fdi2
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gfgfTRANSCRIPT
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PROTEINE
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Definiţie
• Biomacromolecule formate din una sau mai multe catene, rezultate prin policon-densarea aminoacizilor.
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Funcţionalitate extrem de diversă
• Biocataliza enzimatică• Hormoni• Transport, stocare• Formaţiuni motile• Suport structural• Anticorpi• Transmitere de impulsuri nervoase• Reglarea unor procese metabolice
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Determinarea compoziţiei și purificarea proteinelor
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1. Evaluarea conţinutului de proteină brută
• 1. Metoda KjeldahlSe bazează pe corelaţia conţinutului de
azot cu cel de proteină. Se consideră că proteinele contin 16 %
azot
Norg (NH4)2SO4
H2SO4
(NH4)2SO4 NH4OHNaOH
NH4OH NH3
NH3 NH4Cl
HCl
– Mineralizarea probei– Antrenarea amoniacului şi
dozarea acestuia
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1. Evaluarea conţinutului de proteină brută
• 2. Metoda LowrySe bazează pe absorbanţala 660 nm a complexuluireacţiei de tip Biuret (proteină
+ Cu+2) şi formei reduse a reactivului Folin de catre Cu+
rezultat la oxidarea Tyr, Cysşi Trp
• Reactivi:– Soluţie alcalinocuprică– Reactiv Folin Ciocâlteu
(soluţie fosfomolibdo-wolframică NaWO4 2H2O NaMoO4 2H2O)
NO
N
H
R
H
N O
N
HR
H
Cu+2
Lowry et al., J. Biol. Chem. 193: 265-275 (1951)
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1. Evaluarea conţinutului de proteină brută
N
N
CO2H
CO2H
N
N
HO2C
HO2C
Cu+
• 3. Varianta reactivbicinconinic
Acid 2,2’ bichinolin 4,4’ dicar-boxilic
Proteina interacţionează cu ionul Cu2+ într+o reacţie similară cu reacţia Biuret. În urma acestei reacţii, în mediu alcalin are loc reducerea Cu2+ la Cu+ care dezvoltă o coloraţie purpurie prin complexare cu reactivul
562 nmAnal Biochem. 1985 Oct;150(1):76-85
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• 4. Metoda Bradford• Deplasarea maximului de
absorbţie al colorantului în urma ataşării acestuia de proteină (465 595 nm)
CH3H3C
N
N
NH
O SO3-
SO3-
Coomassie Blue G-250
Bradford, M. M. (1976), Anal. Biochem. 72:248-254
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2.Separarea și purificarea proteinelor
• 2.1. Distrugerea membranelor şi pereţilor celulari– Mijloace nechimice
• Utilizarea de abrazivi• Forfecarea prin de destindere• Ultrasonare
– Mijloace chimice• Solvenţi organici• Detergenţi• Agenţi chaotropici• Tratament alcalin• Enzime (lizozim)
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DezintegratoareUltrasonic Forfecare la destindere
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Dezintegratoare (continuare)
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Centrifugarea2.2. Îndepărtarea resturilor de la omogenare
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2.3. Purificarea grosieră a proteinelor
• Precipitare lapunctul izoelectric
• Precipitarea prin efect salin
Solubilitatea carboxihemoglobinei funcţie de tăria ionică și natura ionilor
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Dializa
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Purificarea fină a proteinelor
• Metode cromatografice• Metode electroforetice
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Cromatografia de excluziune sterică
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Electroforeza (principiu)
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Relaţia mobilitate masa la separările electroforetice Gel electroforetic după migrare
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Determinării structurii primare a proteinelor
1. Separarea catenelor (desfacere de legături slabe)– Utilizare de valori extreme ale pH-ului– Agenţi chaotropici (distrugători de structură)
• Uree 8M• Clorohidrat de guanidiună 6M
– Concentraţii ridicate de săruri (sulfat de amoniu)
2. Desfacerea puntilor disulfurice– metode reductive– metode oxidative
3. Stabilirea compoziţiei în aminoacizi– Hidroliza, separare
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Secvenţa determinării structurii primare a proteinelor (continuare)
• 4. Stabilirea aminoacizilor de capăt– N, C.
• 5. Fragmentarea catenelor lungi– Chimic, enzimatic
• 6. Secvenţarea propriuzisă Edman• 7. Reconstituirea secvenţei prin alinierea
fragmentelor– Evaluarea secvenţelor pe tip de fragment
• 8. Relocarea punţilor disulfurice– Electroforeza fragmentelor înainte şi după desfacerea punţilor disulfurice
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2.Desfacerea punţilor disulfurice
1. Căi reductiveMercaptoetanol
Ditiotreitol
2. Căi oxidativeAcid performic
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3.Determinarea compoziţiei in aminoacizi
• Hidroliza acidă (HCl 6N la reflux 72-96h)
1. Separare cromatografica2. Identificare, cuantificare
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O
O
OH
OH
O
O
O
O
O
NCH
R
O
O-H3N
R
CO2-
O
O-
NCH
R
CO2O
O-
NH2
R CHO
HOH
O
O-
N
O
O
OH
H
O
O-
N
O
OPurpura lui RUHEMANN
Ninhidrinã(tricetohidrindenhidrat)
dsdf
Rf=100 x ds/df
Reactia ninhidrinei şi determinarea calitativă prin cromatografie pe strat subţire a aminoacizilor
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4. Determinarea aminoacizilor de capăt
• Aminoacizii N-teminali– a) dansilarea
• Aminoacizii C-teminali• a) Metoda Akabori
+ aa1 +...
hidrolizãacidã
-CH(R)-CO2H
NH3C CH3
SO2NH
Clorurã de dansil
-CH(R)-CO-NH-CH...
NH3C CH3
SO2NH
H2N-CH(R)-CO-NH-CH-....
NH3C CH3
SO2Cl
2,4 dinitrofluorobenzen
NO2
NO2
NH
NO2
NO2
NHNO2
NO2
F
+ aa1 +...
hidrolizãacidã
-CH(R)-CO2H-CH(R)-CO-NH-CH...
H2N-CH(R)-CO-NH-CH-....
a) Metoda Sanger
NH CH CO NH CH CO NH CH CO2-
R1R2R3
H2N NH2H2N NH2
H3N+ CH CO
Ri
NH NH2
H3N+ CH CO2
-
R1
i=2,...
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Enzima Situs preferatTripsina R1: Lys, ArgClostripaina R1: ArgChimotripsina R1: Tyr, Phe, Leu, Ile, Val, TrpTermolisina R2: Tyr, Phe, Leu, Ile, Val, TrpPepsina R1: Phe, Leu, mulţi alţi aminoaciziProtează stafilococică (tampon fosfat) R1: Asp, GluProtează stafilococică (tampon HCO3- ) R1: GluSubtilizina R1: MulţiCarboxipetidaza A R2: Aminoacid C- terminal (exceptie, Arg,
Tyr, Trp)
H3N CH C
R1 O
O COOH3N CH
R2
H3N CH C NH
R1 O
COOCH
R2
5. Fragmentarea catenei polipeptidice
a. Fragmentare enzimatică
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+
bromocian
homoserinolactona
H3NC NH CH C O
O
CH2
CH2
O
HO
H
SCNH3C
C NH CH C N
O
CH2
CH2
O
H
C NH CH C N
O
CH2
CH2
SH3C CN
O
H
C
N
Br
C NH CH C NH
O
CH2
CH2
SH3C
O
b. Fragmentare chimică cu bromocian
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6. Secvenţarea Edman
N
NH
O
R1
S
NC
S
R1
NH
H2N
O
R2O
(H*)
R1
NH
N
O
R2O
S
CNH
H
(H*)
NH2
R2O
R1N
OS
CN
H(H*)
CF3CO2H
pH = 9
mediu apos
feniltiocarbamoil peptidă
peptida fărăaminoacidul N-termina
AnilinotiazolinonăFeniltiohidantoină (PTH)
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7. Reconstitu-irea secvenţei totale
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8. Relocarea puntilor disulfurice
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Alanina A alaArginina R argAsparagina N asnAcidul Aspartic D aspCisteina C cysGlutamina Q glnAcidul Glutamic E gluGlicina G glyHistidina H hisIzoleucina I ileLeucina L leuLizina K lysMetionina M metFenilalanina F pheProlina P proSerina S serTreonina T thrTriptofan W trpTirozina Y tyrValina V val
Aminoacizii naturali, sistematizaţi pe diferite criterii
L-prolinaL-aminoacid
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Structura primară şi secundară a unei proteine
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Diagrama Ramachandran
Unghiurile de torsiune şi
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Conformatiedefavorizata
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Structuri helix
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Principalele tipuri de structuri secundare dispuse pe diagrama Ramachandran
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Modelulalfa helix în diferitereprezentări
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Modele foi plisate (modele beta) paralel si antiparalel
Modelul paralel
Modelul antiparalel
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Foi beta : model antiparalel
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Foi beta : model paralel
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Model paralel
Model antiparalel
FOI BETA
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Model foaie beta antiparalel extins
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Modalităţi de conectare ale structurilor beta:a) Antiparalel, prin buclăb) Paralel, prin buclă de dreapta, cazul comunc) Paralel, prin buclă de stânga, caz extrem de rar
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Modalităţi de formare a structurilor helicoidale din proteine
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Bucle• Buclele se plasează
între helixuri sau foi plisate. Ele nu sunt atât de rigide. De regulă aceste regiuni sunt zone de recunoaştere
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Structuri suprasecundare
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Interacţiuni care stabilizează structurile terţiare proteice