CHNG II. VT LIU SINH HC 2.1.

Protein

1.Khi nim, vai tr ca protein

Khi nim:

l cc hp cht cao phn t bao gm ch yu l cc L-axit amin kt hp vi nhau qua lin kt peptit
Vai tr:

Hemoglobin: 64,5 kDa

Xc tc Vn ti Chuyn ng Bo v

Truyn xung thn kinh iu ho Kin to chng c hc D tr nng lng

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CHNG II. VT LIU SINH HC

2.1.

Protein

2. Axit amin
Khi nim:

l hp cht trong phn t c cha t nht mt nhm amin v mt nhm cacboxyl

trong thin nhin c 150 loi AA, trong c 20 loi tham gia cu to protein = 20 a.a cu trc

Cng thc chung:

R H2N C H

COOH
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CHNG II. VT LIU SINH HC

2.1.

Protein

2. Axit amin
Tn vit tt:

1a) AXIT AMIN KHNG PHN CC,

mch bn l hydrocacbon k nc (1)

Cu & que

Ho lp th

Hnh chiu Fischer

R= Hydro Phn linh ng, mm do Khng cha ccbon bt i

A.a kiu mu Nhm R l -CH3 12

:N

:H

:O

:C

Mch bn k nc Dui thng hoc phn nhnh

Cha lu hunh = Tng tc v kt gn vi ion KL 13 Metaloprotein

Prolin: axit imin duy nht

Cu trc vng: Proline b hn ch v mt hnh th, lm nh hng n cu trc protein

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1b) AXIT AMIN KHNG PHN CC,

mch bn l hydrocacbon thm

Vng phenyl hon ton k nc

Nhm hydroxyl (-OH) Hot ng

Vng Indol Nhm methylen (-CH-) 2 vng lai + Nhm NH

Tyr & Trp do c nhm -OH & -NH nn t k nc hn 15

Nhm II: Axit amin trung tnh

(5)

Alanin c hydroxyl ho

Valin c 1 nhm hydroxyl thay th cho nhm methyl

Tm bt i
OH + ng =Glycoprotein OH + P = c ch, iu ho th 2

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Cysteine
Serine c nhm sulfhydryl, hay thiol (-SH) thay th cho nhm hydroxyl (-OH)

-SH hot ng hn OH. 2 SH = lk disulfide, bn protein

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Asparagine (Asn, N) Cc Amid cua a.a

Glutamine (Gln, Q)

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Nhm III:

Cc mch bn axit tnh (2)

Glutamate hoc axit glutamic(E, Glu) v Aspartat hoc axit aspartic (D, Asp) Lun tch in m pH sinh l(pH 7,4)

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Nhm Iv:

Cc mch bn baz tnh

Histidin (H, His) Lyzin (K, Lys) Arginin (R, Arg)

Nhn mt proton pH sinh l (tch in dng) His b ion ha gn pH sinh l; do c th hot ng nh l mt cht nhn proton hoc cho proton ph thuc vo pH ca mi trng xung quanh. (His l mt a.a c tnh baz yu)

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Cc axit amin c to thnh nh th no? Khi nim axit amin cn thit

Ngi v ng vt khng th tng hp c mt s a.a.

Cc a.a ny c gi l cc a.a cn thit (a.a khng thay th): valine, isoleucine, leucine, methionine,
phenylalanine,tryptophan, threonine, lysine

Arginin v Histidin l cc a.a khng thay th tr nh v mt s ngi mc cc bnh ri lon trao i cht (extreme metabolic stress disease)- y l cc axit amin khng thay th c iu kin.
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tnh cht ca axit amin

(1)

Tnh quang hot

Kh nng lm quay mt phng phn cc ca nh sng

Ch tr a.a n gin nht l glyxin cn tt c cc axit amin u c cha cacbon bt i, nn chng c tnh quang hot. Hng quay ca mt phng nh sng phn cc c k hiu bng du (+): quay phi v (-): quay tri.
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(2)

Tnh cht ion ha (in ly lng tnh)

Tng in tch: +1

-1

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Cc ph.ng th ca nhm -NH2:

(5) P.ng vi 1- Fluoro -2,4 dinitrobenzen

( thay th 1 hidro ca nhm amin bng gc thm dinitrophenyl): 25oC, pH 7-8

SPhm thu c (DNP-aa) c mu khng b thy phn trong mi trng axit. Tnh cht ny l c s ca phng php Sanger, mt phng php dng xc nh trinh t a.a trong protein (trinh bay trong phan tinh chat cua
protein).

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Cc ph.ng th ca nhm -NH2:

(6) Phn ng vi HCHO: phng php chun formol ca Sorensen

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(7) P.ng vi axit nitr

Nh cc amin bc nht khc, axit amin p.ng vi axit nitr gii phng kh nit v oxy axit tng ng c hnh thnh:
R H C - NH2 COOH + HO-N=O R H C - OH COOH + N2 + H2 O

Da vo lng nit thot ra ta c th tnh c lng axit amin c trong dung dch (phng php Van Slyke).

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(8) Phn ng vi ninhydrin

- Nhm cacboxyl, amin u tham gia = CO2 v NH3 u b loi:

H2N

H+

pH 5-7, toC

CO2 + NH3+ RCHO +

COOH
- phn t ninhydrin kh cng vi 1 phn t ninhydrin mi, p. vi NH3b loi ra trn to thnh 1 hp cht c mu tm :

+ NH3+

H
NH4

+ 3 H2 O

+ NH3

-Nguyn t N ca NH3 = cu ni ni 2 phn t nihydrin to thnh hp cht c mu tm 46 ; OD570 nm

- Xem thm: http://www.ecompound.com/Reaction%20reference/reactions/Ninhydrin%20with%20amino%20acid%20mechanism.gif

(9) Phn ng vi reducton

Xem thm: SMOKING TOBACCO PRODUCT AND METHOD OF MAKING THE SAME .United States Patent 3722516

R H2N C H

+R-CO-CO-R RCHO + R-CHNH2-CO-R+CO2

COOH

(10) Phn ng ca axit amin vi ng kh (Phn ng Maillard)

To hp cht c mu nu, hng thm c trng
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(11) Phn ng to lin kt peptid

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CHNG II. VT LIU SINH HC

2.1.

Protein

3. Cu trc ca protein Cu trc bc 1

L trnh t sp xp ca cc axit amin trong mch polypeptide
C k axit amin c k! trnh t sp xp, nhng ch 1 ng phn c hot tnh sinh hc. Lin kt tn ti trong cu trc bc 1: lin kt ng ho tr gia cc axit amin
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 Cu trc bc hai: (1)xon alpha

Dng cu hnh l xo c sinh ra do s t cun xon ca 01 on mch polypeptide c gi bn vng nh cc lin kt hydro ni phn t, c hnh thnh gia nhm CO ca aa th i vi nhm NH ca aa th i+4.

59

 Cu trc bc hai: (2) t giy xp

Cu trc gp np beta; Dng hnh ch chi Sinh ra nh s tng tc gia cc on mch polypeptide

c gi vng nh lin kt hydro hnh thnh gia nhm CO ca on mch polipeptide ny vi nhm NH ca on mch polypeptide gn k.

(A)

(B)
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:N

:H

:O

:C

:R

Cu trc bc ba

Cu hnh ca ton b mch polypeptide

Myoglobin Ch yu l xon alpha, tng = 8 xon (75% mch chnh) Nhm ngoi kt gn vi O2 Nhm Heme l protoporphyrin IX, & nguyn t st tm
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1 loi Protein (Cro) ca thc khun th lambda Dimer ca cc di n v xc nh

Cu trc bc bn, dimer

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4) Tnh cht protein

Hnh cu

4.1. Hnh dng v kch thc

Hnh si
Protein c cu trc bc hai bn chc

V nh hnh

Protein c nhiu aa khng phn cc

Tng tc tt vi nc, to cu hnh ngu nhin

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Kch thc
Ht nano: 1-100 nm; 1 kDa = 1000 Dalton

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4.2. Tnh cht in li lng tnh

pI pH>pI: a anion pH<pI: a cation

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4.3. Cc phn ng mu
Phn ng Biure, Foline

540 nm 5-160 mg/ml protein

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Phn ng axit bicinchoninic (BCA)

