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11

PeptidesPeptides

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22

Codons

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33

Codons

http://libgallery.cshl.edu/items/show/51962

http://libgallery.cshl.edu/items/show/51962http://libgallery.cshl.edu/items/show/51962

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44

Amino acids react with each other toAmino acids react with each other to

!orm di"# tri"# oligo"# and poly"peptides!orm di"# tri"# oligo"# and poly"peptides

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55

A polypeptide has an amino terminusA polypeptide has an amino terminus

and a carbo$yl terminusand a carbo$yl terminus

N-terminal C-terminal

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66

%ame%ame

the AAthe AAresiduesresidues

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&&

'ach protein has'ach protein has

a uni(ue si)e#a uni(ue si)e#composition andcomposition and

se(uence o!se(uence o!

amino acidsamino acids

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**

Molecular weight (Dalton)

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99

Peptide hydrolysisPeptide hydrolysis

Acidic hydrolysisAcidic hydrolysis

+asic hydrolysis+asic hydrolysis

'n)ymatic hydrolysis'n)ymatic hydrolysis

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1,1,

Acidic and +asic -ydrolysisAcidic and +asic -ydrolysis

--224404 or -l 06# boil !or 2, hours#04 or -l 06# boil !or 2, hours#

get all "amino acid# but tryptophan hasget all "amino acid# but tryptophan has

been destroyed.been destroyed.

+asic# %a- 05# boil 1,"2, hours# many+asic# %a- 05# boil 1,"2, hours# many

inds o! amino acid has been destroyed#inds o! amino acid has been destroyed#

produce " " amino acid# but tryptophanproduce " " amino acid# but tryptophan

is stableis stable

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1111

'n)ymatic -ydrolysis'n)ymatic -ydrolysis

Protease: partial hydrolysis# amino acids areProtease: partial hydrolysis# amino acids areintactintact

7rypsin7rypsin

hymotrypsinhymotrypsin

PeptinPeptin

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1212

-ow can the amino acid se(uence-ow can the amino acid se(uence

o! a protein be identi!ied8o! a protein be identi!ied8

Genomic MethodGenomic Method Biochemical MethodBiochemical Method

e(uence the genee(uence the gene

coding !or nsulincoding !or nsulin

e(uence the m;%Ae(uence the m;%A

!or nsulin in pancreatic!or nsulin in pancreaticcellscells

solate nsulin tosolate nsulin to

homogeneityhomogeneity

etermine the aminoetermine the amino

acid se(uence o!acid se(uence o!nsulin using a directnsulin using a direct

chemical methodchemical method

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1313

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1414

nsulin" the !irstnsulin" the !irst

protein to beprotein to be

se(uencedse(uenced

-ow many cysteines8

-ow amino acids

lin together to

!orm insulin8

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1515

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1616

tep 1:tep 1:solate and puri!y the protein o!solate and puri!y the protein o!

interestinterest

tep 2:tep 2:;educe disu!ide bonds with 77:;educe disu!ide bonds with 77:

00ithiothreitolithiothreitol

tep 3:tep 3:

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1&1&

tep 3:tep 3:;eact amino"terminal;eact amino"terminal

residue with

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1*1*

7he amino terminal residue7he amino terminal residue

becomes a %P"deri=ati=ebecomes a %P"deri=ati=e

oncentrated -l hydroly)es alloncentrated -l hydroly)es all

the peptide bondsthe peptide bonds

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1919

%ow we ha=e a%ow we ha=e a >labeled? amino acid>labeled? amino acid

-ow is that help!ul8-ow is that help!ul8

7he7he >phenolic? ring absorbs light at>phenolic? ring absorbs light at2&, nm. Allowing !or detection2&, nm. Allowing !or detection

a!ter chromatographya!ter chromatography

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2,2,

ther common reagents !or labeling o!ther common reagents !or labeling o!

