Cao ng Nguyn(ch bin)

Gio trnh

Cng ngh Protein

NH XUT BN I HC HU Hu-2007

Li ni u

Trong nhng nm gn y cng ngh sinh hc pht trin nh v bo, hng lot cng ngh mi ra i nh genomics, proteomicsv c ng dng vo nhiu lnh vc khc nhau nh nng nghip, cng nghip thc phm v.vc bit l lnh vc y-dc hc. Gio trnh cng ngh protein c bin son vi thi lng ch trong hai n v hc trnh, trn c s cp nht nhng kin thc hin i, nhng thnh tu mi nht v proteomics trong nghin cu c bn v ng dng thc tin trn th gii v Vit nam, nhm phc v cho vic ging dy v hc tp cho cc ngnh sinh hc v cng l ti liu tham kho ca nhng ngnh hc lin quan khc. Gio trnh bin son c 6 chng vi hai ni dung chnh: - Nhng kin thc c bn v protein nh thnh phn, cu trc, tnh cht ha -l, cc phng php tch, tinh sch v xc nh protein. - Cng ngh sn xut mt s loi protein. Gio trnh bin son c phn cng c th nh sau: Chng 1. M u Cao ng Nguyn Chng 2. Amino acid - n v cu to protein Cao ng Nguyn Chng 3. Peptide - cu trc v chc nng Cao ng Nguyn Chng 4. Cu trc v tnh cht l-ho ca protein Cao ng Nguyn Chng 5. Cc phng php chit rt, tinh sch Qu Hai v xc nh protein Chng 6. Cng ngh sn xut mt s protein Cao ng Nguyn Chng ti chn thnh cm n Qu nng cao cht lng-D n gio dc i hc i hc Hu h tr chng ti bin son gio trnh ny; cm n GS.TSKH L Don Din, PGS.TS L c Ngc gp cng chi tit, GS. TS Ngc Lin c bn tho v ng gp nhng kin qu bu. Gio trnh c xut bn ln u tin chc chn khng trnh khi nhng thiu st. Cc tc gi xin chn thnh cm n v rt mong c s gp ca cc ng nghip v bn c khi ti bn s c hon thin hn Cc tc gi

MC LCChng 1. M u I. Khi qut chung v protein 1.1 Nhng c trng chung ca nhm cht protein 1.2. ngha khoa hc v thc tin ca nhm cht protein II. Phn loi protein 2.1. Protein n gin 2.2. Protein phc tp III. Chc nng xc tc sinh hc 3.1. Xc tc v enzyme 3.2. Isozyme 3.3. Cc enzyme di lp th (allosteric enzyme) v phc h enzyme (multienzyme). 3.4. Nhng quan im y hc v protein Chng 2. Amino acid- n v cu to protein I. Thnh phn v tnh cht l-ho cua amino acid 1.1. Thnh phn v cu to amino acid 1.2. Phn loi amino acid 1.3. Mu sc v mi v ca amino acid 1.4. Tnh tan ca amino acid 1.5. Biu hin tnh quang hc ca amino acid 1.6. Tnh lng tnh ca amino acid 1.7. Cc phn ng ho hc cua amino acid II. Thu nhn amino acid bng thu phn protein 2.1. Thu phn bng acid 27 28 30 31 31 8 22 22 22 22 26 Trang 9 9 9 9 10 10 11 11 11 14 16

2.2. Thu phn bng kim 2.3 Thu phn bng enzyme 2.4. Cc phng php theo di v xc nh tc thu phn III. Cc phng php phn tch amino acid 3.1. Phng php l -ho 3.2. Phng php sc k 3.3. Phn tch bng my t ng 3.4. Phng php in di 3.5. Phn tch bng quang ph khi 3.6. Phng php ng v 3.7. Phng php enzyme 3.8. Phng php vi sinh vt Chng 3. Peptide - cu trc v chc nng I. Tnh cht chung ca peptide 1.1.Cu to ca peptide 1.2. Cch gi tn v phn loi peptide 1.3. Cc phn ng c trng ca peptide II Cc phng php tch phn lp v xc nh peptide III. S tn ti t nhin v vai tr chc nng ca peptide 3.1. Khi nim chung 3.2. Glutation v cc cht tng t 3.3. Cc hormon c bn cht peptide v protein 3.4. Cc peptide khng sinh 3.5. Cc peptide c ngha sinh l t bo 3.6. Cc peptide c chc nng bo v Chng 4. Cu trc v tnh cht l ho ca protein

32 32 32 33 33 33 34 35 35 36 36 36 38 38 38 40 41 41 42 42 42 43 49 51 51 53

I Cc quan im khc nhau v nghin cu protein II. Thnh phn v lin kt trong cu trc protein 2.1. Lin kt ccng ho tr 2.2. Lin kt yu III. Hnh dng kch thc v cu trc phn t protein 3.1. Hnh dng kich thc 3.2. Cu trc s cp 3.3. Cu trc th cp 3.3 Cu trc khng gian. IV. Tnh cht l -ho ca protein V. Bin tnh protein Chng 5. Cc phng php chit rt, tinh sch v xc nh protein I. Khi nim II. Cc bin php cn thit nhn protein nguyn th 2.1. Nhit 2.2. Nng proton 2.3. Tc nhn ho hc III. Ph v t bo v chit rt protein 3.1. Ph v t bo 3.2. Chit rt protein IV. Tinh sch protein 4.1 Loi tp cht 4.2. Cc k thut thng thng trong tinh sch protein V. nh lng v nh gi sch ca ch phm protein 5.1.Cc phng php xc nh hm lng protein

53 53 53 54 57 57 57 58 62 67 72 67

75 75 75 75 77 79 79 79 80 80 80 89 89

5.2. nh gi tnh ng th ca protein Chng 6. Cng ngh sn xut mt s protein I. Mc ich yu cu 1.1. ngha ca protein i vi qu trnh sinh hc 1.2. Nhu cu khoa hc v x hi i vi protein 1.3 Tnh hnh nghin cu v sn xut protein c hot tnh sinh hc II. Sn xut protein bng con ng vi sinh vt 2.1. Sn xut protein n bo 2.2. Sinh tng hp cc hormon bng vi sinh vt 2.3.Sn xut cc cytokin 2.4. Sn xut cc cht min dch v cc vaccine III. Sn xut protein tg ngun thc vt 3.1. C s sinh ho v ngha ca nui cy mt v t bo 3.2. Cc thnh tu sn xut protein thc vt tren th gii IV. Sn xut protein t ngun ng vt 4.1. Cc nguyn tc v iu kin sn xut protein 4.2. Sn xut khng th n dng 4.3. Sn xut cc hormon, enzyme v protein khc V. Sn xut protein t ngun ph thi 5.1. Sn xut cystine v cc amino acid 5.2. Sn xut protein n bo t b ma

92 95 95 95 96 97 100 100 104 107 107 111 111 113 114 114 115 118 125 126 127

Chng 1

M uI. Khi qut chung v protein1.1. Nhng c trng chung ca nhm cht protein Protein c pht hin ln u tin th k XVIII (1745 bi Beccari); mi u c gi la allbumin (lng trng trng). Mi n nm 1838 , Mulder ln u tin a ra thut ng protein (xut pht t ch Hy lp proteos ngha l u tin, quan trng nht. Bit c tm quan trng v nhu cu x hi v protein, n nay nhiu cng trnh nghin cu v sn xut hp cht ny c cng b, em li nhiu ngha ht sc to ln phc v cho nhn loi. V vy, nhiu nh khoa hc trn th gii vinh d nhn c gii thng Nobel v cc lnh vc nghin cu lin quan n protein. Nh bit protein l hp cht hu c c ngha quan trng bc nht trong c th sng. V mt s lng, n chim khng di 50% trng lng kh ca t bo. V thnh phn cu trc, protein c to thnh ch yu t cc amino acid qua lin kt peptide. Cho n nay ngi ta thu c nhiu loi protein dng sch cao c th kt tinh c v xc nh c thnh phn cc nguyn t ho hc, thng thng trong cu trc ca chng gm bn nguyn t chnh l C H O N vi t l C 50%, H 7%, O 23% v N 16%. c bit t l N trong protein kh n nh. Nh tnh cht ny nh lng protein theo phng php Kjeldahl, ngi ta tnh lng N ri nhn vi h s 6,25. Ngoi ra trong protein cn gp mt s nguyn t khc nh S 0-3% v P, Fe, Zn, Cu... Khi lng phn t, k hiu l Mr (c tnh bng Dalton)* ca cc loi protein thay i trong nhng gii hn rt rng, thng thng t hng trm cho n hng triu. V d: insulin c khi lng phn t bng 5.733, glutamat-dehydrogengenase trong gan b c khi lng phn t bng 1.000.000 (bng 1.1). 1.2. ngha khoa hc v thc tin ca nhm cht protein T lu, bit rng protein tham gia mi hot ng sng trong c th sinh vt, t vic tham gia xy dng t bo, m, n tham gia hot ng xc tc v nhiu chc nng khc v.v...Ngy nay, khi hiu r vai tr to ln ca protein i vi c th sng, ngi ta cng thy r tnh cht duy vt v ngha ca nh ngha thin ti ca Anghen F. : sng l phng thc tn *1 Dalton = 1/1000 Kilodalton v c k hiu l kDa

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ti ca nhng th protein. Vi s pht trin ca khoa hc, vai tr v ngha ca protein i vi s sng cng c khng nh. Cng vi acid nucleic, protein l c s vt cht ca s sng.

II. Phn loi proteinProtein gm hng trm, hng ngn amino acid ni vi nhau bng lin kt peptide to nn mt hay nhiu chui polypeptide c cu trc rt phc tp. Cn c s c mt hay vng mt ca mt s thnh phn c bn cht khng phi protein m ngi ta chia protein thnh hai nhm ln:Bng 1.1 Khi lng (Mr) v cu trc phn t ca mt s protein protein Khi lng (Dalton) s gc amino acid s chui polypeptide

Glucagon Insulin Ribonuclease (ty b) Lysozyme (lng trng trng) Myoglobin (tim nga) Chymotripsin (ty b) Hemoglobin (ngi) Albumin (huyt thanh ngi) Hexokinase (men bia) Tryptophan-synthetase (E.coli) -globulin (nga) Glycogen-phosphorylase (c th) Glutamate-dehydrogengenase (b) Synthetase ca acid bo (men bia) Virus khm thuc l

3482 5733 12.640 13.930 16.890 22.600 64.500 68.500 96.000 117.000 149.000 495.000 1.000.000 2.300.000 40.000.000

29 51 124 129 153 241 574 550 800 975 1.250 4.100 8.300 20.000 336.500

1 2 1 1 1 3 4 1 4 4 4 4 40 21 2.130

2.1. Protein n gin Protein n gin l nhng phn t m thnh phn cu to ca n gm hon ton amino acid. Th d mt s enzyme ca tu b nh

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ribonuclease gm hon ton amino acid ni vi nhau thnh mt chui polypeptide duy nht (c 124 gc amino acid, khi lng phn t 12.640), chymotripsin gm ton amino acid ni vi nhau thnh chui polypeptide (c 241 gc amino acid, khi lng phn t 22.600)v.v...Da theo kh nng ho tan trong nc hoc trong dung dch m mui, kim hoc dung mi hu c ngi ta c th chia cc protein n gin ra mt s nhm nh nh: -Albumin: tan trong nc, b kt ta nng mui (NH4)2SO4 kh cao (70-100%). -Globulin: khng tan hoc tan t trong nc, tan trong dung dch mui long ca mt s mui trung tnh nh NaCl, KCl, Na2SO4..., v b kt ta nng mui (NH4)2SO4 bn bo ho. -Prolamin: khng tan trong nc hoc dung dch mui long, tan trong ethanol, isopanol 70-80%. -Glutein: ch tan trong dung dch kim hoc acid long. -Histon: l protein c tnh kim d tan trong nc, khng tan trong dung dch amoniac long. 2.2. Protein phc tp Protein phc tp l nhng protein m thnh phn phn t ca n ngoi cc - amino acid nh protein n gin cn c thm thnh phn khc c bn cht khng phi l protein cn gi l nhm thm (nhm ngoi). Tu thuc vo bn cht ca nhm ngoi, ngi ta chia cc protein phc tp ra cc nhm nh v thng gi tn cc protein theo bn cht nhm ngoi: -Lipoprotein: nhm ngoi l lipide. -Nucleoprotein: nhm ngoi l acid nucleic. -Glycoprotein: nhm ngoi l carbohydrate v dn xut ca n. -Phosphoprotein: nhm ngoi l acid phosphoric. -Cromoprotein: nhm ngoi l hp cht c mu. Tu theo tnh cht ca tng nhm ngoi m c nhng mu sc khc nhau nh ( hemoglobin), vng ( flavoprotein)...

III. Chc nng sinh hc ca protein3.1. Xc tc v enzyme 3.1.1. Quan im v xc tc enzyme Hu ht tt c cc phn ng xy ra trong c th u do cc protein c bit ng vai tr xc tc, nhng protein c gi l cc enzyme.

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Mc d gn y ngi ta pht hin c mt loi RNA c kh nng xc tc qu trnh chuyn ho tin RNA thng tin (pre-mRNA) thnh RNA thng tin (mRNA), ngha l enzyme khng nht thit phi l protein. Nhng enzyme xc tc sinh hc c bn cht l acid nucleic c gi l ribozyme. Nhng nh ngha c tnh cht kinh in: enzyme l nhng protein c kh nng xc tc c hiu cho cc phn ng ho hc, l cht xc tc sinh hc vn c ngha c bit quan trng. Hin nay ngi ta bit c khong 3.500 enzyme khc nhau, nhiu enzyme c tinh sch, kt tinh v nghin cu cu trc. 3.1.2. Nhng khc bit v c tnh xc tc, gia xc tc v c v xc tc enzyme. Enzyme l cht xc tc sinh hc, ngoi kh nng xc tc ging nh cht xc tc v c bnh thng n cn th hin mt s tnh cht sau y: a) Hiu sut xc tc rt ln. S chuyn ho c cht khi s dng emzyme xc tc ln hn nhiu so vi cht xc tc v c thng thng. V d: 1mol Fe3+ ch xc tc phn ly c 10-6 mol H2O2/pht. Trong khi mt phn t catalase c mt nguyn t Fe xc tc phn ly 5.106 mol H2O2/pht;1 gam pepsin trong 2 gi thu phn c 5 kg protein trng luc nhit bnh thng. Tng t 1gam phn t -amilase sau 1 giy c th phn gii 4.000 lin kt glucoside trong phn t tinh bt, cao hn nhiu so vi xc tc bng cht v c. b) Tnh c hiu cao. Tnh c hiu cao l mt trong nhng khc bit ch yu gia xc tc bng enzyme v xc tc bng cc cht v c khc. Mi enzyme ch xc tc cho s chuyn ho mt hay mt s cht nht nh, theo mt kiu phn ng nht nh. Da vo s tc dng c tnh chn lc, ngi ta c th chia ra mt s kiu c hiu sau: - c hiu kiu phn ng. c hiu ny th hin ch mi enzyme ch xc tc cho mt trong cc kiu phn ng chuyn ho mt cht nht nh. V d: phn ng oxy ho kh, chuyn v, thu phn, v.v... - c hiu c cht. L kh nng kt hp ca c cht vo trung tm hot ng ca enzyme v b chuyn ho di tc ng ca chng. Da vo mc c hiu ngi ta li chia ra mt s kiu nh: c hiu tuyt i, l enzyme ch tc dng trn mt c cht duy nht; c hiu tng i, l enzyme c kh nng tc dng ln mt kiu lin kt ho hc nht nh ca phn t c cht m khng ph thuc vo cu to ca cc phn t tham gia to thnh mi lin kt ; c hiu nhm, l enzyme c kh nng tc

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dng ln mt kiu lin kt ho hc nht nh vi iu kin mt trong hai phn tham gia to thnh lin kt phi c cu to xc nh. Hot tnh ca enzyme ph thuc vo nhiu yu t nh: nng enzyme, nng c cht, nhit v ion kim loi v.v... 3.1.3. C ch xc tc ca mt vi enzyme. Giai on u tin

Ser hoc Thr peptide

Giai on trung gian

Giai on cui cng

H nh 1.1

C ch chuyn nhm phosphate ca protein kinase A

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Hin nay ngi ta bit r c ch hot ng ca nhiu enzyme nh carboxypeptidase, chymotripsin v.v..., mt enzyme cng c nghin cu kh k thuc nhm phosphotransferase xc tc qu trnh chuyn v nhm phosphate l protein kinase A (hnh 1.1) 3.1.4. S phn b enzyme trong t bo. Trong c th enzyme c mt mi m, mi t bo. Nhng ty theo chc nng ca m m cc t bo trong m thng c nhng h thng enzyme ring bit. S khc nhau khng ch gia cc loi t bo m ngay trong mt t bo cng tu theo chc nng ca tng bo quan (organell) ring bit c nhng h thng enzyme c hiu. S sp xp cc enzyme mt cch hp l trong t bo to ra s phi hp nhp nhng trong h thng phn ng dy chuyn lin tc cho hot ng sng ca c th: - Trong nhn t bo c cc enzyme nh ATP-ase, nucleosidase, nicotinic-mononucleotide adenylyltransferase, 5-nucleotidase. - Trong ty lp th c cha hu ht cc enzyme lin quan n qu trnh chuyn ho nng lng nh h enzyme ca chui h hp t bo v qu trnh phosphoryl ho to ATP, cc enzyme ca chu trnh Krebs,v.v... -Trong lysosome c cha nhiu enzyme thu phn (hydrolase). -Trong ribosome c cha cc enzyme cho qu trnh tng hp protein. - Trong bo tng cha nhiu loi enzyme, c bit c tt c cc enzyme ca qu trnh ng phn. - Trn mng t bo cng nh mng ca nhiu bo quan c nhng loi enzyme gip cho s vn chuyn mt s cht qua mng v mt s hot ng khc ca mng. 3.2. Isozyme 3.2.1. c tnh phn t Isoyme l cc dng phn t khc nhau ca cng mt enzyme, xc tc cho mt phn ng sinh ha. Mc d cng xc tc cho cng mt loi phn ng sinh ho, nhng do s tn ti cc dng phn t khc nhau nn c mt s tnh cht l, ho v min dch khc nhau. V d: lactatdehydrogengenase (LDH) l enzyme kh hydrogen thun nghch gia lactate v pyruvate c khi lng phn t 130.-140.KDa, cu trc gm 4 tiu n v (subunit), cc tiu n v ny c dch m t 2 gene khc nhau c k hiu l H v M (t ch ting Anh H- Heart v MMuscle).

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Hai loi chui polypeptide to nn 5 dng phn t khc nhau l: LDH1: HHHH LDH2: HHHM LDH3: HHMM LDH4: HMMM LDH5: MMMM Trong in di theo chiu t m sang dng th th LDH1 chy nhanh nht ri n LDH2, LDH3, LDH4 v LDH5 chy chm nht. 3.2.2. Vai tr chc nng ca cc isozyme. Cc kt qu nghin cu thu c ch ra rng, t l cc dng isozyme c th thay i tu theo tui tc, trng thi sinh l v bnh l. S tn ti ca nhiu dng phn t khc nhau ca cng mt enzyme l mt hin tng sinh hc rt quan trong c v mt l lun v thc tin. Chng hn s phn b khc nhau v LDH trong c th ca cc loi c xng sng, loi chui H ch yu tm thy c tim v loi M ch yu tm thy c xng. Bi v loi chui H thng b c ch bi pyruvat vi nng qu tha. Nhng acid pyruvic trong m tim c oxy ho hiu kh mt cch u n trong ty th cung cp nng lng cho m ny, v thng thng khng c s ng ca acid pyruvic. Tri li tuy m c xng c rt nhiu LDH dng M v m ny c nhiu hot ng bt thng gy nn nhng ng pyruvat nht thi, nhng dng M li khng b c ch bi acid pyruvic. 3.3. Cc enzyme di lp th (allosteric enzyme) v phc h enzyme (multienzyme). Trong c th sng cn gp nhng enzyme ngoi trung tm hot ng xc tc cn c mt loi trung tm khc lm nhim v iu chnh hot tnh ca enzyme cn gi l enzyme di lp th. Tuy trung tm iu chnh v trung tm xc tc c tc dng h tr nhau, nhng l hai loi cu trc khc bit nhau. Trong nhiu trng hp c th kho trung tm d lp th m vn gi c hot ng xc tc ca trung tm hot ng. Cc enzyme ny thng c cu to t cc n v nh kt hp vi nhau bng cc lin kt yu , nn rt mm do c th tn ti di nhiu dng khc nhau v c th bin i thun nghch t trng thi ny sang trang thi khc mt cch d dng. Mt trong nhng loi enzyme ny c nghin cu k nht l aspartate-transcarbamylase (ATC-ase) xc tc qa trnh tng hp carbamylaspartate. Enzyme ny gm hai n v nh xc tc v 3-4 n v nh iu ho.

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Ngoi enzyme di lp th cn gp phc h a enzyme (multienzyme). l nhng phc hp gm nhiu enzyme c lin quan n nhau trong mt qu trnh chuyn ho nht nh. V d phc hp enzyme trong qu trnh to acetyl-CoA t acid puruvic c gi l phc h pyruvatedehydrogenase. Phc hp ny gm ba loi enzyme l pyruvate dehydrogengenase, dihydrolipoyl transacetylase v dihydrolipoyl dehydrogenase. 3.4. Nhng quan im y hc v protein 3.4.1. Protein l nhng phn chc nng ca c th. Ngoi vai tr l thnh phn chnh trong cu trc ca t bo v m, protein cn c nhiu chc nng phong ph khc quyt nh nhng c im c bn ca s sng nh s truyn t thng tin di truyn, s chuyn ho cc cht l cc enzyme, cc khng th chng li bnh tt, cc hormon dn truyn cc tn hiu trong t bo v.v... u c bn cht l cc protein. 3.4.2. Hnh thnh chc nng mi trn c s cu trc protein. S pht trin ca sinh hc phn t da trn l thuyt trung tm DNA RNA Protein. Nh vy, s bin i DNA s dn n s bin i cu trc ca phn t protein v do chc nng sinh hc ca n s b bin i ko theo nhng thay i c lin quan n ton b c th. Trong qu trnh tin ho ca sinh vt s hnh thnh v thch nghi mt chc nng mi din ra mt giai on lch s lu di. S xut hin mt protein mi bin dng (mt hot tnh hoc t bin cu trc) thng lun i km bnh tt. 3.4.3. S xut hin cc protein bnh l. Y hc l ngnh khoa hc v s sng, ngy nay vi tin b ca khoa hc, nhng hiu bit v bnh l mc phn t vt ra khi gii hn ca gii phu t bo hoc c quan. Sinh hc phn t ra i to ra cuc cch mng trong cc quan nim v bnh. T , s pht trin ca bnh hc phn t lun lun i km vi sinh hc phn t. S bin i cu trc ca mt protein hay s xut hin cc enzyme c cu trc bt thng u do yu t di truyn gy nn. Da theo cc biu hin di truyn ngi ta chia cc protein bnh l ra lm hai loi ln: a) Nhng bin i v s lng ca protein: l s thay i do s tng hoc gim protein no , thm ch xut hin nhng protein m t bo bnh thng khng tng hp mt cch thng xuyn. Nhng protein ny vn c cu trc bnh thng v nh vy khng c nhng bin i ca gene cu trc. Nhng lch lc ny do ri lon qu trnh iu ho sinh tng

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hp protein. Do protein vn c cu trc bnh thng m ch thay i v s lng nn chng vn c chc nng bnh thng v ch thay i v mc hot ng. Trong trng hp l protein enzyme th nhng lch lc v s lng enzyme s dn n nhng ri lon dy chuyn chuyn ho. b) Nhng bin i v cht lng protein: l nhng ri lon v cu trc protein do gene bi bin i, dn n cu trc protein thay i ko theo s thay i chc nng sinh hc ca protein . V d, s bin i cu trc ca hemoglobin (Hb) l protein c chc nng vn chuyn oxygen trong mu dn n bnh thiu mu, hay nh bnh thiu mu do hng cu hnh li lim... 3.4.4. Cu trc v chc nng ca protein min dch. Tham gia vo h thng min dch c nhiu c quan, nhiu loi t bo v c bit nhiu loi protein thc hin cc chc nng ring bit to nn hiu qu min dch c hiu v khng c hiu. Cc protein min c nhc n nhiu hn c l cc khng th, b th v cc cytokine. a) Cc khng th (antibody). Tt c cc phn t khng th c chng minh l cc globulin c chc nng min dch (vit tt l Ig: Immunoglobulin) v c bn cht l glycoprotein. Cc khng th c chia thnh 5 lp. Tu theo cu trc v chc nng min dch l IgG, IgA, IgM, IgD, IgE. Cu trc ca phn t khng th c hnh thnh t hai loi chui polypeptide l chui nng (k hiu: H=Heavy chain) c khi lng phn t t 53-59 kDa v chui nh (k hiu:L=Light chain) c khi lng phn t 22-26 kDa. C bn chui c gn vi nhau bng cu disunfid (S-S). Trung tm hot ng l phn lin kit vi khng nguyn (hnh 1.2) nm vng tn cng N ca chui nng v chui nh c cu trc ch khong 8-10 amino acid. Cc phn t Ig c c tnh hot ng min dch theo hai chc nng: - C kh nng lin kt vi khng nguyn t nht hai v tr tip nhn i vi khng nguyn nh s bin i k diu ca phn tn cng NH2 trn phn t khng th. - Phn tn cng COOH ca phn t khng th c kh nng thc hin mt s ln cc hot ng sinh hc di nh hng ca s lin kt vi th th trn b mt ca t bo. Tt c cc khng th u c cng mt cu trc phn t nhng khc nhau mc ca vng lin kt vi khng nguyn. Nhn chung phn t khng th c chia lm hai phn: phn Fab l phn lin kt vi khng nguyn, phn Fc l phn d kt tinh phn ng vi cc t bo ca h thng min dch qua th th ca cc t bo. Dng

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enzyme papain hay pepsin c th ct khng th thnh hai mnh Fab v Fc, hoc F(ab)2 tng ng.chui nh V tr lin kt Papain ct Pepsin ct

chui nng

V tr lin kt

H nh 1.2

Cu trc chung ca phn t khng th (Ig)

b) B th (complement). L nhng protein huyt tng phn ng vi nhau nhm tn cng cc dng tc nhn gy bnh. H thng b th bao gm khong 40 protein c chc nng p ng min dch, chng vi sinh vt v p ng vim. V chc nng, cc khng th tng tc c hiu vi tc nhn truyn nhim bnh, cn h thng b th c c nh ln tt c cc khng th thc hin chc nng min dch. Cc thnh phn ca b th tng tc gia chng vi nhau v cc yu t khc ca h thng min dch. c) Cc cytokine. L ton b cc phn t c tit ra bi cc t bo ca h thng min dch, tham gia vo hot ng tn hiu gia cc t bo trong hot ng p ng min dch. Tt c cc cytokine u c bn cht protein hay glycoprotein v c phn loi nh sau: - Cc interferons (IFN): c cc dng -IFN, -IFN v -IFN, c chc nng ngn nga ca mt s virus gy bnh. - Cc interleukin (IL): c cc dng t IL1 n IL 13, chng c nhiu chc nng, nhng ch yu l kim tra s bit ho v sinh sn t bo. - Cc yu t kch thch qun lc (CSF): c chc nng kim tra s phn chia v sinh sn ca cc t bo ngun v cc t bo mu s khai.

18

- Cc cht dn truyn sinh hc (mediator): l nhng protein ca giai oi p ng min dch cp tnh. 3.4.5. Cu trc v chc nng ca protein vn chuyn. Trong c th c nhng protein lm nhim v vn chuyn nh hemoglobin, mioglobin, hemocianin vn chuyn O2, CO2 v H+ i khp cc m, cc c quan trong c th. Ngoi ra cn c nhiu protein khc nh lipoprotein vn chuyn lipid, ceruloplasmin vn chuyn ng (Cu) trong mu v.v...Mt trong nhng protein lm nhim v vn chuyn c nhc n nhiu nht l hemoglobin. Phn t c cu to t bn tiu n v (subunit), hai tiu n v v hai tiu n v .

Hnh 1.3

Cu trc ca phn t hemoglobin

Tiu n v ca Hemoglobin

H nh 1.4 So snh cu trc tiu n v ca hemoglobin vi leghemoglobin v myoglobin.

19

Mi tiu n v ni vi mt heme bng lin kt khng phi cng ho tr. Khi so snh vi protein cng chc nng nh myoglobin c v leghemoglobin thc vt l nhng protein c cu trc ch mt tiu n v (monomer) thy rng cc tiu n v cu trc kh ging nhau (hnh: 1.3, 1.4 ) 3.4.6. Cu trc chc nng v vai tr ca lectin. Lectin l nhng protein hay glycoprotein khng phi ngun gc min dch, lectin c kh nng ngng kt vi nhiu loi t bo, cng nh nhiu loi ng hoc cc hp cht cha ng c tnh cht chn lc. Hu ht lectin c cu trc bc 4, vi khi lng phn t giao ng trong phm vi kh rng t hng ngn cho n hng trm ngn Dalton. V d: lectin t r cy Urtica dioica (h gai Urticaceae) c Mr=8,5 KDa trong khi loi sam bin chu (Tachypleus tridentatus) c Mr=700.KDa. V chc nng, ngi ta thy rng mc d lectin khng phi l khng th chng li tc nhn gy bnh nhng chng c vai tr bo v c th nh tng tc vi mng t bo v gy ngng kt t bo ca chng. H khng nh rng lectin c kh nng gn cc t bo vi khun v khng nguyn l vi cc i thc bo, do vy m vi khun v khng nguyn l b o thi ra khi c th. Ngoi ra c nhng lectin cn c kh nng kch thch s phn chia v bit ho t bo. ng thi ngi ta cng pht hin c nhiu lectin c c hot tnh ca enzyme, v d lectin hot tnh kh mnh, c tch ra t ht u mng, c khi lng phn t khong 16.KDa c c hot tnh ca enzyme -galactosidase. 3.4.7. Nhng chc nng khc ca protein. Trong c th ngoi cc protein m nhn chc nng xc tc nh enzyme, chc nng vn chuyn nh hemoglobin, mioglobin, lipoprotein, v chc nng bo v nh cc khng th min dch, cc protein c t nh enzyme nc rn, lectin v.v..., protein cn tham gia nhiu chc nng quan trng khc nh: - Cc protein lm nhim v kch thch iu ho qu trnh trao i cht nh cc hormon - Cc protein lm nhim v cu trc nh v virus, mng t bo, colagen da, fibrolin t - Cc protein lm nhim v co rt nh myosin, actin si c - Cc protein lm nhim v d tr nh casein ca sa, ovalbumin ca trng, v.v...