Giai on 1: Phn ng Biure

Giai on 2: Phn ng gia Cu+ vi BCA

Mu tm m, 562 nm 5.10-2 -160.10-2 mg/ml

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Phn ng Folin-Ciocalteu
Giai on 1: Phn ng Biure, 10 pht-RT Giai on 2: Phn ng gia Cu+/axit amin thm (Tyr, Trp) vi thuc th Folin- Ciocalteu (dung dch axit phosphomolybdic-phosphotungstic) ? 30 pht, mu xanh, 750 nm, 10 g/ml

http://www.life.illinois.edu/biochem/355/articles/LowryJBC193_265.pdf http://web.itu.edu.tr/~dulekgurgen/Proteins.pdf
http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Datasheet/6/47641dat.Par.0001.File.tmp/47641dat.pdf

86

1 - 1500g/mL www.freepatentsonline.com/6558717.html
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4.4. Kh nng bin tnh

Di tc dng ca cc tc nhn vt l hoc cc tc nhn ho hc, protein b mt i cc tnh cht ban u, protein b bin tnh.

Luc trng

Kem trng

u ph

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Khi protein b bin tnh:

Cc cu trc bc 2,3,4 b thay i Mt hoc gim hot tnh sinh hc Phn t protein b dui ra
xut hin cc nhm k nc

kh nng ho tan gim kh nng gi nc km mt kh nng kt tinh.

phi by cc lin kt peptide ng vi vng c hiu ca protease, protein bin tnh d tiu ho.
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4.5. Kh nng ho tan v kt ta

Cc protein c kh nng ho tan khc nhau Protein hnh si khng tan. Protein hnh cu tan Protein c nhiu nhm phn cc bn b mt phn t ha tan tt Protein c nhiu axit amin axit tnh tan tt hn Trong mi trng mui long, kim long protein tan tt, trong mi trng c nng mui cao protein khng tan. Bi ????
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 ho tan ca protein ph thuc vo 2 yu t:

1. Mc hydrate ho (lp v nc) 2. Mc tch in ca protein (lp v in tch)

Protein G vi lp v nc dy 5 A

http://binf.gmu.edu/~greck/Server/intro.php

98

Ha tan v Kt ta (tip)

Di tc dng ca cc yu t vt l, ho hc lm trung ho in tch ca cc phn t protein, ph v mng nc, phn t protein s kt hp vi nhau to thnh khi ln, tch khi dung dch kt ta.

Hnh: Kt ta protein trong nc tiu bng sulfosalicylic acid (SSA) http://ahdc.vet.cornell.edu/clinpath/modules/ua-rout/protssa.htm

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TNH CHT CA PROTEIN (tip)

4.6. Tnh cht thu phn:
Bng axit: mt s axit amin thm b tn tht Bng kim: axit amin c lu hunh b ph hu, dng L b ng phn ho thnh dng D, gi tr dinh dng gim 50%.; dng khi thu phn protein bm xng trnh ho tan xng nh khi dng axit. Bng enzym: lu (nc mm, tng an ton khng 3-MCPD)
It guards against hair loss, dandruff and environmental damage. Ingredients: Aqua, Isopropyl Alcohol, Propylene Glycol, Caffeine, PEG-40 Hydrogenated Castor Oil, Parfum, Carbomer, Menthyl Lactate, Tocopheryl Acetate, PEG-35 Castor Oil, Hydrolyzed Wheat Protein, Benzyl Alcohol, Sodium Hydroxide, Panthenol, Synthetic Thymus Hydrolysate, Sodium Benzoate, Phenoxyethanol, Methylparaben, Propylparaben

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http://news.socbay.com/nuoc_cham_o_tphcm_kem_chat_luong_mat_an_toan_-600244572-268632064.html

4.7. To gel, to mng: Khi protein b bin tnh, tp hp li hnh thnh cu trc mng li khng gian 3 chiu c trt t gel protein. Nt cc mng l lin kt a bo hnh thnh gia cc gc R.

www.chembio.uoguelph.ca/.../page6.shtml

104

Lm bn nh tng, to bt
Nh tng=h phn tn ca 2 cht lng ko trn ln, 1 cht dng git nh (0,1-1 mcm) ca pha phn tn, cn cht kia pha lin tc Bt=h phn tn ca cht kh trong pha lin tc l cht lng hoc bn rn

4.8. Tnh cht b mt:

Protein hp ph trn cc b mt, lm bn nh tng v bt theo cc c ch khc nhau Protein thng b bin tnh

Tng tc tnh in

Hiu ng khng gian

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