amino acidsamino acids

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2121

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2222

In amino acid chromatography, more thanIn amino acid chromatography, more than

one physical property is used to separateone physical property is used to separate

molecules one from the othermolecules one from the other

et electric chargeet electric charge

!ydropho"icity!ydropho"icity

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2323

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2424

Polystyrene ulphonatePolystyrene ulphonate

%egati=ely charged at a wide p- range%egati=ely charged at a wide p- range

-ydrophobic bacbone-ydrophobic bacbone

Amino acids separate based on theirAmino acids separate based on theirpartitioning between the hydrophilic#partitioning between the hydrophilic#

uncharged mobile phase# and theuncharged mobile phase# and the

hydrophobic# charged# stationary phasehydrophobic# charged# stationary phase

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2525

Polystyrene ulphonatePolystyrene ulphonate

%egati=ely charged%egati=ely chargedamino acids !low throughamino acids !low through

Positi=e amino acidsPositi=e amino acidsinteract with the resininteract with the resin

and there!ore mo=e more slowlyand there!ore mo=e more slowly

-ydrophilic-ydrophilic amino acids ha=e lower a!!inity !oramino acids ha=e lower a!!inity !or

the resin thanthe resin than hydrophobichydrophobiconesones

! net charge is the same# hydrophilic amino! net charge is the same# hydrophilic aminoacids will elute earlier !rom the columnacids will elute earlier !rom the column

compared to hydrophobic onescompared to hydrophobic ones

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2626

t is possible to resol=e most o! the 2,t is possible to resol=e most o! the 2,

amino acids in one runamino acids in one run

#lution time, min$

%"sor"ance,&

$D$

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2&2&

eparations areeparations are highly reproduci"lehighly reproduci"le

#lution time, min$

%"sor"ance,&

$D$

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2*2*

7his procedure allows identi!ication

o! %"terminal amino acid o! a polypeptide

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2929

e(uencing bye(uencing by >'dman egradation?>'dman egradation?

n=ented by 'dman Pehrn=ented by 'dman Pehr

;e(uires an isolated single polypeptide;e(uires an isolated single polypeptide

7he peptide is immobili)ed by its7he peptide is immobili)ed by its carboxylcarboxyl""terminus to a solid supportterminus to a solid support

7he ma$imal practical length !or se(uencing7he ma$imal practical length !or se(uencing

by this method is @5, residuesby this method is @5, residues

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3,3,

;eact;eact aminoamino""terminus withterminus with

phenyl"phenyl"

isothiocyanateisothiocyanate0P70P7

tep 4:tep 4:>'dman>'dman

egradation?egradation?

Alaline conditionsAlaline conditions

'

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3131

IC*adduct

IC*adduct

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3232

Proteolytic clea=age o! polypeptidesProteolytic clea=age o! polypeptides

!P t l ti l ! l tid

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3333

Proteolytic clea=age o! polypeptidesProteolytic clea=age o! polypeptides

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3434

7he action o!7he action o!

yanogenyanogen

+romide+romide

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3535

Peptide appingPeptide apping

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3636

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A nonapeptide was determined to have the following amino acid

composition: (Lys)2, (Gly)2, (Phe)2, His, Leu, et! "he native

peptide was incu#ated with $%&uoro%2,'%dinitro#enene (FDNB)and then hydrolyed 2,'%dinitrophenylhistidinewas identi*ed

#y HPL+! hen the native peptide was e-posed to cyanogen

#romide (CNBr), an octapeptide and free glycine were

recovered! .ncu#ation of the native peptide with trypsingave a

pentapeptide, a tripeptide, and free Lys! 2,'%

/initrophenylhistidine was recovered from the pentapeptide, and

2,'%dinitrophenylphenylalanine was recovered from the

tripeptide! /igestion with the enyme pepsin produced a

dipeptide, a tripeptide, and a tetrapeptide! "he tetrapeptide wascomposed of (Lys)2, Phe, and Gly! "he native se0uence was

determined to #e: