a peptide of one amino acid
covalently (dehydration) from the a - carboxyl group
e Two atnino acid molecules are rermoval of the elements of water
dipeptide. Such a linkage is formed by
of another.
and thea- amino group
Gyoine
Glycine
C

HO

Pupie bond
conditions, the equilibrium for the
of a condensa tion reaction.
Under standard biochemical
Dantide bond fomation is anexample
amino acids over the dipeptide.
ireattion favars theformation of
 BIOCHEMISTRY
d. This reaction, which is catalyzed at the ribosome by a
biosynthesis. The peptidyl-transferase activity is catalyzedpeptidyitransferase
by the 22S rRNA
and is repeated many times, forms the basis of protein
Iprokaryotes) and28S rRNA [eukaryotes] in the larger
subunit of ribosome.
e. Peptide bond formation can be described as a nucleophilic attack of the
another amino acid. Apart from peptide bond, redox reactions comprise aa-aminosecond
group of one amino acid on carboxyl group of
important class of reactions of amino acid side
chains. The sulfur-containing amino acids cysteine and
methionine are most responsive to oxidation.
d. Three amino acids can be joined by two peptide bonds to form a
tetrapeptides, pentapeptides, and so forth. tripeptide; similarly, amino acids can be linked to form
e. In a peptide, the amino acid residue at the end with a free
residue at the other end, which has a free carboxyl group, is the a-amino group is the amino-terminal (or N-terminal) residue; the
carboxyl-terminal (C-terminal) residue.
f. The peptide bond is acid labile.
Cytocin hormon
B. Biologically Active Peptides
Dipeptide Lcaspartyl-L-phenylal anine methyl ester, the artificial sweetener better known as
Oxytocin (9 residues) - aspartame or NutraSweet
Bradykinin (9)- inhibitsstimulates uterine contractions
inflamnatien.of fissues
Thyrotropin-releasing factor (3), Amanitin (8), Insulin (51) and Glucagon (29).
3. POLYPEPTIDES AND
PROTEINS
a. Scientists commonly differentiate between
proteins and polypeptides based on their size and structure.
b. Regarding size, a polypeptide composed of more
than 50 amino acids is generally classified as a protein.
c. Secondly, polypeptides shorter than about 40-50. amino
to fold into a three-dimensional stable fixed acids in length do not fold into a fixed structure. Proteins,
structure. Proteins tend to have a fixed structure however, are able
for a certain function (i.e. hemoglobin,
protein responsible for transporting oxygen in the blood). Polypeptides shorter than a
do not have enough cooperative interactions to form a 40-50 amino acids, on the other hand, generally
stable native structure.
d. Some proteins consists a single polypeptide chain, but
associated noncovalently. others are multisubunit proteins, have two or more polypeptide chains
Hemoglobin has four polypeptide subunits: two identical a chains and two identical ß chains, all
interactions. So it is a tetramer. four held together by noncovalent
e. Average molecular weight of an amino acid in
the peptide/proein is 110 Daitons. Assuming a protein with
molecular weight of the protein is 110 x 100 = 11000 Daltons. 100 amino acids,
f. Theosmotic pressure is in more wide use than
other coligative techniques [vapor pressure, freezing point and
used to determine molecualr weight proteins in solution. boiling point)
Acid Hydrolysis of Proteins
used for liberate amino acids from protein substrate and
quantitatively recover them in the hydrolysate.
It is perfornied in 6M HCI at i10 °C for 24 hours.
Amino acids are chemically diverse
alanine, leucine, phenylalanine,group
of compounds and only few of them
histidine (aspartic acid, glutamic acid, proline, glycine.
and arginine) can be quantitatively
aminoacids may undergo transformations during hydrolysis. determined during acid hydrolysis. The other
Asparagine and glutaminesuffer deamination reactions during acid hydrolysis
being converted in aspartic acid and glutamicacid.
Serine and threonine are two aminoacids which are partially
destroyed during acid hydrolysis. Cysteine and methionine also suffer
transformation during acid hydrolysis, as cysteine can be destroyed
methionine sulfone. while methionine can be oxidized to methionine
sulfoxide and
Valine andisoleucine contain peptide bonds with very
hydrophobic residues are quite difficult to eleave with acid. thus. when using
acid hydrolysis in 6M HCI for 24 hoursat 1i0 °C,the obtained quantíties are often low.
Tyrosine is an amiuo acid that can undergo halogenation during acid
the 6M HCI hydrolysis using HCIL. To prevent this, phenol is often added to
Lvsine is stable under standard acid hydrolysis conditions, and in
pure proteins and foods thermally untreated can be
deternmined using HCl hydrolysis. readily
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Tryptophan is an amino acid that can be destroyed during acid hydrolysis.

4. EXTRA PONTS ON PROTEINS HILk mF
tvmLT UL Path
sennhad Aa
1, Asparagine, the first amino acid to be discovered.
2. The
3. WATSON does rotamino
9 essential represent
acidsavalid
are:amino acidsequence,
histidine, when compared
isoleucine, leucine,to lysine,
EINSTEIN, CRICK andphenylalanine,
methionine, FARADAY. threonine,
tryptophan, and valine.
4. Tyrosine, arginine and histidine are semiessential amino acids.
5. Valine, leucine, and isoleucine are branched-chain amino acids.
6. Glycine doesnot have a B-carbon. Threonine and isoleucine have handed B-carbons.
7. Tryptophan has highest fluorescence quantum yield () in aqueous solution.
8. Proline has a higher propensity for cis peptide bond formation.
9. Threonine and isoleucine, containtwo asymmetric carbòn atoms and thus, exist as four stereoisomers forming two enantiomer
and fouY diastereomer pairs, respectively. on Hydropathy index values).
10. Ala, Leu, Val and Ile residues are arranged in the ascending order of hydrophobicity [based
in the interior of protein structure.
11. Asparticacid andglutamic acid of a membrane protein are most likely to be buried
12. Val, Ile, Phe and Met find inside of a typical globular protein at pH 7.
13. After site-directed mutagenesis, Lys ’ Ala mutant has the largest difference in the number of atoms compared to the wild type.
14. Ser... Léu pair of amino acids represènits the yeakest interaction between their side chains in proteins.
15. Lós- Glu side chains of amino acids interact with each other form asalt bridge. detectioy
16. Disulphide bonds in proteins could be formed by cysteines oxidized glutathione.
17. pKa of a-amino group is lower than the pKa of -aminogroup.at physiologic pH, since its pl value close to pH 7.e
18. Histidine in a protein would have a higher buffering capacity
19. Sakaguchi test used for the detection of arginine. tost e t avgini
20. Ninhvdrin test detection of amino acids.
2i. Pauly's diazo
iydin
Test is specific for the detectionof Tryptophan or Histidine..
test
Sakagehi
22. Millon's test for detection of phenolic amino acids such as Tyrosine and itsderivatives.
23.Proline is also called N-alkylated alpha-amino acid.
24. Gramicidin is a polypeptide made up from mixture of D- and L-amino acids.
25. Tryptophan is a precursor of the neurotransmitter serotonin.
26. Tyrosine (phenylalanine) are precursors of neurotransmitters dopamine, epinephrine.
27. In plants, it is a precursor of various phenylpropanoids, which are important in plant metabolism.
28. Glycine is a precursor of porphyrins such as heme.
29. Arginine is a precursor of nitric oxide.
30. Ornithine and S-adenosylmethionine are precursqrs of polyamines.
31. B-Mercaptoethanol and DTT are reducing agents.
32. Aspartate, glycine, and glutamine are precursors of nucleotides.
polymer
33. Polyaspartate, a water-soluble biodegradable SECIS
34. Selenocysteine is encoded by stop codon and element.
35. The hydrolysis of a peptide bond is an exergonie reaction, it occurs slowly because of its high activation energy.
36. The peptide bonds in proteins are quite stable, with an average half-life (tña) of about 7ycars.
97, Serine plays an important role in the catalytic function of of chymotrypsin, trypsin, and many other enzymes.
38. Disulfide bonds are introduced co- and Dost-translatiohally in endoplasmic reticulum (ER) eequires Oxy gen.
called Isoelectrofocussing.
39. plvalue of the protein determned by a technique
40. Pyrrolysine(0) encoded by_the amber' stop cudon UAG is an a-amino acid that is used in the biosynthesis of proteins in
some methanogenic archaea and bacteria; it is notpresent in humans.
41. Selenocysteine (U) is encoded by the 'opal' stop UGACodon.
42. In the unproonated state the functional side chains of arginine, lysine, histidine, cysteine, aspartic acid, giutamic acid and
tyrosine are potent nucleophiles.
43. The relative order of nucleophilicily of functional groups in amino acids is R-S > R-NH, > R-C00- R-O-.