20

CU HI N TP 1. Nu nhng c trng chung v ngha khoa hc thc tin ca nhm cht protein. 2. Phn loi v chc nng sinh hc ca protein? TI LIU THAM KHO 1.Trn Th n, i Duy Ban, Nguyn Hu Chn, nh H, L c Trnh. 1980. Ho sinh hc. NXB Y hc 2.Phm Th Trn Chu, Trn Th ng. 1999. Ho sinh hc. NXB Gio dc 3.Phm Th Trn Chu, L Minh Chu, Lm Chi, Nguyn Ln Dng, nh H, L Ngc T. 1983. Nhng hiu bit mi v enzim. Tp 8. NXB KH & KT H ni. 4. inh H, i Duy Ban, 1977. Sinh hc phn t v cuc cch mng trong sinh hc, NXB KH& KT. H ni 5. Ngc Lin. 2004. Min dch hc c s. NXB i hc Quc gia H ni (in ln th 2). 6.Fersht A.,1998, Structure and Mechanism in Protein Science, W. H. Freeman, 3rd Rev Edit. 7.Lehringer A.L., 2004. Principle of Biochemistry, 4th Edition. W.H Freeman, 2004 8. Liebler D.C., 2002. Introduction to proteomics. Humana Press Inc. Totuwa, New Jersey. 9. Lodish H., 2003. Molecular Cell Biology. 5th ed.W.H Freeman. 10. Virella G.,1998. Introduction to medical immunology, Marcel Dekker, Inc. New York. Basel. Hong kong. 4th. ed.

21

Chng 1

Trao i cht v nng lng sinh hc1.1. Khi nim chung v trao i cht Mi c th sng u tn ti trong mi trng v lin h mt thit vi mi trng . Hin tng c th ly mt s cht t mi trng kin to nn sinh cht ca mnh v thi ra ngoi nhng cht cn b c gi l s trao i cht. S trao i cht gii v sinh khc vi gii hu sinh. gii v sinh, trao i cht lm cho cc cht hu c v v c b phn hu. V d, vi (canxi carbonate) b xi mn v H2CO3 c trong nc tc dng vi vi thnh canxi bicarbonate, m b i ho thnh mt s cht khc l do tc dng vi oxy. th gii sinh vt, mi c th sng lun lun trao i cht vi mi trng, ly thc n vo chuyn ho thnh cc cht s dng cho c th v thi ra ngoi cc cht cn b. Qu trnh c thc hin l do cc bin i ho hc lin tc xy ra trong c th. Ton b cc bin i ho hc c gi l s trao i cht. Qu trnh trao i cht gm nhiu khu chuyn ho trung gian. Mi chuyn ho l mt mt xch ca mt trong hai qu trnh c bn: ng ho v d ho. ng ho v d ho l hai qu trnh i lp, nhng li thng nht vi nhau trong mt c th: chng xy ra ng thi v lin quan mt thit vi nhau. Cc cht c tng hp nn trong qu trnh ng ho l nguyn liu cho qu trnh d ho (v d gluxit l sn phm ca qu trnh quang hp, l nguyn liu cho qu trnh h hp). Nng lng gii phng ra trong qu trnh d ho c s dng mt phn cho qu trnh tng hp. 1.2. Nng lng sinh hc H thng sng cn nng lng chuyn ng, ln ln, tng hp cc phn t sinh hc v vn chuyn ion, phn t qua mng. Cc c th ly nng lng t mi trng sng v s dng nng lng thc hin cc qu trnh sng c hiu qu. nghin cu nng lng sinh hc i hi phi c hiu bit v nhit ng hc, mt s nh lut, nguyn l m t ngun, trao i nhit, nng lng v vt cht trong h thng nghin cu.

1

Nhit ng hc cho chng ta xc nh qu trnh ho hc v phn ng c th t xy ra hay khng. Mc d nhit ng hc l khi nim phc tp, nhng n da trn ba nh lut tng i n gin v d hiu. Mt vi nguyn l ca nhit ng hc c bn c a ra trong chng ny bao gm phn tch ngun nhit, sn sinh entropy, hm nng lng t do v mi lin quan gia entropy v thng tin. Chng ny cng cp n ATP v nhng hp cht cao nng khc. Khi nim v nhit ng hc c bn Bt k s quan tm no ca nhit ng hc cng phi phn bit gia h thng v mi trng. H thng l mt phn ca v tr m chng ta quan tm, trong khi mi trng l gm tt c nhng g cn li. C ba trng thi c bn: h thng c lp, h thng ng v h thng m. H thng c lp: Khng c s trao i cht v nng lng vi mi trng. H thng ng: C trao i nng lng, nhng khng c trao i cht vi mi trng. H thng m: C trao i cht v nng lng vi mi trng. C th sng l h thng m in hnh c trao i cht (dinh dng v sn phm thi ra) v nng lng (nhit t trao i cht) vi mi trng. nh lut 1: Nhit, cng v cc dng nng lng khc Trc y trong s pht trin ca nhit ng hc ngi ta cho rng nhit c th bin i thnh nhng dng nng lng khc v tt c cc dng nng lng mt cch c bn c th bin i thnh mt s dng khc. nh lut 1 ni rng: tng nng lng ca mt h thng c lp l khng thay i. Cc nh nhit ng hc m phng thnh mt hm ton hc nghin cu s bin i nhit v s dng cng trong nhng h thng nhit ng hc. Hm ny c gi l nng lng ni nng, thng k hiu l E hoc U. Nng lng ny ch ph thuc vo trng thi hin ti ca mt h thng v v vy c coi l hm trng thi. Nng lng ni nng khng ph thuc vo h thng xy ra nh th no v v vy khng ph thuc vo ng hng. Ni mt cch khc l chng ta c th thay i h thng bng bt c con ng no v cho n khi no h thng tr v trng thi ban u, nng lng ni nng s khng thay i. Nng lng ni nng, E ca h thng c th thay i nu ngun nng lng vo hoc ra khi h thng dng nhit hoc cng cho qu

2

trnh no bin i mt trng thi ny (1) sang mt trng thi khc (2) thay i nng lng ni nng l: E = E2 - E1 = q + w (1.1) q l lng nhit c h thng hp th t mi trng w l cng thc hin trn h thng do mi trng Cng c hc c nh ngha l s chuyn ng t ch ny n ch khc, gy ra do s dng lc. C hai phi xy ra cng mi c thc hin. V d: Mt tu ch khch cha y khch nhng khng di chuyn, theo nh ngha nhit ng hc cng khng c thc hin. Trong h thng ho sinh hc v ho hc cng thng lin quan vi p sut v th tch ca h thng. Cng c hc c xc nh w = -P V Trong P l p sut, V l s thay i th tch, V = V2-V1 Cng c th c thc hin nhiu dng: c hc, in, t v ho hc. E, q, w phi c cng n v: calorie (cal) v kilocalorie (kcal) c s dng theo truyn thng, nhng theo n v SI: Joule c ngh nn dng. Enthalpy: Hm c nhiu tin li cho h thng sinh hc Nu nh ngha cng c gii hn bi cng c hc, trong trng hp ny E ch l thay i nhit th tch khng i. V vy nu V khng i, cng khng c thc hin. E = q. V vy E l mt nh lng rt tin li trong qu trnh th tch khng thay i. E khng cn thit bng bin i nhit. V l do ny cc nh ho sinh hc, ho hc xc nh mt hm c bit ph hp cho qu trnh p sut khng i. N c gi l enthalpy, H c nh ngha: H = E + PV (1.2) Nu p sut khng thay i chng ta c: H = E + P V = q + w + P V = q - P V + P V = q (1.3) R rng H tng ng vi bin i nhit trong qu trnh p sut khng i. V cc phn ng ho sinh thng xy ra trong th lng hoc rn hn l th kh nn thay i th tch l nh v enthalpy v nng lng ni nng thng l nh nhau. thun li khi so snh cc ch s nhit ng hc ca cc phn ng khc nhau th ngi ta xc nh iu kin tiu chun. Mt dung dch ho tan trng thi tiu chun, thng s dng n v n gin l nng 1M. Enthalpy, nng lng ni nng v nhng nh lng nhit ng hc khc thng a ra hoc xc nh cho nhng iu kin tiu chun v c k hiu l H0, E0... Enthalpy thay i cc qu trnh ho sinh c th c xc nh bng vic o nhit hp th (hoc to ra) bng mt calorimeter.

3

Mt khc cho bt k qu trnh no A B trng thi cn bng, s thay i enthalpy trng thi tiu chun c xc nh t s ph thuc vo nhit v h s cn bng: d (ln Keq) 0 H = ---------------(1.4) d (1/T)

30 20 10 RlnKeq 0 -10 -20 -30

54,5 C -3,21-(-17,63) =14,42 3,04-3,067 =-0,0272,98 3,00 3,02 3,04 3,06 3,08 1000 -1 (K ) T 3,10

o

Hnh 1.1 S thay i enthapy, H0 ca 1 phn ng c xc nh dc ca s RlnKeq ngc vi 1/T. minh ha phng php ny nhng gi tr hai bn ca 327K (54,50C) c nu ra. S liu c s dng tnh H0 54,50C.

y R l hng s kh = 8.314 J/mol K V d: trong s bin tnh nhit ca protein chymotripsinogen (qu trnh thun nghch). Trng thi nguyn thu (N) Trng thi bin tnh (D) Keq = D / N John F. Brandts o hng s cn bng cho s bin tnh ca mt s protein mt s gi tr pH v nhit khc nhau (bng 1.1). Gi tr H0 c ngha g i vi bin tnh ca protein? Gi tr dng ca H0 biu din s b gy lin kt hydro cng nh gii phng

4

nhng nhm a nc t bn trong phn t protein ban u trong qa trnh bin tnh, nh vy s nng nng lng ca dung dch protein. Bng 1.1 Cc ch s nhit ng hc cho s bin tnh proteinProtein (v iu kin) Chymotrypsinogen (pH 3; 250C) b- Lactoglobulin (5 M urea; pH 3; 250C) Myoglobin (pH 9; 250C) Ribonuclease (pH 2,5; 300C) H0 kJ/mol 164 -88 180 240 S0 kJ/mol.K 0,440 -0,300 0,400 0,780 G0 kJ/mol 31 2,5 57 3,8 Gp kJ/mol.K 10,9 9,0 5,9 8,4

nh lut th hai v entropy: nh lut th hai ca nhit ng hc c m t v th hin trong nhiu cch bao gm nhng im sau: H thng c xu hng tin t trng thi trt t sang trng thi khng trt t (tng entropy). Entropy ca h thng + mi trng l khng i bi qu trnh thun nghch. Entropy ca h thng + mi trng tng do qu trnh khng thun nghch. Tt c cc qu trnh xy ra trong t nhin hng ti trng thi cn bng, l trng thi nng lng nh nht. Mt s im ca nh lut 2 dn n khi nim entropy, l thc o s mt trt t ca h thng, trong trng thi mt trt t l trng thi c entropy cao. Entropy c th c xc nh theo mt vi cch. Nu W l s cch sp xp thnh phn ca mt h thng m khng thay i nng lng ni nng hoc enthalpy ( l s lng ca trng thi knh hin vi c a ra nhit , nh sng v tng vt cht). Entropy c tnh: S = klnW (1.5) k l hng s Boltzmann = 1,38.10-23 J/K nh ngha ny tin li cho tnh ton thng k, nhng dng ph bin hn lin quan entropy n s bin i nhit trong mt qu trnh l: dQ dSthun nghch = (1.6) T

5

dSthun nghch l thay i entropy ca h thng trong mt qu trnh thun nghch, q l nhit c bin i, T l nhit s bin i nhit xy ra. nh lut 3: Ti sao0 tuyt i quan trng nh vy? nh lut 3 ca nhit ng hc ni rng: entropy ca bt k cht no hon ton c trt t, tinh th phi tin n 0. nhit tin n 0 K v T= 0 K entropy chnh xc = 0. Da trn iu ny c kh nng thit lp mt h thng t l entropy tuyt i, s lngT

S = Cp dlnT0

(1.7)

Cp: kh nng bin i nhit p sut khng i. Kh nng nhit ca mt cht l tng s nhit ca 1M c th d tr khi nhit ca cht c nng ln 1 . i vi qu trnh p sut khng i n c m t bng ton hc dH Cp = (1.8) dt Nu kh nng nhit c th c tnh tt c nhit gia 0 K v nhit no , entropy tuyt i c tnh i vi qu trnh sinh hc thay i entropy c nhiu tin li hn entropy tuyt i. Thay i entropy cho mt qu trnh c th c tnh nu thay i enthalpy v nng lng t do bit. Nng lng t do: Mt gi thuyt nhng l cng c tin li Mt cu hi quan trng i vi nh ho hc, c bit i vi nh ho sinh hc l: Phn ng s xy ra theo hng t phi sang tri? Gibbs, mt trong nhng ngi xy dng nn nhit ng hc nhn thy cu tr li cho cu hi ny nm trong s so snh thay i enthalpy v thay i entropy mt nhit no , nng lng t do Gibbs c nh ngha nh sau: G = H - TS (1.9) Cho bt k qu trnh A B nhit v p sut khng i. S thay i nng lng t do c tnh: G = H - T S (1.10) Nu G gn = 0 qu trnh cn bng, khng i theo hng thun hoc ngc li khi G = 0 S = H/T v thay i enthalpy v entropy l cn bng chnh xc. Bt k qu trnh vi G khc 0 thc hin t ng n trng thi cui cng

6

c nng lng t do thp. Nu G m th qu trnh xy ra theo hng t tri sang phi. Nu G 0 phn ng xy ra theo hng ngc li (k hiu v gi tr ca G cho php xc nh qu trnh s xy ra nhanh nh th no). Nu qu trnh c G m th qu trnh t xy ra, nu G dng th qu trnh khng t xy ra (hay t xy ra theo chiu nghch). Thay i nng lng t do tiu chun Thay i nng lng t do G cho bt k phn ng no ph thuc vo cht tham gia phn ng v sn phm phn ng v cng b nh hng bi iu kin phn ng k c nht , p sut v pH v nng ca cht phn ng v sn phm. Nu thay i nng lng t do cho mt phn ng l nhy cm vi iu kin ho tan, iu g c bit c ngha cho s thay i nng lng t do trng thi tiu chun. tr li cho cu hi ny xem xt mt phn ng gia hai cht A v B to nn sn phm C v D A + B C + D (1.11) Thay i nng lng t do cho nng khng trng thi tiu chun l [C] [D] G = G0 + RT ln [A] [B] (1.12)[C] [D] [A] [B] = Keq

trng thi cn bng G = 0 Chng ta c G0 = - RT ln Keq (1.13) hoc logarit c s 10 G0 = - 2,3 RT/ log 10 Keq (1.14) N c bin i Keq =10 - G/2,3 RT (1.15) Trong bt c dng no mi lin h cho php xc nh thay i nng lng t do tiu chun cho bt k qu trnh no nu hng s cn bng c bit. Quan trng hn, iu ni rng cn bng thit lp cho mt phn ng trong dung dch l mt hm ca s thay i nng lng t do tiu chun cho qu trnh, ngha l G0 l cch vit khc ca hng s cn bng.

7

V d hng s cn bng xc nh bi Brandts mt s nhit vi s bin tnh ca chymotrypsinogen c th c dng tnh s thay i nng lng t do cho qu trnh bin tnh. V d h s cn bng 54,50C l 0,27, nh vy G0 = - (8,314 J/mol. K (327,5K) ln (0,27) G0 = - (2,72 kJ/mol . ln (0,27) G0 = 3,56 kJ/mol K hiu dng ca G0 ngha l qu trnh bin tnh khng u th. Dng gp l dng bn ca protein 54,50C. Mt khc ln tng i nh ca G0 ngha l dng gp chim u th nh.

10 8 6 4 2

G, kj/mol

0 -2 -4 -6 -8 -10 50 52 54 56 Nhit Co

58

60

62

8

Hnh 1.2 S ph thuc ca bin tnh ca chymotrypsinogen

G0 vo nhit nhit trong qu trnh

Hnh 1.2 ch s ph thuc ca G0 vo nhit bin tnh pH = 3. Tnh c H0 v G0 ca s bin tnh chymotrypsin, c th tnh S0 s dng phng trnh (3.10). ( G 0 H 0 ) S0 = (1.16) T 54,50C (327,5 K) S0 = - (3560 - 533,000 J/mol) / 327,5 K S0 = 1,620J/mol.K Hnh 1.3 biu din s ph thuc ca S0 vo nhit bin tnh ca chymotrypsin pH = 3. Gi tr dng S0 ch rng dung dch protein tr nn khng trt t khi protein b bin tnh. So snh gi tr 1,62 kJ/mol.K vi gi tr S0 bng 1.1 ch ra rng gi tr hin ti cho chymotrypsin 54,50C l hon ton ln. ngha vt l ca ch s nhit ng hc cho s bin tnh ca chymotrypsin s r hn trong phn sau. ngha vt l ca c tnh nhit ng hc Nhng ch s nhit ng hc cho ta bit nhng hin tng sinh ho g? Cch tt nht tr li cu hi ny l mt ch s ring r (v d H hoc G ) khng c nhiu ngha. Mt gi tr dng H0 cho s bin tnh ca mt protein c th phn nh hoc l s gy cc lin kt hydro trong protein hoc s xut hin cc nhm a nc ra bn ngoi. Tuy vy s so snh mt s ch tiu nhit ng hc c th cung cp nhng hiu bit bn trong c ngha v mt qu trnh.2,4 2,3 2,2 2,1

G, kj/mol-K

2,0 1,9 1,8 1,7 1,6 1,5 1,4 52 54 56 Nhit Co

58

60

9

Hnh 1.3 S ph thuc ca S0 vo nhit trong qu trnh bin tnh ca chymotrypsinogen

nh hng ca nng n thay i nng lng t do thc t Phng trnh (3.12) ch ra rng thay i nng lng t do i vi mt phn ng rt khc nhau so vi gi tr trng thi tiu chun nu nng ca cht phn ng v sn phm khc vi nng hot ng (1M cho dung dch). Xem xt s thu phn ca phosphocreatine: Phosphocreatine + H2O Creatine + Pi Phn ng ny to nhit rt mnh v G0 370C l -42,8 kJ/mol Nng sinh l ca phosphocreatine, creatine v Pi thng l gia 1 mM v 10 mM. Cho rng nng 1 mM v s dng phng trnh (3.12) G cho thu phn phosphocreatine l

G = - 42,8 kJ/M + (8.314J/M) (310 K) ln

[0,001][0,001] [0,001]

G = - 60,5 kJ/M 370C s khc nhau gia trng thi tiu chun v nng 1 mM cho mt phn ng nh vy l khong -17,7 kJ/mol Tm quan trng ca cc qu trnh kt hp trong c th sng Nhiu phn ng cn thit gi t bo v c th chng li th nhit ng hc, l theo hng G dng. Trong c s tng hp ATP v nhng phn t cao nng khc v to nn gradient ion trong tt c t bo ng vt c v. Nhng qu trnh ny c thc hin theo hng bt buc nhit ng hc.

10

Bng s kt hp vi cc qu trnh c kh nng t xy ra nhiu qu trnh gn lin vi nhau c cp phn sau ca chng ny. Chng rt quan trng trong trao i cht trung gian, phosphoryl ho oxy ho v vn chuyn qua mng. Chng ta c th on nhng cp phn ng kt hp s xy ra t ng bng s thay i tng nng lng t do cho mi phn ng. V d, xem xt phn ng t qu trnh ng phn lin quan n s bin i phosphoenolpyruvic acid n pyruvat. S thu phn PEP gii phng nng lng v c s dng phosphoryl ho ADP thnh ATP, mt qu trnh m v mt nng lng khng t xy ra. 1.2.1 c tnh nng lng ca s trao i cht Nng lng ca cc qu trnh trao i cht (nng lng sinh hc) khc vi nng lng c thc hin trong bn cht khng sng ba c im sau y: c tnh th nht l s chuyn ho nng lng thnh cng v thnh nhng dng khc m khng km theo s chuyn ho s b nng lng ny thnh nhit nng. Xut pht t nguyn tc ny chng ta cn xem h thng sng nh l mt ng c ho ng hc ch khng phi l ng c nhit. c tnh th hai l vic gii phng nng lng trong cc qu trnh oxy ho sinh hc sinh ra t t, tng phn mt, trong mt chui di cc qu trnh k tip nhau cho n khi no tt c cc nguyn t hydro v cacbon u bin thnh cc sn phm cui cng l CO2 v H2O. V d s oxy ho mt phn t gam ng gii phng ra 686 kcal. Nu nng lng ny c gii phng ra cng mt lc th s gy ting n v h thng sng khng th s dng ton b nng lng trong mt khong thi gian ngn nh vy.

11

Giai on 1: B gy cc i phn t thnh cc n phn t

Protein Aminoacid

Polysaccharide ng n Glucose

Lipid Glycerin v acid bo

Giai on 2: Phn gii cc n phn t thnh Acetyl CoA, to ra ATP v NADH mc nht nh

Pyruvate

Acetyl CoA

Mng ty th Giai on 3: Oxy ha hon ton Acetyl CoA thnh H2O v CO2, gii phng mt lng ln NADH v ATP ty th

Chu trnh citric acid

Mng t bo sinh vt nhn chun

Hnh 1.4 Tin trnh gii phng nng lng ho hc trong s trao i cht c chia lm 3 giai on

c tnh th ba l nng lng ho hc c gii phng ra khi phn gii glucid, lipid v nhng hp cht cao phn t khc u c th c tch lu trong nhng hp cht tch tr nng lng c th, c gi l hp cht cao nng. Tin trnh ca vic gii phng nng lng ho hc c bn c chia lm 3 giai on (hnh 1.4): Giai on th nht cc hp cht cao phn t (tinh bt, glycogen, proteine, lipid...) b thu phn thnh cc cht c phn t b

12

(monosaccharide, amino acid, axit bo, glycerine...). Nng lng gii phng ra trong giai on ny khng ng k, ch bng gn 1% d tr nng lng t do ca cc cht ny c gii phng ra di dng nhit. Giai on th hai l qu trnh ng phn, oxy ho cc axit bo v cc amino acid. Nng lng gii phng ra trong giai on ny gn bng 1/3 nng lng t do d tr trong cc cht . Sn phm chnh ca giai on ny l acetyl-CoA, -xetoglutaric acid v oxaloaxetic acid. Giai on th ba l oxy ho tip tc cc cht trn trong chu trnh Krebs. Khong 2/3 nng lng c gii phng ra giai on ny. 1.2.2 Cc hp cht cao nng Nhng hp cht cao nng: Tt c s sng trn tri t ph thuc vo nng lng mt tri, trong nhng dng sng c mt h thng th bc v nng lng. Mt s tip nhn nng lng mt tri trc tip, mt s khc nhn nng lng t nhm trn trong nhng qu trnh tip theo. Nhng sinh vt hp thu nng lng nh sng trc tip c gi l c th t dng. Nhng c th ny d tr nng lng mt tri trong cc phn t hu c khc nhau. Nhng sinh vt s dng nhng phn t , gii phng nng lng d tr trong mt lot cc phn ng oxy ho kh c gi l sinh vt ho dng. Mc d khc nhau c hai loi u c c ch chung v ti sinh mt dng nng lng ho hc, nng lng c th c gii phng trong nhng phn ng to nhit thc hin cc qu trnh sng a dng (cn nng lng). Mt nhm nh cc phn t l cht trung gian chuyn nng lng t cc phn ng gii phng nng lng n cc phn ng cn nng lng ca c th. Nhng phn t ny l coenzyme dng kh, nhng hp cht phosphate c gi l cao nng nu chng gii phng ra nng lng t do c gi tr m ln khi thu phn ( G 0 c gi tr m ln hn -25 kJ/M). bng 1.2 sau y l danh sch nhng hp cht cao nng quan trng nht, nhng phn t nh phosphoric anhydric (ATP, ADP), enol phosphate (PEP), acyl phosphate (acetyl phosphate), guanidinophosphate (creatine phosphate). C nhng hp cht thioeste, nh acetyl CoA khng cha phospho nhng gii phng mt nng lng t do ln khi thu phn.

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Bng 1.2 Nng lng t do gii phng khi thy phn mt s cht cao nngCc cht G (kJ/mol)o

Cng tc cu to

Phosphoenolpyruvat (Pyruvate + Pi)

-62,2

3,5-Cyclic adenosin monophosphate (5-AMP)

-50,5

P

1,3-Bisphosphoglycerate (3-phosphoglycerate + Pi)

-49,6

Creatine phosphate (creatine + Pi) Acetyl phosphate (acetate + Pi) Adenosine-5 triphosphate (ADP + Pi)

-43,3

-43,3 -35,7

Adenosine-5 triphosphate ++ (ADP + Pi) (nng Mg qu cao)

-30,5

Adenosine-5 diphosphate (AMP + Pi)

-35,7

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Cc cht phosphate nng lng thp hn

G (kJ/mol)

o

Cng tc cu to

Glucose-1-P (glucose+ Pi)

-21,0

Fructose-1-P (fructose+ Pi)

-16,0

Glucose-6-P (glucose+ Pi)

-13,9

Glycerol-3-P (glycerol+ Pi)

-9,2

Adenosine-5 monophosphate (adenosin + Pi)

-9,2

Tng s nng lng chnh xc gii phng ra khi thu phn ph thuc vo nng , pH, nhit ... nhng gi tr G 0 khi thu phn nhng hp cht ny c gi tr dng ln hn ng k so vi nhng cht trao i khc. Chng c hai c im quan trng: Nhng cht phosphate cao nng (high- energy phosphate compounds) khng phi l cht d tr nng lng lu di, chng l nhng cht chuyn tip nng lng d tr, l cht mang nng lng t im ny sang im khc, t mt h thng ny n mt h thng khc. Nng lng hot ho c cung cp ng k t ATP khi thu phn nhm -phosphat. Nng lng lm gy lin kt O-P thng l 200-400 kJ/M, ln hn ng k so vi 30,5 kJ/M khi thu phn ATP. Cc nh ho sinh hc quan tm nhiu n nng lng gii phng thc t. Vai tr trung tm ca ATP trong nng lng sinh hc ATP cha hai pyrophosphoryl (hnh 1.4). Nhng phn t c lin kt anhydric, ADP, GTP, GDP v cc nucleoside triphosphate khc, nucleotide-ng nh UDP-glucose v pyrophosphate v c th hin nng

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lng t do G0 ln khi thu phn. Nguyn nhn ho hc ca gi tr G0 m ln l do s khng bn vng ca cht phn ng do s cng lin kt gy ra bi s y tnh in. S bn vng ca sn phm phn ng do s ion ho, s cng hng v nhng yu t entropy gy ra do thu phn v s ion ho tip theo. Mc d PEP, cyclic AMP, 13 DPG, phosphocreatine, acetylphosphate v pyrophosphate u c gi tr G0 ln hn, nhng ATP l duy nht nh v gia cc cht phosphate cao nng, (ATP c tng hp khi phn gii cc cht hu c) v cc cht nhn nng lng (khi cc cht ny c phosphoryl ho tham gia cc phn ng tip theo trong trao i cht). Ni mt cch khc ATP l mt xch ni lin hai qu trnh ngc nhau, l ng ho v d ho. Vic hnh thnh tt c cc hp cht cao nng khc cng xy ra do s tiu ph nng lng vn tch lu trong ATP. ADP c th nhn c phosphate v nng lng t cc phosphate cao nng. ATP cho c gc phosphate v nng lng i vi cc phn t c nng lng thp. Nh vy ATP c vai tr d tr nng lng cng nh tiu hao nng lng. Xt v c ch bin i v chuyn ho nng lng trong s phn gii ATP v cc hp cht cao nng tng t ATP ta thy nng lng cn thit thc hin phn ng ho hc c gii phng ra mt im, c th c chuyn n mt im khc, y nng lng c s dng mt cch trc tip. iu ny c ngha l trong c th sng khng nht thit phi tip xc vi nhau bng cch va chm (c trng cho ngoi c th sng) gia cc phn t cho v nhn nng lng.

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Adenosine AMP ADP ATP

Lin kt Phosphoric anhydride

ATP (adenosin-5-triphosphate)

Hnh 1.5 Chui ba gc phosphat ca ATP cha 2 lin kt pyrophosphat, c hai lin kt ny gii phng nhiu nng lng khi thu phn

Cu hi 1. Nng lng sinh hc? 2. Cc ng hng tng hp ATP trong thc vt ? 3. Cc dng hp cht cao nng, cho v d?

17

Chng 2

Amino acid - n v cu to ProteinI. Thnh phn tnh cht l- ho ca amino acid1.1. Thnh phn v cu to ca amino acid Protein l polymer ca cc amio acid ni vi nhau bng cc lin kt cng ho tr l lin kt peptide. Protein c th b thu phn to thnh cc amino acid t do bng nhiu phng php khc nhau. Ngi ta xc nh protein c cu trc t 20 loi amino acid khc nhau. Amino acid l cht hu c m phn t cha t nht mt nhm carboxyl (COOH) v t nht mt nhm amino (NH2), tr prolin ch c nhm NH (thc cht l mt acid imin). Trong phn t amino acid u c cc nhm COOH v NH2 gn vi carbon v tr . Hu ht cc amino acid thu nhn c khi thu phn protein u dng L- amino acid. Nh vy cc protein ch khc nhau mch nhnh, hay cn gi l chui bn (thng c k hiu: R).

Hnh: 2.1

Cng thc cu to chung ca cc amino acid

1.2. Phn loi amino acid 1.2.1. Cc quan im v phn loi amino acid. Hin nay c nhiu ngi phn loi amino acid theo nhiu kiu khc nhau, mi kiu sp xp u c ngha v mc ch ring. Tuy nhin, h u da trn cu to ho hc hoc mt s tnh cht ca gc R. V d: c ngi chia cc amino acid thnh 2 nhm chnh l nhm mch thng v nhm mch vng. Trong nhm mch thng li tu theo s c mt ca s nhm carboxyl hay s nhm amino m chia ra thnh cc nhm nh, nhm amino acid trung tnh (cha mt nhm COOH v mt nhm NH2); nhm amino acid c tnh kim (cha mt nhm COOH v hai nhm NH2); nhm amino acid c tnh acid (cha hai nhm COOH v mt nhm NH2). Trong nhm mch vng li chia ra thnh nhm ng vng hay d vng v.v... C ngi li da vo tnh phn cc ca gc R chia cc amino acid thnh 4 nhm: nhm khng phn cc hoc k nc, nhm phn cc nhng khng tch in, nhm tch in dng v nhm tch in m.

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y xin c gii thiu cch phn loi cc amino acid mt cch chung nht. Theo cch ny da vo gc R cc amino acid c chia lm 5 nhm: Nhm I. Gm 7 amino acid c R khng phn cc, k nc, l: glycine, alanine, proline, valine, leucine, isoleucine v methionine.

Hnh: 2.2

Cng thc cu to cc amino acid nhm I

Nhm II Gm 3 amino acid c gc R cha nhn thm, l phenylalanine, tyrosine v tryptophan.

Hnh: 2.3

Cng thc cu to cc amino acid nhm II

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Nhm III. Gm 5 amino acid c gc R phn cc, khng tch in, l serine, threonine, cysteine, aspargine v glutamine.

Hnh: 2.4

Cng thc cu to cc amino acid nhm III

Nhm IV. Gm 3 amino acid c R tch in dng, l lysine, histidine v arginine.

Hnh: 2.5

Cng thc cu to cc amino acid nhm IV

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Nhm V. Gm 2 amino acid c gc R tch in m, l aspartate v glutamate.

Hnh: 2.6

Cng thc cu to cc amino acid nhm V

1.2.2. Cc amino aicd thng gp. Cc amino acid thng gp l nhng amino acid thng c mt trong thnh phn ca cc loi protein. Chng c khong 20 loi v c thu nhn khi thu phn protein. Cc loi amino acid ny c tn gi, khi lng phn t v k hiu c trnh by trn bng 2.1. 1.2.3. Cc aminno acid khng thay th, hay cn thit. Cc amino acid c hnh thnh bng nhiu con ng khc nhau. Nh bit, trong phn t protein c khong 20 loi amino acid, tuy nhin trong c th ngi v ng vt khng tng hp c tt c cc loi m phi a t ngoi vo qua thc n. Nhng amino acid phi a t ngoi vo gi l cc amino acid khng thay th. Ngy nay ngi ta bit c c khong 8-10 loi amino acid khng thay th bao gm: Met, Val, Leu, Ile, Thr, Phe Trp, Lys, Arg v His, ngy nay ngi ta cn xem Cys cng l mt amino acid khng thay th. 1.2.4. Cc amino acid t gp. Ngoi cc amino acid thng gp trn, trong phn t protein i khi cn c mt s amino acid khc, l nhng loi t gp. Cc amino acid ny l dn xut ca nhng amino acid thng gp nh: trong phn t colagen c cha 4-hydrogenxyproline l dn xut ca proline, 5hydrogenxylysine l dn xut ca lysine v.v... Mt khc, mc d khng c trong cu trc protein, nhng c hng trm loi amino acid khc chng c th tn ti dng t do hoc lin kt vi hp cht khc trong cc m v t bo, chng c th l cht tin thn hay l cc sn phm trung gian ca qu trnh chuyn ho trong c th.

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Bng 2.1 Cc amino acid thng gpTn amino acid Glycine Alanine Proline Valine Leucine Isoleucine Methionine Phenylalanine Tyrosine Tryptophan Serine Threonine Cysteine Aspargine Glutamine Lysine Histidine Arginine Aspartate Glutamate Tn amino acid gi theo danh php ho hc -aminoacetic -aminoprpionic -pirolidincarboxylic -aminoiaovaleric -aminonoisocaproic -amino- -metylvaleric -amino- -metyltiobutiric -amino- -phenylpropionic -amino- -hydrogengenxypheny propionic -amino- -idolylpropionic -amino- -hydoxypropionic -amino- -hydrogengenxybutiric -amino- -tiopropionic amid ca aspartate amid ca glutamate , diaminocaproic -amino- -imidazolpropionic -amino- -guanidinvaleric -aminosucinic -aminoglutarate Tn vit tt Gly Ala Pro Val Leu Ile Met Phe Tyr Trp Ser Thr Cys Asn Gln Lys His Arg Asp Glu K hiu G A P V L I M F Y W S T C B Q K H R D E Khi lng (Mr) 75 89 115 117 131 131 149 165 181 204 105 119 121 132 146 146 155 174 133 147

1.3. Mu sc v mi v ca amino acid Cc amino acid thng khng mu, nhiu loi c v ngt kiu ng nh glycine, alanine, valine, serine histidine, triptophan; mt s loi c v ng nh isoleucine, arginine hoc khng c v nh leucine. Bt ngt hay cn gi l m chnh l mui ca natri vi acid glutamic (monosodium glutamate). 1.4. Tnh tan ca amino acid Cc amino acid thng d tan trong nc, cc amino acid u kh tan trong alcohol v ether (tr proline v hydrogenxyproline), chng cng d ho tan trong acid v kim long (tr tyrosine).

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1.5. Biu hin tnh quang hc ca amino acid Cc amino acid trong phn t protein u c t nht mt carbon bt i (tr glycine) v th n u c biu hin hot tnh quang hc, ngha l c th lm quay mt phng ca nh sng phn cc sang phi hoc sang tri. Quay phi c k hiu bng du (+), quay tri c k hiu bng du (-). Gc quay c hiu ca amino acid ph thuc vo pH ca mi trng. Tu theo s sp xp trong cu trc phn t ca cc nhm lin kt vi carbon bt i m cc amino acid c cu trc dng D hay L (hnh 2.7) gi l ng phn lp th. S ng phn lp th c tnh theo 2n (n l s carbon bt i)

Hnh 2.7

ng phn lp th ca alanine

Hu ht cc amino acid khc hp th tia cc tm bc sng ( ) khong t 220 - 280 nm. c bit cng nng 10-3M, trong bc sng khong 280 nm tryptophan hp th nh sng cc tm mnh nht, gp 4 ln kh nng hp th ca tyrosine (hnh 2.8) v phenylalanine l yu nht. Phn ln cc protein u cha tyrosine nn ngi ta s dng tnh cht ny nh lng protein

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(nm)Hnh 2.8 Ph hp th nh sng cc tm ca triptophan v tyrosine

1.6. Tnh lng tnh ca amino acid Trong phn t amino acid c nhm carboxyl -COOH nn c kh nng nhng proton (H+) th hin tnh acid, mt khc c nhm amin- NH2 nn c kh nng nhn proton nn th hin tnh base. V vy amino acid c tnh cht lng tnh. Trong mi trng acid, amino acid dng cation (tch in dng), nu tng dn pH amino acid ln lt nhng proton th nht chuyn qua dng lng cc (trung ho v in), v tip tc tng pH amino acid s nhng proton th hai chuyn thnh dang anion (tch in m). V vy i khi ngi ta coi n nh mt di-acid.

cationHnh 2.9

lng cc

anion

Tnh lng tnh ca amino acid

Tng ng vi phn ly H+ ca cc nhm COOH v NH3+ c cc tr s pK1 v pK2 (biu th phn ly ca cc nhm c 1/2). T ngi ta xc nh c pHi (pI= pH ng in) = pK1 + pK2 / 2. V d: khi ho tan glycine vo mi trng acid mnh th hu nh glycine u dng

28

cation. Nu tng dn lng kim, thu c ng cong chun . Trn ng cong chun thy rng: glycine ln lt nhng 2 proton trc tin chuyn sang dang lng tnh v sau cng chuyn thnh dng anion

phn ly ca H+

Hnh 2.10

ng cong chun ca glycine nng 1 M 25OC

Tng ng phn ly ca nhm COOH c mt na c tr s pK1= 2,34 v phn ly ca NH3+ c mt na c tr s pK2= 9,60. Nh vy ta c 2,34 + 9,60 pHi = = 5,97 2 Mt khc ti pK1 + 2 s phn ly H+ ca nhm COO- glycine l 99%, ch 1% dng COOH v pK2 -2 dng NH3+ l 99%, ch 1% dng NH2. Nh vy trong vng pH t pK1 + 2 n pK2 -2, phn t glycine ch yu dng lng tnh v kt qu ta c mt vng ng in. Ngoi ra cc amino acid trong gc R c thm nhm COOH hay NH2 s phn ly ca chng s c thm mt tr s phn ly na-pKR (xem bng 2.2)

29

1.7. Cc phn ng ho hc ca amino acid Cc amino acid u c nhm NH2 v COOH lin kt vi C , v vy chng c nhng tnh cht ho hc chung. Mt khc cc amino acid khc nhau bi gc R, v vy chng c nhng phn ng ring bit. Ngi ta chia cc phn ng ho hc ca amino acid thnh 3 nhm:Bng: 2.2 Cc tr s pK ca cc amino acid thng gp Tn cc amino acid Glycine Alanine Proline Valine Leucine Isoleucine Methionine Phenylalanine Tyrosine Tryptophan Serine Threonine Cysteine Aspargine Glutamine Lysine Histidine Arginine Aspartate Glutamate pK1(ca COOH) 2,34 2,34 1,99 2,32 2,36 2,36 2,28 1,83 2,20 2,38 2,21 2,11 1,96 2,02 2,17 2,18 1,83 2,17 1,88 2,19 Cc tr s pK pK2(ca NH+3) 9,60 9,60 10,96 9,62 9,60 9,68 9,21 9,13 9,11 9,39 9,15 9,62 10,28 8,80 9,13 8,95 9,17 9,04 9,60 9,67 pKR(ca R) pI 5,97 6,01 6,48 5,97 5,98 6,02 5,74 5,48 5,66 5,89 5,68 5,87 5,07 5,41 5,65 9,74 7,59 10,76 2,77 3,22

10,07

8,18

10,53 6,00 12,48 3,65 4,25

a) Phn ng ca gc R. Do cc amino acid c cu to gc R khc nhau, nn ngi ta c th dng xc nh tng amino acid ring r nh phn ng c trng ca n, v d phn ng oxy ho kh do nhm SH ca cysteine, phn ng to mui

30

do cc nhm COOH hay NH2 ca glutamte hay lysine, phn ng to ester do nhm OH ca tyrosine v.v... b) Phn ng chung. L phn ng c s tham gia ca c hai nhm - COOH v - NH2 . Khi phn ng vi ninhydrin trong iu kin un nng to thnh CO2 , NH3 aldehyde v nihydrin bi kh, cui cng to nn sn phm c mu xanh tm. c) Phn ng ring bit C th chia cc phn ng ring bit theo hai nhm - COOH v NH2 -Cc phn ng ca nhm - COOH . Ngoi cc phn ng ca nhm COOH thng thng to ester, to amid, to mui ...th n cn c nhng phn ng c trng khc nh c th b kh thnh hp cht ru amino di s xc tc ca NaBH4. R-NH2CH-COOH R-NH2CH-CH2OH Nhm COOH c th to thnh phc aminoacyl-adenylate trong phn ng hot ho amino acid tng hp protein, hay c th loi CO2 gp rt nhiu trong qu trnh thoi ho amino acid, to cc dn xut amin c hot tnh sinh hc cao nh histamine, sevtnine. - Cc phn ng ca nhm - NH2 . Nhiu phn ng ca nhm amino c dng xc nh cc ch tiu ca amino acid nh: nh lng amino acid ngi ta cho phn ng vi HNO2 gii phng N2 v nh lng nitrogen R-NH2CH-COOH + HNO2 R-OHCH-COOH +N2 +H2O nh lng amino acid ngi ta cho phn ng vi formaldehyde to thnh base schif. R-CH-COOH R-CH-COOH NH2 + HCHO N = CH2 + H2O + H Sau dung NaOH (hoc KOH) chun nhm COOH ca aminoacid xc nh amino acid u N-tn cng ngi ta cho tc dng vi 24 dinitrofluobenzen (phn ng sanger) hay phenyliothiocyanate (phn ng Edman). Sau xc nh amino acid N-tn cng tch bit khi chng dng dn xut vi hai loi ho cht trn.

II. Thu nhn amino acid bng thy phn protein2.1. Thy phn bng acid

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thu nhn cc amino acid phng php thng c dng nhiu nht l thu phn bng acid HCL 6N d tha nhit 100-120oC trong khong 24 gi. Sn phm thu c ch yu l cc amino acid t do di dng hydrogenclorate. Mt s amino acid nh serine v threonine b ph hu mt phn, tryptophan b ph hu hon ton, glutamine v asparagine phn ly thnh acid glutamic, acid aspartic v NH4+. 2.2. Thu phn bng kim Ngi ta cng c th thu nhn cc amino acid bng phng php thu phn vi NaOH, bng cch un nng trong nhiu gi. Sn phm thu c hu ht l cc amino acid nhng u b racemic ha, cc amino acid cysteine, serine v treonine b ph hu nhng tryptophan khng b ph hu. V vy, phng php thu phn bng kim thng ch dng xc nh tryptophan. 2.3. Thu phn bng enzyme thu nhn ch phm amino acid ngy nay vic thu phn bng enzyme c ng dng rng ri trong nhiu lnh vc khoa hc khc nhau bi s dng enzyme c nhiu u im nh ni trn. Cc enzyme thu phn protein to thnh cc amino acid hay cc cc peptid c phn t thp c gi chung l peptidhydrogenlase. C nhiu loi peptidhydrogenlase, chng c phn bit nhau bi tnh c hiu khc nhau vi lin kt peptid. Mt s loi ct t lin kt peptid u tn cng ca chui polypeptide c gi l exopeptidase nh carboxypeptidase phn gii lin kt peptide u C tn cng, aminopeptidase phn gii lin kt peptide u N tn cng. Mt s khc ch ct t cc lin kt peptide gia ca chui polypeptide c gi l cc endopeptidase nh pepsin, tripsin, v.v... 2.5. Cc phng php theo di v xc nh tc thu phn bng enzyme Tc thu phn protein ca enzyme ph thuc vo nhiu yu t nh nng enzyme, nng c cht, cc cht km hm, cc cht kch hot, nhit v pH ca mi trng phn ng. xc nh tc thy phn ca enzyme ngi ta khng nh lng enzyme mt cch trc tip m thng xc nh gin tip thng qua hot ca enzyme. Khi thc hin phn ng, enzyme c th lm thay i cc tnh cht vt l, ho hc, v.v... ca hn hn hp phn ng. V vy, theo di nhng bin i thng qua s nh lng c cht b mt i hay sn phm ca phn ng enzyme c to thnh c th bit c chnh xc mc hot ng ca enzyme. Ngi ta chia ra thnh ba nhm phng php: a) Xc nh lng sn phm to thnh hay lng c cht b mt i trong mt thi gian nht nh, ng vi lng enzyme nht nh.

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b) Xc nh thi gian cn thit thu nhn c mt lng bin thin nht nh ca c cht hay sn phm ng vi mt lng enzyme nht nh. c) Chn nng enzyme nh th no thu c s bin thin nht nh v c cht hay sn phm trong mt thi gian nht nh. Ngi ta thng s dng mt s n v tnh hot nh sau: - n v enzyme Quc t (UI -) l lng enzyme c kh nng xc tc lm chuyn ho c mt micromol c cht sau mt pht iu kin tiu chun. 1 UI = 1 mol c cht (10-6 mol)/pht. - Katal (Kat) l lng enzyme c kh nng xc tc lm chuyn ho c mt mol c cht sau mt giy iu kin tiu chun. 1 Kat = 1 mol c cht/giy 1 1 UI = 10-6 Kat = 16,67 nKat (nanokatal) 60 - Hot ring ca mt ch phm enzyme l s n v UI (hoc s n v Katal) ng vi 1 ml dung dch hoc 1mg protein ca ch phm. Nu ch phm enzyme tinh sch hot c th c biu th bng s UI (hoc Kat) trn 1mg enzyme. - Hot ring phn t l s phn t c cht c cht c chuyn ho bi mt phn t enzyme trong mt n v thi gian.

III. Cc phng php phn tch amino acid3.1. Phng php ho l t lu vic phn tch nh tnh v nh lng amino acid thng l s kt hp ca nhiu phng php ho l v sc k. C th da vo ph hp ph cc amino acid khng ging nhau phn tch. Hoc da v cu trc ca amino acid t do sau khi chui polypeptide b thu phn, c th nh lng amino acid u N-tn cng bng phn ng Sanger hoc Edman. Cng nh vy, c th xc nh amino acid u C tn cng nh phn ng kh nhm carboxyl vi tc nhn kh NaBH4, hoc s dng enzyme carboxypepitdase v.v... 3.2. Sc k Ngi ta c th dng nhiu phng php sc k khc nhau, y ch gii thiu nguyn tc ca mt s phng php thng dng phn tch amino acid. a) Sc k giy.

33

Nguyn tc ca phng php ny l da vo s phn b gia hai pha dung mi: dung mi c nh v dung mi di ng. Dung mi c nh thng l nc gi giy sc k (trong iu kin bo ho hi nc, giy c th gi 15-22% nc tnh theo trng lng giy). Dung mi di ng thng l mt dung mi hu c bo ho nc di chuyn trn t giy theo mao dn ko theo cc cht trong dung dch. Tc di chuyn ca tng cht khng ging nhau v mi cht c c trng bi mt tr s nht nh gi l Rf. aRf =

b Trong : - a l khong cch chuyn dch ca cht phn tch - b l khong cch chuyn dch ca dung mi C nhiu kiu sc k giy khc nhau l sc k mt chiu i ln, sc k mt chiu i xung, sc k vng nm ngang v sc k hai chiu. Loi giy thng dng l Whatman s 1 v Schleicher-Schull 2044 a v b, dung mi gm cc cht nh 4 Butanol:1 Acetic acid: 5 Nc (dng cho chiu th nht); 3 Phenol: 1 Nc (dng cho chiu th hai). b) Sc k lp mng. Nguyn tc ca phng php ny da trn l thuyt ca sc k giy, ngha l cng da trn s phn b cc cht gia hai pha: cht hp ph c trng rng trn mt phin knh to thnh mt lp mng v pha di ng l dung mi thch hp. Dung mi di chuyn lm dch chuyn cc cht trong mu th. Cc cht hp ph thng dng l silicagel, alumin oxyt, sephadex ,v.v...c kt hp vi thch cao (gypse) dn vo phin knh c) Sc k kh. Nguyn tc ca phng php ny l li dng tnh cht kh bay hi ca cc amino acid nn c th s dng chng trnh nhit chuyn chng thnh cc dn xut (thng l N-acetyl-amin). Cho tc dng amino acid vi cn amylic v HBr khan, sau cho tc dng hn hp ny vi anhydrit acetic. Ct thng dng l loi Chromosorb W (60-80 mesh) c mt lp polyetylenglycol 1% (carbowax 1564 hoc 6000). Chng trnh nhit gia 125o C v 155oC, tc chy 60-240 ml/pht. 3.3. Phn tch bng my t ng a) Xc nh trnh t (sequence) amio acid trong chui polypeptide

34

My phn tch amino acid trong chui polypeptide t ng da trn nguyn tc ca phng php Edman, ngha l tch tch ln lt tng amino acid u N tn cng v xc nh ln lt theo th t tng amino acid c tch ra , v vy c th xc nh chnh xc trnh t sp xp ca cc amino acid trong chui. b) Xc nh thnh phn amio acid trong chui polypeptide bng my sc k lng cao p-HPLC (High Liquid Pressor Chromatography ) Trc ht protein hay peptide phi c thu phn bng HCl 6 N 0 110 C trong ng hn kn cha nitrogen ( trnh s oxy ho ph hu amino acid) trong thi gian 12-15 gi. Sau trung ho hn hp dch thu phn amino acid v cho vo my phn tch t ng HPLC thnh phn amino acid cng vi mu chun amino acid. My t ng s cho bit hm lng (t l %) ca tng amino acid da theo cc nh (peak) ca amino acid chun. cn nh my HPLC ch cho bit thnh phn (composition) ca tng amion acid ch khng cho bit trnh t cc amino acid 3.4. in di Da vo tnh cht tch in ca cc amino acid trong mi trng c pH nht nh, m c th phn tch amino acid bng k thut in di. Di tc dng ca in trng cc amino acid tch in dng (+) s chy v pha cc (-), cc amino acid tch in m s chay v cc (+). Do kh nng tch in khng ging nhau gia cc amino acid v vy chng di chuyn khng ging nhau trong in trng. Kt qa cc amino acid phn b tri ra trn gi th (giy hoc tm polyamide). 3.5. Phn tch bng quang ph khi Quang ph khi (MS- mass spectrophotometer) l mt cng c phn tch vi chnh xc cao. Nguyn tc ca phng php ny l: Dng mt chm electron bn vo mt lng cht th rt nh, cc phn t cht thKnh mao dn Dung dch mu

in th cao

Gii hn chn khng

Hnh: 2.11

S hot ng ca quang ph khi

35

trc ht c ph thnh nhiu mnh ion mang in dng trong iu kin chn khng. Cc mnh ion nh mt b phn pht hin v ghi thnh pic vi cng khc nhau tng ng vi khi lng ca mi ion - l khi ph. C nhiu loi my quang ph khi vi mc phn gii khc nhau, my c phn gii cao l my c kh nng tch c hai mnh ion c khi lng ch chnh nhau phn trm n v khi. 3.6. Phng php ng v Phng php ng v c th s dng xc nh mt hoc vi amino acid trong hn hp c nhiu loi amino acid khc nhau, hay mt amino acid cn c xc nh trong nhiu mu ca mt lot th nghim. Nguyn tc ca phng php ny l da vo hot tnh phng x ca amino acid cng mt loi nh du xc nh v tr v s lng ca amino acid trong chui polypeptide. Ngoi ra ngi ta c th dng phng php ny nghin cu tnh c hiu ca cc amino-acyl-tRNAsynthetase v tRNA 3.7. Phng php enzyme C th xc nh amino acid bng phng php enzyme da trn nguyn tc mi mt loi enzyme c kh nng phn gii c hiu mt loi L-amino acid nht nh. Xc nh sn phm to thnh sau phn ng vi enzyme tng ng c th bit c amino acid trong hn hp. 3.8. Phng php vi sinh vt Mt s vi khun sinh trng trong mt iu kin thch hp, chng rt nhy cm vi pH sn xut ra cc enzyme L-amino aciddecarboxylase c hiu vi tng loi amino acid v hot ng ca chng gii phng CO2 trong mi trng. Xc nh nng CO2 bng p k Warburg suy ra thnh phn ca amino acid. Phn ln trng hp iu kin pH thch hp thng nm trong vng acid, v d: pH 4,5 to ra L-glutamate 1-carboxy-lyase;EC 4.1.1.15 xc tc chuyn ho L-glutamic -amino butyric; pH 5,5 to ra L-tyosinecarboxy-lyase EC 4.1.1.25 xc tc chuyn ho L-tyrosine Tyramine v.v...

36

CU HI N TP 1. Nu thnh phn cu to v tnh cht l ha ca amino acid. 2. C th phn chia cc amino acid thnh my nhm theo cch phn loi chung nht.Ti sao gi cc amino acid l mt di-acid ? 3. C th thu nhn v phn tch cc amino acid bng nhng phng php no? TI LIU THAM KHO 1.Trn Th n, i Duy Ban, Nguyn Hu Chn, nh H, L c Trnh. 1980. Ho sinh hc. NXB Y hc 2.Phm Th Trn chu, Trn Th ng. 1999. Ho sinh hc. NXB Gio dc 4. Nguyn Vit Tu, 1980. Phng php nghin cu ho hc cy thuc. nh xut bn Y hc. TP H Ch Minh. 5. Copeland R. A., 2000. Enzymes; A Practical Introduction To Structure; Mechanism & Data Analysis. Willey-VCH. A John Willey & Sons, INC., Pub. 2nd ed. 6. Daniel L.C., 2002. Introduction to proteomics. Humana Press Inc. Totuwa, New Jersey. 7. Fersht A.,1998, Structure and Mechanism in Protein Science, W. H. Freeman, 3rd Rev Edit. 8. Hans U. B., 1974. Methods of Enzymatic Analysis. Second English Edition Acdemic Press, Inc., New York San Francisco London, Vol., 4. 9. Hollas J. M., 2004. Modern spectroscopy. John Willey & Sons. Ltd. 4th ed. 10. Lehninger A.L., 2004. Principle of Biochemistry, 4th Edition. W.H Freeman, 2004 11. Lodish H., 2003. Molecular Cell Biology. 5th ed.W.H Freeman. 12. Walker J. M., 1996. The Protein Protocols Hand book. 2nd ed. Humana Press Inc. Totuwa, New Jersey.

37

Chng 3

Peptide - cu trc v chc nngI. Tnh cht chung ca peptidePeptide l nhng protein thng c cu trc on ngn khong t hai n vi chc amino acid ni vi nhau, c khi lng phn t thng di 6.000 Dalton. Chng c th c tng hp trong t nhin hoc c hnh thnh do thoi ho protein. Mc d c cu trc nh nhng nhiu peptide c vai tr kh quan trng trong hot ng trao i cht ca c th. 1.1. Cu to ca peptide. Nh gii thu trn cc amino acid l nhng n phn t xy dng nn cc chui polypeptide. Trong cc chui cc amino acid c lin kt vi nhau thng qua lin kt peptide (hnh 3.1).

Hnh 3.1

S to thnh lin kt peptide

Dng cng ho tr

Dng ion +

Dng lai (hybrid)Hnh 3.2 S tn ti cc dng ca lin kt peptide

38

Lin kt peptide (peptide bond) c bn cao bi cu trc ca n c 4 e , 2e/ thuc v lin kt C=O cn 2e/ thuc v b i e/ t do ca nguyn t N. Lin kt gia C-N l lin kt phc tp, n c th chuyn t dng n dng lai (trung gian) th b mt phn ghp i ca lin kt (hnh 3.2). Ngi ta cho rng t l ca lin kt kp ny l khong 30% i vi lin kt C-N v 70% vi lin kt gia C v O. Nh vy u ca mt chui peptide l amino acid c nhm -amino ( -NH2) t do c gi l u N-tn cng v u kia c nhm - carboxyl ( -COOH) t do c gi l u C tn cng. Lin kt peptide to nn b khung chnh ca chui polypeptide, cn cc gc R to nn mch bn ca chui (hnh 3.3)./

Mch chnh

Mch bn

Hnh 3.3

Mch bn v khung ca mt chui polypeptide

Nh vy lin kt peptide gia C-N khng ging nh lin kt gia C-N ca cc cht thng thng, v vy ko theo mt s hu qu sau: - Cc nguyn t C O N H nm trn mt mt phng v khng xy ra s quay t do xung quanh lin kt C-N nu khng c thm nng lng, trong H ca nhm NH lun v tr trans so vi O ca nhm COOH v cu hnh trans ca lin kt peptide l cu hnh n nh.

Nhm peptide

Hnh: 3.4

M hnh cu trc ca lin kt peptide

39

- Mt khc kh nng quay t do gia C v C gia N v C (hnh 3.4) l rt ln, lm cho mch peptide c khuynh hng hnh thnh cu trc xon. - Ngoi ra lin kt peptide c tnh cht ion mt phn lm tng tnh linh ng ca nguyn t N. Mt khc lin kt n gia C-N trong lin kt peptide c chiu di (1,33 Ao) hi ngn hn so vi a s cc lin kt C-C, C-N ( 1,47Ao) v cc lin kt khc nn to cho chui peptide mt cu hnh khng gian c bn cao. 1.2. Cch gi tn v phn loi peptide Cn c vo s amino acid trong peptide gi tn ca peptide, nu c 2 amino acid gi l dipeptide, 3 amino acid gi l tripeptide, 4 amino acid gi tetrapeptide, 5 amino acid gi l pentapeptide v.v...cch gi tn cc peptide theo gc amino acid bng cch tn bt u t amino acid u tin ln lt n amino acid cui cng. Tr amino acid cui cng cn tt c ui ca cc amino acid u b thay bng ui - yl.

tn cng

tn cng

Hnh 3.5 Cu to v cch gi tn ca mt pentapeptide Seryl-glycyl-tyrosyl-alnyl-leucine

Ngi ta cng c th dng k hiu vit tt 3 ch hoc 1 ch theo th t cc amino acid biu th thnh phn v th t cc amino acid trong chui v d pentapeptide trn (hnh 3.5), c th vit Ser-Gly-TyrAla-Leu hoc SGYAL. Thng thng ngi ta vit amino acid u N tn cng pha bn tri v amino acid u C tn cng pha bn phi v cng c th ghi r u no ca chui peptide l u N-tn cng hay u C- tn cng v nh vy cng pentapeptide trn c th vit H2N-Ser hay LeuCOOH. Trong trng hp c nhng amino acid mt on no trong chui peptide cha xc nh c r ngi ta c th cc amino acid trong ngoc chng hn Ala- Ser-Gly-(Ala,Leu, Val) Glu-Arg-...

40

1.3. Cc phn ng c trng ca peptide Ngoi phn ng ca nhm NH2 v COOH u tn cng, cc gc R ca peptide cng cho nhng phn ng mu c trng ca cc amino acid t do tng ng. Mt trong nhng phn ng mu c trng nht cho lin kt peptide l phn ng Biure, phn ng ny khng xy ra vi amino acid t do. Trong mi trng kim mnh, lin kt peptide phn ng vi CuSO4 to thnh phc cht mu tm v c kh nng hp th cc i bc sng 540 nm. y l phn ng c s dng rng ri nh lng protein. Phng php xc nh protein theo Lowry cng da trn nguyn tc ca phn ng ny bng cch thm thuc th Folin-Ciocalteau lm tng nhy ca phn ng sau khi thc hin phn ng biure, ng thi da vo cc gc Tyr, Trp nh thuc th to phc mu xanh da tri. OC =NH HN C NHHnh 3.6

OHN = C Cu O C HNS to phc mu tm trong phn ng Biure

O

NH

II. Cc phng php tch phn lp v xc nh peptideC mt s phng php tch phn lp v xc nh thnh phn, s lng v trnh t amino acid trong peptide. Nguyn tc chung ca cc phng php tch phn lp v xc nh peptide v c bn cng nh i vi protein. Tuy nhin peptide l nhng on ngn ca chui polypeptide v th c th b qua giai on ct chui polypeptide thnh cc peptide nh m c th tch phn lp ngay bng phng php in di hay sc k tch ring tng peptide. Sau khi tch ring cc peptide ngi ta tin hnh thu phn n hon ton thnh cc amio acid t do. T xc nh cc amino acid u N-tn cng v amino acid u C-tn cng. Cc d liu thu c qua phn tch s c so snh i chiu v tng hp li. V d, Puppy v Bodo phn tch mt peptide ca dch khi thu phn Cytocrom C thu c cc d kin sau y: - Thnh phn amino acid ca peptide sau khi c thu phn hon ton v sc k l 2Cys, 1 Ala, 2 Glu,1His, 1Thr, 1Val,v 1Lys.

41

- Dng phng php Sanger xc nh c amino acid u N-tn cng l Cys v phng php carboxypeptidase xc nh c amino acid u C-tn cng l Lys. - Cu to ca peptide nh (bng cch thu phn tng phn ban u v xc nh cc amino acid , amino acid u N-tn cng v amino acid u C-tn cng ca mi peptide nh): Cys- Ala Glu- Cys (Val- Glu) Cys-(Ala,Glu) Cys- His Thr (Val, Glu) Ala- Glu Glu (Cys, His) Glu- Lys Thr (Val, Glu, Lys) Tng hp cc d kin trn, h xc nh c trnh t cc amino acid ca peptide nghin cu l: H2N-Cys-Ala-Glu-Cys-His-Thr-Val-Glu-Lys-COOH. y l nguyn tc chung xc nh mt trnh t trong peptide. Tuy nhin i vi nhng peptide di vic xc nh rt phc tp.

III. S tn ti t nhin v vai tr chc nng ca peptide3.1. Khi nim chung Trong t nhin tn ti nhiu dng peptide c chc nng quan trong lin quan n hot ng sng ca c th nh l cc hormon, cc cht khng sinh hay nhng cht tin thn ca t bo vi khun v.v... Bn cnh cng c nhng peptide chc nng cha r rng, c nhng peptide l sn phm thu phn ang cn dang d ca protein. Trong phm vi ca chng trnh ny xin c gii thiu mt s peptide quan trng, c nhiu ngha cho hot ng sng ca sinh vt. 3.2. Glutathion v cc cht tng t Glutation l mt tripeptide -glutamyl-cysteyl-glycine vi cng thc cu to nh sau: NH2 CH2 SH HOOC-CH-CH2-CH2-CO-HN-CH-CO-NH-CH2-COOH Trong cu trc nhm SH ca cysteine l nhm hot ng, v vy ngi ta thng vit tt ch glutation l G-SH. Trong mi trng hot ng glutation c th nhng hydrogengen (H) thnh dng oxy ho v ngc li c th nhn H thnh dng kh: Nh phn ng trn, glutathion ng vai tr ca mt h thng oxy ho kh (vn chuyn hydrogen). Glutathion l mt trong nhng peptide

42

ni bo ph bin nht, n phn b nhiu trong cc m v cc c quan nh: gan, thn, lch, tim, phi, hng cu v.v...

G - SH G - SHDng kh

-2H

G-S G-SDng oxyha

+2H

3.3. Cc hormon c bn cht peptide v protein 3.3.1. Adrenocorticotropic hormone (corticotropin, ACTH). ACTH hay cn gi l kch t v thng thn kch thch s tng hp steroid, adrenocortic v thng thn. L hormon polypeptide, cu trc phn t bao gm 39 amino acid, c khi lng phn t khong 4.500. Nghin cu cu trc v chc nng thy rng, cc on trinh t amino acid trong chui peptide c nhng chc nng hot ng sinh hc khc nhau: - on 24 amino acid u tin l phn hot ng sinh hc trong 13 amino acid u tin tng ng vi chui -MSH (melanin stimulating hormone- kch hc t) v 5 amino acid t 6-10 c hot ng ca MSH, 16 amino acid u tin ca ACTH mang tnh cht hot ng to ra steroid. - on trong chui t amino acid s 25 n 39 c s thany i tu theo loi ng vt. Trong c th ACTH kch thch ch yu t bo v thng thn bi tit ra hormoon chuyn ho ng(corticosteron,cortisol), ACTH xc tc phn ng hydrogenxyl ho v c bit l phn ng ct chui ngang ca cholesterol (tng hp steroid), phn ng 11 -hydrogenxyl ho. 3.3.2. Cc hormon kch thch bi tit melanin (MSH). MSH c bi tit thu gia tuyn yn. Ngi ta chia MSH thnh hai loi: - MSH gm 13 amino acid, ging vi 13 amino acid u ca ACTH nn c tc dng yu ca ACTH v - MSH. Hin nay ngi ta tng hp c MSH hon ton. S iu ho tng hp MSH c thc hin bi hai yu t: mt yu t c ch gii phng MSH l MIF (melanocyte release inhibiting factor) c cu to l mt trpeptide gm pro-leu-gly-NH2; mt yu t kch thch gii phong MSH l MRH (melnocyte release stimulating hormone), cu to l mt peptide gm 5 amino acid.

43

MSH gy tng sc t ln, nhng MSH khng tn ti nh mt s hormon ring bit ngi. Hin tng sm da ca nhng con bnh nghin (addision) c th do ch yu l s tng tit ca MSH. 3.3.3. Oxytocin, Vasopressin Vasotocin. Oxytocin l hormon thc , gy co d con, l mt peptide c 9 amino acid. ng vt c v, oxytocin ch khc s thay i ca 2 amino acid nh sau: amino acid v tr th ba l isoleucine v amino acid v tr th tm l leucine (Bng 3.1). Vasopressin ca loi ch nhi c cu trc trung gian gia vasopresin v oxytocin ca ng vt c v (amino acid th ba l isoleucin v amino acid th tm l arginin). Vasopressin l hormon gy co mch, l mt peptide c cu trc gm 9 amino acid. Phn ln ng vt c v amino acid th 8 ca vasopressin l lysine(lys-Vasopressin), tr ln v h m, amino acid th 8 l lysine (lys-vasopressin).Bng 3.1 So snh cu trc ho hc gia oxytocin v vasopressin ca mt s loi ng vt

Va-

Lysin 1 2 3 4 5 6 7 8 9 Vaso- Cys-Tyr-Phe-Glu-Asn-Cys-Pro-Lys-Gly-NH2 pressin Arginin vasopressin 1 2 3 4 5 6 7 8 9 Cys-Tyr-Phe-Glu-Asn-Cys-Pro-Arg-Gly-NH2

Ln, H m Phn ln ng vt c v ng vt c xng sng, khng c v

so-

pres-

sin

Vasotocin

1 2 3 4 5 6 7 8 9 Cys-Tyr-Ile-Glu-Asn-Cys-Pro-Arg-Gly-NH2

Oxytocin

ng vt c 1 2 3 4 5 6 7 8 9 xng Cys-Tyr-Ile-Glu-Asn-Cys-Pro-Leu-Gly-NH2 sng, ( ch khc vasopressin chui bn amino acid c v, th ba v th tm) chim

44

ng vt c v amino acid th 3 l isoleucine; vasopressin c tn l la arginin-vassotocin. Oxytocin c tc dng trn c trn ca t cung v tuyn v, gy co khi t cung sinh con v kch thch s tit sa khi cho con b. Vasopressin c tc dng chng li niu, tng cng ti hp thu nc thn, ng thi lm co mch, do c tc dng tng huyt p. 3.3.4. Cc hormon sinh trng (HGH). Hormon sinh trng ca ngi (human growth hormone) cn c tn gi STH (somatotropin hormone) l mt chui polypeptide bao gm 191 amino acidc khi lng phn t 21 kDa (L. Stryer, 1998) . Trong cu trc c hai cu disulfua c to thnh gia amino acid 53-165 v gia amino acid 182-189 (hnh 21). Hot ng sinh hc ca HGH l chui gm 134 amino acid. HGH c cu to rt ging vi hormon lactogen ca rau thai (85% amino acid ging nhau) v gn ging prolactin ca ngi (32% amino acid ging nhau) . Hormon sinh trng c tc dng s tng trng ni chung, kch thch s to sn hn l to xng, n cng l mt hormon chuyn ho. Hormon sinh trng kch thch s tng hp protein t nhng amino acid c vn chuyn d dng vo trong t bo nh HGH, v l hormon gy tng ng huyt, sinh i tho ng ( kch thch s bi tit glucagon), ng thi kch thch s thoi ho lipid m bo nhu cu v nng lng cho c th, gy tng acid bo t do trong huyt tng. S thiu ht HGH nu xy ra trc tui dy th s dn n chng ngi ln, s d tha HGH nu xy ra trc tui dy th s xy n chng ngi khng l, nu xy ra sau tui dy th s dn n chng ngi b to d thng (pht trin chiu dy ca u, xng v mt). 3.3.5. Prolactin (PRL- kch nh t) Prolactin hoc LTH (luteotropic hormon), l mt polypeptide c khi lng phn t 23.000. ngi c cu trc 198 amino acid. cc loi ng vt s lng amino acid trong chui ging nhau khong 70%. Cu trc bc 1 v hot ng ca PRL c tnh cht tng t HGH v c hormon to sa ngun gc rau thai. Prolactin c bi tit lin tc khi c thai, chng kch thch th vng bi tit ra progesteron trc khi progesteron c bi tit bi nhau thai. PRL chun b cho tuyn v bi tit sa. Sau khi , khi t cung rng PRL m bo cho s bi tit sa. 3.3.6. Hormon tit (thyrotropin-TRH).

45

TRH l vit tt ca hormon gii phng thyrotropin (tiotropin releasing hormone). Cu trc ho hc ca TRH d c Schally v Guilemin xc nh (nm 1969 v t gii thng Nobel 1977) l mt peptid ngn ch 3 amino acid l pyroglutamyl-histidyl-proline-NH2. TRH c chc nng tham gia vo qu trnh tng hp v bi tit TSH (kch gip trng t). TRH c chc nng va nh mt hormon gii phng, va nh mt hormon kch thch. 3.3.7. Insulin. tuyn tu, c bit i vi qu T 1953, Sanger (gii thng Nobel 1958) nghin cu, tinh ch v xc nh hon ton cu trc ca phn t insulin. Phn t insulin bao gm 51 amino acid, c cu trc gm 2 chui polypeptide, vi khi lng phn t 5.700: Chui A c 21 amino acid. Chui B c 30 amino acid . Hai chui c ni vi nhau bng 2 cu disulfua. Trong chui A cung hnh thnh 1 cu disulfua gia amino acid th 6 v amino acid th 11. Phn c hiu (c trng ca mt loi) ch tp trung vo cc amino acid th 89-10, 12-14 ca chui A v c bit l amino acid th 30 ca chui B (hnh3.7). Ngi ta cng xc nh c cu trc ba chiu ca insulin v thy rng cu trc phn t insulin c gi vng bi nhiu lin kt mui, lin kt hydro v lin kt cu disulfua gia chui A v chui B. Insulin c tc dng r nht trong tt c cc hormon caHnh 3.7 Cc amino acid ca chui A v B insulin b

46

trnh chuyn ho glucid, n c tc dng h ng huyt. Insulin cn kch thch qu trnh tng hp v c ch qu trnh thoi ho glycogen c, gan v m m. c bit insulin tng cng tng glucose hp acid bo, protein v kch thch s ng phn. Tc dng quan trng nht ca insulin l kch thch s xm nhp glucose, mt s ng monose, amino acid trong t bo c v m. Do vy insulin lm gim lng glucose trong mu. Ngoi ra insulin cng lm gim s tn to glucose do lm gim nng enzyme nh pyruvat carboxylase v fructose 1-6 diphosphatase. 3.3.8. Glucagon. Glucagon l mt peptide c tit ra bi t bo alpha ca o langerhans, cu trc phn t glucagon ngi gm 29 amino acid, vi khi lng 3.500. Lc u glucagon c hnh thnh di dng tin hormon, preproglucagon ri n proglucagon c 37 amino acid . Sau khi loi i 8 amino acid tr thnh glucagon. Glucagon lu thng trong mu di dng t do (khng kt hp vi protein), chng c tc dng lm tng nng glucose trong mu bng cch kch thch s phn hu glycogen gan qua trung gian ca AMP vng. Glucagon kch thch s tn to glucose v c ch s phn hu glucose bng cch c ch pyruvate kinase v phospho fructokinase. N cn c tc dng c ch tng hp lipid gan, nhng kch thch s to thnh cht cetonic. i vi protein, glucagon c tc dng thoi ho. 3.3.9. Calcitonin (CT) Calcitonin c pht hin t nm 1962, ngi ta xc nh c cu trc ca n l mt chui peptide gm 32 amino acid c mt cu disulfua gia amino acid th 1 v amino acid th 7 (hnh 3.8). S S Cys1-Ser2-Asn3-Leu4-Ser5-Thr6-Cys7-Val8-Leu9-Ser10-Ala11-Tyr12-Met13-Arg24-Asn23-Leu22-Asn21-Asn20-Phe19-His18-Arg17-Phe16-Ser15-Gly14-Trp25-Gly26-Phe27-Gly28-Pro29-Glu30-Thr31-Pro32 -NH2Hnh 3.8 S cu trc calcitonin ca ln

Calcitonin c tc dng iu ho calci mu. Calci huyt tng c duy tr nng 10 mg/100 ml nh calcitonin (cn c c PTH: parathormon v vitamin D). Cht i trng sinh l chnh ca calci l phosphate. ho tan ca calci phosphate rt thp v s tng ca mt ion

47

ny s gy gim mt ion khc, nu khng phosphate calci s kt ta. Trong mu mt na calci kt hp vi protein, mt na dng t do v c hot ng sinh hc. Xng cha trn 99% calci ca c th v l kho d tr chnh ca calci khi c th cn n. 3.3.10. Angiotensin. Angiotensin c cu trc l nhng peptide c 7 amino acid ( AIIAngiotensin II) hoc 8 amino acid (AIII-Angiotensin III), chng c bi tit bi t bo thn. Chng c tc dng kch thch lp v ca t bo thng thn bi tit aldosteron lm tng lng natri trong mu khi th tch mu hoc natri gim. 3.3.11. Cholecystokinin-pancreozymin(CCK-PZ). L mt peptide c cu trc gm 33 amino acid. Hot ng sinh hc ca n gn lin ch yu C tn cng. Cholecystokinin c bi tit bi nim mc t trng khi c s tiu ho lipid v protein. Trong thc t nhiu t bo bi tit nhiu kiu CCK khc nhau v s lng amino acid (c th l 58; 32; 8 hoc 4 amino acid). CCK c tng hp t mt hormon chung (pre-pro-cholecystokin) c 114 amino acid. Trong c th CCK c tc dng kch thch co bp ti mt, kch thch t bo nang ty bi tit enzyme amylase. 3.3.12. Secretin. c pht hin bi Bayliss v Starling vo nm 1902. l mt peptide gm c 27 amino acid, secretin c bi tit bi t trng nh kch thch ca pH acid. Hin nay ngi ta tng hp c hon ton phn t secrectin Secretin c tc dng kch thch bi tit bicarbonate nhm trung ho acid ca d dy (pH t 1,5-2,5 tng ln 7,0). Ngoi ra secretin cn c nhiu tc dng khc nh kch thch tit mt v bi tit pepsin. 3.3.13. Gastrin. Gastrin c bi tit bi t bo vng hang v ca nim mc d dy. Gastrin c cu trc l mt peptide bao gm 17 amino acid, c bi tit t mt hormon c 34 amino acid. Gastrin tng hp nhn to ch gm 4 amino acid cui cng (t 14 n 17) c hot ng y ca gastrin v c ng dng rng ri trong lm sng (phng php gastrin). Gastrin c tc dng kch thch t bo thnh ca tuyn d dy bi tit HCl v t bo chnh bi tit pepsinogen. Ngoi ra gastrin cn kch thch s vn ng ca d dy, rut, lm gin c vng ca mn v.

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3.4. Cc peptide khng sinh l nhng peptide do vi vi khun hoc nm sn sinh ra. Nhiu peptide khng sinh l peptide vng cha cc amino acid dng D v L. 3.4.1. Penicillin. Penicillin c chit ra t dch nui cy nm Penicillium chrysogenum . Penicillin gm nhiu loi, chng c cu to gn ging nhau, bao gm mt vng tiasolidin, mt vng -lactam, mt nhm amino c gn vi CO2 v mt mch bn (R), hnh 3.9.mch bn vng thiazolidine

vng -lactam Hnh 3.9 Cu to chung ca phn t penicillin

Ngy nay, ngi ta bit c nhiu loi penicillin chng ch khc nhau bi mch bn.V d: - mch bn ca penicillin F l CH3-CH2-CH=CH2- mch bn ca penicillin K l CH3-(CH2)3-CH2- mch bn ca penicillin O l CH2=CH-CH2-S-CH2- mch bn ca penicillin G l - mch bn ca penicillin G l HO- mch bn ca penicillin V l -CH2-CH2-O-CH2-

Penicillin ln u tin pht hin c tc dng chng vi khun gram dng Staphylococcus, Diplococcus...nhng hu nh khng c tc dng

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chng vi khun gram m v nm men. Vi thp nin tr li y pht hin ra nhiu loi penicillin mi trong mt s c hiu qu chng li vi khun gram m v nm men, v d: nng cao (10 mg/ml) pencillin c kh nng chng cc ni nm men Saccharomyces cerevisae n bi v E. coli. 3.4.2. Gramicidin. Gramicidin c pht hin t nm 1942 v bao gm hai loi l gramicidin S c 5 amino acid v gramicidin J c 24 amino acid, chng u l nhng peptide c cu trc vng. Vi nng vi mg/ml gramicidin c tc dng chng c Diplococcus pneumoniae, Streptococcus hemolyticus, Meningococcus, Staphylococcus aureus v Neiseria. Cc cht gramicidin c dng iu tr cc bnh nhim trng do t cu, lin cu qung, nhim trng mu hu sn, vim hng, vim mng no v.v... 3.4.3.Tyrocidin. Tyrocidin c pht hin vo nm 1939 bi Dubos khi ln men vi khun B. brevis. Tyrocidin kt hp vi gramicidin to thnh tyrothricin. Khi thu phn tyrocidin trong mi trng acid thy c cc amino acid ca dang L nh sau: ornitine, valine, leucine, proline, tyrosine, phenylalanine, tryptophan, glutamic, asparaginic. Trocydin lai tip tc phn hu thnh cc tyrocidin A, B v C. Cng ging nh gramicidin nng vi mg/ml tyrocidin u c tc dng chng cc loi vi khn nh Diplococcus pneumoniae, Streptococcus hemolyticus, Meningococcus, Staphylococcus aureus v Neiseria. 3.4.4. Bacitracin. Bacitracin c Johnson v cng s pht hin t nm 1945 t dch chit ca vi khun Baccillus licheniformi (thuc nhm B. subtilis). Bacitracin bao gm 10 loi khc nhau l : bacitracin A, A1, B, C, D, E, F1, F2, F3 v G. Trong bacitracin A chim nhiu nht (37%). Bacitracin A cu trc l mt peptide nh ch gm khong 10 amino acid sau: L-leucine, L-lysine, -L-isoleucine, L-cysteine, L-asparaginic, glutamic, D-asparaginic D-phenylalanin, D-ornitin. Bacitracin tuy khng c hot tnh chng vi khun gram m, nhng li c hot tnh mnh m chng vi khun gram dng, 1n v /ml c th chng c S. pyogenes, S. hemolyticus, S. albus, Clostridium welchii, ...Bacitracin c dng nhiu trong chn nui v cng nghip thc phm. 3.4.5. Polymycin.

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Polymycin c pht hin cng mt lc vo nm 1947 bi ba nhm nh khoa hc ca M v Anh t dch chit ca vi khun Bac. Polymixa. Polymycin l mt hn hp bao gm cc cht gn ging nhau gi l polymycin A, B, C, E v M. Chng u l nhng peptide c tnh cht kim, d to thnh mui vi acid hu c v v c. Polymycin c hot tnh mnh m chng vi khun gram dng v m, c bit c kh nng chng cc vi khun m xanh (Ps.aeruginosa) khng li cc cht khng sinh khc. 3.5. Cc peptide c ngha sinh l t bo Cc peptide c ngha sinh l t bo thng ch yu l cc peptide hormon c gii thiu trn. Trong phn ny ch mang tnh cht nu ln nhng tnh cht c bn trong hot ng sinh l t bo. l nhng peptide c t 3 n khong 200 amino acid. N gm nhng hormon ca cc tuyn vng di i, tuyn yn, tuyn tu, v.v...S tng hp hormon peptide xy ra li ni cht nguyn sinh di dng mt chui polypeptide di hn, l mt tin hormon (pro-hormon) chng hn nh pro-insulin i vi insulin, proglucagon i vi glucagon...Nhng hormon peptide lu thng trong mu di dng t do, c na i sng ngn (thng di 60 pht), thi gian p ng ngn (vi giy cho tc dng tng ng huyt ca glucagon hoc chng li niu ca vasopressin). Cc hormon peptide khng vo trong t bo ch m tc dng trn b mt ca th th (receptor) c hiu mng t bo. 3.6. Cc peptide c chc nng bo v Trong c th ng, thc vt c nhng peptide lm nhim v bo v cho c th. Loi peptide u tin phi k n ng vt c xng sng l cc peptide khng th trong mu. Chng l nhng yu t nhn bit c lc cc tc nhn vi khun, virus v cc vt l xm nhp vo c th v loi tr chng ra khi c th. Ngoi ra trong mu ca ng vt trn cn c cc interferon vi mt nng nh c kh nng chng li s xm nhim ca virus. Trong mu ca ng vt cn c cc peptide chng chy mu nh fibrin v.v... thc vt c nhiu loi to ra nhng peptide c t, chi vi mt liu lng nh cng c kh nng git cht ngi v ng vt. Ngoi ra trong c th ng vt, thc vt v cc sinh vt khc ni chung u tn ti mt hp cht c bn cht peptide l nhim v bo v l lectin. V c kh nng lin kt ng mt cch c hiu v chn lc, nn lectin c th kt ta cc tc nhn hay t bo l c cu trc ng xm nhp

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vo c th bo v c th. V th nhiu ngi cn cho lectin l khng th thc vt (xem chng 1).

1. 2.

CU HI N TP Th no l peptide, tnh cht chung, cc phng php tch phn lp v xc nh peptide? Trnh by v s tn ti t nhin, vai tr v chc nng ca peptid

TI LIU THAM KHO 1.Trn Th n, i Duy Ban, Nguyn Hu Chn, nh H, L c Trnh. 1980. Ho sinh hc. NXB Y hc 2.Phm Th Trn Chu, Trn Th ng. 1999. Ho sinh hc. NXB Gio 3. Nguyn Ln Dng, Phm Vn Ty, Nguyn nh Quyn. 1999. Vi sinh vt hc. NXB Gio dc. 4. L c Trnh. 1998. Hormon. NXB Y hc. H ni 5. Nguyn Vit Tu,1980. Phng php nghin cu ho hc cy thuc. nh xut bn Y hc TP H Ch Minh. 6.Copeland R. A., 2000. Enzymes; A Practical Introduction To Structure; Mechanism & Data Analysis. Willey-VCH. A John Willey & Sons, INC., Pub. 2nd ed. 7. Daniel C. L., 2002. Introduction to proteomics. Humana Press Inc. Totuwa, New Jersey. 8. Dennison C., 2002. A Guide To Protein Isolation. Kluwer Academic Publishers. New York, Boston, Dordrecht, Lodon, Moscow. 9. Hans U. B. 1974. Methods of Enzymatic Analysis.Second English Edition Acdemic Press, Inc., New York San Francisco London, Vol., 4. 10. Lehninger A.L., 2004. Principle of Biochemistry, 4th Edition. W.H Freeman. 11. Lodish H ., 2003. Molecular Cell Biology. 5th ed, W.H Freeman.

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Chng 4

Cu trc v tnh cht l-ho ca protein

I. Cc quan im khc nhau v nghin cu cu trc proteinNh bit, protein l nhng phn chc nng ca c th, s hnh thnh chc nng mi da trn c s thnh phn cu trc ca protein, hay ni cch khc gia chc nng v thnh phn cu trc trong protein c lin h mt thit vi nhau. T s nghin cu cu trc protein c th da trn cc quan im: nghin cu thnh phn cu trc hoc da vo chc nng sinh hc tm hiu cu trc ca chng. Chng hn, vic nghin cu xc nh cu trc bc I ca phn t protein l bc u tin quan trng xc nh phn t hot tnh sinh hc v tnh cht ho l ca protein, l c s xc nh cu trc khng gian ca phn t protein. Da vo cu trc khng gian ca cc phn t protein tng ng, c th d on s nh v cu disunfua, cu trc khng gian ca protein nghin cu. Ngc li s xut hin mt bnh l c trng no lin quan n thay i chc nng ca protein m nguyn nhn ch l thay i 1gc amino acid trong phn t. V d: bnh thiu mu hng cu hnh li lim, khi nghin cu cu trc bc I ca hemoglobin bnh thng v bnh l xc nh c l do gc amino acid glutamic v tr th 6 trong chui ca hemoglobin A (bnh thng) b thay th bng gc amino acid valine

II. Cc kiu lin kt trong cu trc protein2.1. Cc lin kt cng ho tr Trong phn t protein ngoi cc lin kt cng ho tr bnh thng, ngi ta thng nhc n hai kiu lin kt cng ho tr c bit c ngha quan trng i vi cu trc v chc nng ca chng l: - Lin kt peptide (xem chng 3). - Lin kt disunfua (-S-S-). Lin kt disunfua l lin kt ng ho tr to thnh do s kt hp gia hai phn t cysteine vi nhau loi i 2H (hnh 4.1). Lin kt disunfua (cn gi l cu disunfua) c th hnh thnh gia hai phn t cysteine trong cng mt chui polypeptide hoc gia hai cysteine thuc hai chui polypeptide khc nhau. Cu disunfua c vai tr quan trng trong vic duy tr cu trc bc III ca phn t protein. Nhng phn t protein cng cha nhiu cu disunfua th cng cht ch, vng bn. nhng protein khng tan nh protein ca lng, mng, tc, sng rt vng bn vi cc tc nhn ho hc, cha ti 12% l cysteine

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Oxy ha

Hnh 4.1 S hnh thnh cu disulfua gia hai phn t cysteine

2.2. Cc lin kt yu lm n nh cu trc protein 2.2.1. Lin kt hydro L tng tc yu hnh thnh gia mt nguyn t mang in tch m (gi l nguyn t nhn A-acceptor) v mt nguyn t hydro (H) ang nm D H+A D H ALin kt hydro

aHnh 4.2

b

c

Mt s lin kt hydro quan trng trong h thng sng

Ghi ch: a) gia hydro ca mt ancohol v oxy ca nc; b) gia nhm carbonyl keto v nc; c) gia nhm peptide trong polypeptide;

54

trong mt ni cng ho tr vi mt nguyn t khc (gi l nguyn t cho D- donnor). Ni cng ho tr gia D v H phi l ni phn cc v m my in t ca A phi mang nhng in t khng lin kt c kh nng thu ht in tch + ca H. Nng lng cn ph v mt lin kt hydro l khong 5 kcal mol-1. Mt c im quan trng ca cc lin kt hydrogengen l H, nguyn t nhn A, nguyn t cho D u xp trn mt ng thng. Nguyn t N trong lin kt N-H cng nh O trong lin kt O-H u l nhng nguyn t cho chnh. Trong h thng sng l cc nhm amine (-NH2) v hydroxyl (-OH), s hin din ca cc nhm ny khin cho cc phn t c mang chng d ho tan trong nc do c s hnh thnh cc lin kt hydrogengen gia chng. c bit, cc phn t nc H2O - nhn t ch yu ca vt cht sng lun lun hnh thnh mt mng li u n nhng hnh t din d th lng hay th rn.

Hnh 4.3 S cu trc mng li hnh thnh bi cc phn t H2O- Nguyn t oxy biu th bng cc vng trn ln - Nguyn t hydro c biu th bng cc vng trn nh

2.2.2. Lin kt ion L tng tc tnh in gia hai nhm c in tch ngc du. Trong nhiu trng hp cht v c, in t lin kt lun lun b ht v

55

pha nguyn t c m in cao hn gy ra s phn li cation (nguyn t tch in tch m) v anion (nguyn t tch in dng), v d: NaCl Na+ + ClV in t lin kt khng dc phn chia ng u cho hai nguyn t nn lin kt ny khng c xp vo loi cc lin kt cng ho tr. Khc vi cc lin kt cng ho tr hay lin kt hydro, cc lin kt ion khng c nh hng trong khng gian v in trng l khng ng u quanh ion. Trong mi trng nc, cc anion v cation lun lun c vy bc bi cc phn t H2O to thnh mt lp v bc ngoi nn khng th lin kt trc tip vi cc anion v cation khc. Do , ngi ta cho rng tng tc tnh in ny khng ng vai tr quan trong quyt nh cu hnh khng gian ca cc phn t hu c. 2.2.3. Lin kt Van der Waals L cc tng tc khng c hiu xut hin gia hai nguyn t khi chng tin li gn nhau. Tng tc ny khng do s phn phi lch ca cc in t gia hai phn t m do cc bin ng thong qua ca m my in t gy ra s phn cc nht thi trn phn t. Lin kt Van der Waals l kt qu ca lc ht v lc y. Hai lc ny cn bng mt khong cch nht nh, c trng cho tng loi nguyn t. Khong cch ny c gi l bn knh Van der Waals. y l lc lin kt yu nht, vi gi tr ch khoang 1 k cal mol-1. lin kt ny tht s c ngha, n phi tn ti mt s lng ln, ngha l b mt tip xc gia hai phn t phi cc i. in hnh l khi mt phn t c mang mt hc c hnh dng ph hp vi ch li trn phn t kia nh trng hp tng tc gia khng nguyn - khng th, gia enzyme - c cht. 2.2..4. Lin kt k nc (tng tc k nc)