2025年生物化学入门指南探索生命科学的基石

BIO107IntroductiontoBiochemistryGuoxiaHan

Lct.l

Questions

Chemicalstructure,interaction,extractenergy,storeandtransmittheinformation,chemical

changes,controlled

3principleareas

StructuralChemistry,

Metabolism,

Storage,transmission,andexpressionofgeneticinformation

Lct2,ChO3

AminoAcids,Peptides,andProteins

AminoAcids

COO-Isomers

Structuralisomers构造异构

体

H3N—C—HCis-transisomers顺反异构

体

REn-antionmers光学异构

山HM口4-/.3

Stereoisomers空间异构体（Glycer-aldehyde甘油醛、Ala丙氨酸）

,CHOCHO

i

-H一OH

CHQH

L-Glyceraldehydeo-Glyceraldehyde

COO-COO-

♦1

H3N-£-HH-C-

CH3CH,

L-Alanineo-Alanine

蛋白质中出现的氨基酸都是Lisomers

>AminoAcidsClassifiedbyRGroups

Nonpolar,aliphaticRgroups

COO"COO-coo-coo-非极性脂肪基

♦1♦1l/H♦1R

HN—C—HHN—C-HHN—C—H

333Glycine甘氨酸Gly,G

11HNCH1

HCH22CH

3AlanineAla,A

HCCH/\丙氨酸

22CHCH

33Proline脯氨酸Pro.P

GlycineAlanineProlineValine

Valine缴氨酸Val,V

COO'COO-coo-

♦♦♦Leucine亮氨酸Leu,L

n3nvriH3N—C—HH3N—C—H

w3Isoleucine异亮氨酸

3He,I

/火CH2

AA…L'TccRATH

CH3CH3CH3s

1

CH3

LeucineIsoleucineMethionine

Polar,unchargedRgroups

coo-coo-coo-

♦I.1

H3N—C-HH3N—C-HH3N—C—H

CH20HH—C—OH

I胪

CHSH

3极性不带电R基

SerineThreonineCysteine

Serine丝氨酸Sens

coo-coo-

♦IThreonine苏氨酸Thr,T

HN—C—HHN—c—H

33Cysleine半胱氨酸

段Cys,C

心Asparagine天冬酰胺

/%Asn.N

H2NO

AsparagineGlutamine

PositivelychargedRgroups

coo-coo

正电R基

Lysine赖氨酸Lys,K

Arginine精氨酸Arg,R

Histidine组氨酸His,H

Guanidino服基

Imidazole咪嘤

C=NH,

NH2

LysineArginineHistidine

AromaticRgroups

芳香族R基

Phenylalainine苯丙氨酸Phe,F,

非极性

Tyrosine络氨酸Tyr,Y,极性无电

Tryptophan色氨酸Trp,W,非极性

Rotate

ring

r1.I-:nainn-rr

PhenylalanineTyrosineTryptophan

1、非极性氨基酸（疏水氨基酸）8种

丙氨酸（Ala）缀氨酸（Vai）亮氨酸（Leu）异亮氨酸（He）脯氨酸（Pro）苯丙氨酸

（Phe）

色氨酸（Trp）蛋氨酸（Met）

2、极性氨基酸（亲水氨基酸）：

1）极性不带电荷：7种

甘氨酸（Gly）丝氨酸（Ser）苏氨酸（Thr）半胱氨酸（Cys）

酪氨酸（Tyr）天冬酰胺（Asn）谷氨酰胺（Gin）

2）极性带正电荷的氨基酸（碱性氨基酸）3种赖氨酸（Lys）精氨酸（Arg）组氨酸

（His）

3）极性带负电荷的氨基酸（酸性氨基酸）2种天冬氨酸（Asp）谷氨酸（Glu）

AlanineAlaAnonpolarNeutral1.8

GlycineGlyGnonpolarNeutral-0.4

IsoleucineHeInonpolarNeutral4.5

LeucineLeuLnonpolarNeutral3.8

MethionineMetMnonpolarNeutral1.9

PhenylalaninePheFnonpolarNeutral2.8257.206,1880.2,9.3,60.0

ProlineProPnonpolarNeutral-1.6

TryptophanTrpWnonpolarNeutral-0.9280,2195.6,47.0

ValineVaiVnonpolarNeutral4.2

positive(10%)

HistidineHisHPolar-3.22115.9

neutral(90%)

ArginineArgRPolarPositive-4.5

LysineLysKPolarPositive-3.9

AsparagineAsnNPolarNeutral-3.5

CysteineCysCPolarNeutral2.52500.3

GlutamineGinQPolarNeutral-3.5

SerineSerSPolarNeutral-0.8

ThreonineTb.rTPolarNeutral-0.7

TyrosineTyrYPolarNeutral-1.3274.222,1931.4,8.0,48.0

AsparticacidAspDPolarNegative-3.5

Glutamicacid（；h：EPolarNegative-3.5

Reversibleformationofdisulfidebond

两个半胱氨酸的端基形成二硫键

H3N

Cysteine

Cysteine

Absorptionofultravioletlightbyaromaticaminoacids

色氨酸、络氨酸的紫外线吸取图

f,0lW

•rXZW'»1*.h”

Lambert-BeerLaw

log(iyi)=8CL

Uncommonaminoacidsalsohaveimportantfunctions

aPo*d'fp

•NH

ptonlcalval.3

y<arboxygluUmirte

■■*anHE/OO

4*Hydroxyprolin€CH

片“刈

-CH>-CH3〉CHTCH、KM》,

OH

SHydroxytysine

2网2

整~2

6-N-MethyllysineDesmosine

ResiduescoatedbyModificatioitofCOMMonresiduesalreadyincotpora^dintoapolypeptide

HSe-CH,—CH—COO*

rare,dufingpeQ力〜300additionalaminoadds

*NHsyniNsisratherthanceaied

thavebeenfoundincells

S«leno<yst«inethmughapostsy泄论如modification

植物细胞壁胶原质collagen,胶原质co壁gen,肌浆球蛋白myosin,prothrobim,elastin弹性蛋白

细胞中坏发现了其他〜300种氨基酸

这些残基residue通过调整已经组合成多肽的氨基酸的常见残基而形成

Reversibleaminoacidmodificationsinvolvedinregulationofproteinactivity

氨基酸的可逆修饰控制蛋白质的活动

-o-r—0—CMi-^w-coo

产一!—・

-0p-o-CHTH-COQ-

)E.GluUmat#riMfhyl<st«r

H>o9pH<Mhrvonene

1-fM-COO-

PhMfriMtyrOMn«

-NH<CH)>CHa-O«1-CH-COO*

-CH,-O«i-CM,-CH；-,"TOO-

Aminoacidscanactasacidsandbases

氨基酸的酸碱两性

HH

I

R-C-COO*—COO-+H+R-C-C二0F・。

HNOHH,N+O-

*NH.NH22

ZwitterionNonk)nkformZwitterionicform

Macid

HHR-C—COO-+H*—,*

♦NH,

*NHj♦NH,Zwittarion

Zwitterion«sbase

asbase

Non-ionicform非离子态；Zwitter-ionicform两性离子态;Amphoteric两性的

对Gly的titration滴定

Isoelectricpoint(orisoelectricpH)

pl=%(p/+pfc2)=%(2.34♦9,60)=5.97

对比Gly滴定与乙酸、甲按滴定成果

P*a24681012

Methyl-substituted

carboxyland

aminogroupsCH,-COOH

H*

AceticacidMethylamine

Th«normalpKaforaThenormalpK.foran

carboxylgroupisabout4.8.aminogroupisabout10.6.

Carboxyland

aminogroupsH-i-COOH&

inglycine

HH*

<t*Aminoacid(glycine)u-Aminoocid(glycine)

叱・234叫片9g

RepulsionbetweentheaminoElect/onefatlveoxygenatoms

groupandthtdefxartingprotoninthecarboxylgrouppullelectrons

lowerstheforthecarboxylawayfroentheaminogroup,

group,andoppositelychargedloweringHspK.,

groupslowerthepKabystabi"

lizicg1h<zwitterion.

Aminoacidsdifferintheiracid-baseproperties

of-COOHgroup:1.8〜2.4

R基donotionize的氨基酸

of一NHjgroup:8.8〜11.0

IonizableR基的氨基酸e

三个ionization电离阶段f3个p口值

Peptidesarechainsofaminoacids

RHR2

H3N—CH—C—OH+H-N-CH-COO'F

IICNjOMHHHCM2HCKtH

OHJM-C--^-H-^-COOT

型检附sHq］卜电。condensationHOH0M0HON

AminoCarboxyl-

terminal<ndterminalend

R1HR2Serylglycityros\1alanylleudne

♦IIIor

H3N—CH-C—N-CH-COO'Ser-GIy-Tyr-ZMa-Leu

Hor

°SGYAL

Hydrolysis&CondensationPeptidebond肽键Di-peptide二肽Penta-peptide三肽

寡肽、低聚肽oligo-peptidepolypeptide多肽

Multi-subunitproteins有两个或更多的多肽链(noncovalently)结合

假如至少有两条链是iden:ical完全,致的-则这个蛋白质是oligomeric低聚物的，

而这些同样的units(包括一种多种链)被称为protomers原聚体

自然产生的蛋白质的residues残基一般少「2,000

Polypeptides有独特的氨基酸构成

某些蛋白质包括不是氨基酸的chemicalgroups（conjugatedproteins并合的蛋白）

TABLE3-4ConjugatedProteins

CfassProsthebcgroucExample

LipoproteinsLipids/3t-Lipoprotein

ofblood

GlycoproteinsCarbohydratesImmunoglobulinG

PhosphoprotensPfiosphate©oupsCaseinofmilk

HemoproteinsHeme(ironporfhynn)Hemoglobin

FlBvoprotemsRavinnucleotidesSuccinate

dehydrogenase

MetalloproteinsIronFemtin

ZincAlcohol

dehydrogenase

CalciumCalmodulin

MolybdenumDlnttrogenase

CopperPlastocyanin

这些非氨基酸的部分一般被称为prostheticgroup辅基

>ProteinSeparationandPurification

ColumnChromatography色层分离法

Crudeextract粗提取今今今Fractionation分播

Ion-ExchangeChromatography

离子互换色层分离

•Largenetpositivecharge

。Netpositivecharge

cNetnegativecharge

•Largenetnegativecharge

包括负电官能团的聚合物球珠

蛋白质混合物与阳离子互换剂加入试管

蛋白质下渗的速度由他们在目前ph下的净电量决定。

阳离子互换剂使得负净电量高的蛋白质移动更快被更

早洗提elute出来。

Proteinmixtureisadded

tocolumncontaining

cationexchangers.

Proteinsmovethroughthecolumnatratesdeterminedbytheir

netchargeatthepHbeingused.Withcationexchangers,proteins

withamorenegativenetchargemovefasterandeluteearlier.

ExclusionChromatography(gelfiltration)

排斥色层分离(胶体分离)

多孔渗水的聚合物球

蛋白质混合物与cross-linked聚合物加入试管

蛋白质分子根据大〃环一样而被分离

更大的分子更轻易通过，出目前更早的（分）储（部）分

fraction

Proteinmoleculesseparate

bysize;largermolecules

passmorefreely,appearingn

intheearlierfractions.123456

AffinityChromatography

同类色层分离

Proteinof

interest

ligand

蛋白质混合物

惴/爆

MixtureSolution蛋白质混合物与结合苕配体ligand的聚合物加入试

七

ofproteinsofligand管，

；配体可以与想要的蛋白质结合

Proteinmixtureb

addedtocolumn杂质雷白被冲出试管

containinga了

polymer-bound配体热溶液

ligandspecificfor

proteinofinterest.

4)

UnwantedproteinsProteinofinterest

arewashedthroughiselutedbyligand

column.solution.

TABLE3-5APurificationTableforaHypotheticalEnzymeI

FractionvolumeToialoroteinActMtySpecificactivity

Procedureorstep(ml)(mg)(units)(unlts/ngj

1.Crudecellularextract40010000100,00010

2.Precipitationwithammoniumsulfate2803.00096,00032

翼

3.Ion-exchangechromatography即40080,0002C0

4.Size-exclusionchromatography10060,0006CO

5.Affinitychromatography345,00015.0CO

Note:AlldatareoresentthestatusofttesampleaftermedesignatedprocedurehasbeenearnedoutActivityandspecificactivityarede

finedonpage94..

Activityfumts，表达一份溶液中酶的总单彳立数量

Specificactivity(units/mg)表达每毫克总蛋白质酹的单位置；酶的纯度

1.0unitofenzymeactivity=在25℃和最佳测量条件下,每分钟引起1.0nmol底物转化的酶

的数展(amountofenzymecausingthetransformationof1.0pmolofsubstrateperminuteat

25℃underoptimalconditionsofmeasurement)

Electrophoresis电泳

SDS-polyaeryIamidegel

Crosslinkedpolymer

polyacrylamide

letsasamolecularsie^e,sbwingthemigration

proteinsappradmateJ^inproportiontotheir

harge-to-massratio.

Relativemigration

IsoelectricfocusingTwo-dimensionalelectrophoresis

TABLE3-6TheteoeloctrkPointe

ofSomeProteins

A-oewpl

<1.0

照altxiE力4.6

Seamalbunvn4.9

Urease5.0

/Racto^obuln5.2

Hemogjotxn68

MyogioDtn7.0

Chymotrypsinogen9.5

C»tocfiron>ec10.7

Lysaome11.0

Thereareseverallevelsofproteinstructure

PrimarySecondaryTertiaryQuaternary

structurestructurestructurestructure

AminoacidaHelixPolypeptidechainAssembledsubunits

residuesf.

1氨基酸残基的线性序列

（氨基酸残基residues,包括二硫键）

2局部稳定的氨基酸残基序列所形成反复出现的构造模型，包括a-螺旋，B•折叠，B•转

角，无规卷曲四种构造

3在二级构造基础上深入折叠成紧密的三维形式

（多肽链，多种3d折叠后的多肽）

4由两个或多种多肽链（亚基）互相作用形成的空间构造。

（由蛋白质亚基构造形成为多于一条多肽链的蛋白质分子的空间排列）

（组合在一起的subunit,形成Multi-subunitProtein,Arrangementisspaceofpolypeptide

subunits。）

Determinationofaminoaddsequence

Theaminoacidsequencesofmillionsofproteinshavebeendetermined.

数百万种蛋白质的氨基酸序列已经被确定

Shortpolypeptidesaresequencedusingautomatedprocedures

Sanger'smethod

测定amino-terminal残基

TheEdmandegradationprocedure

降级过程

由一种测序仪进行

Largeproteinsmustbesequencedinsmallerfragment

打断disulfidebond

Osulfidebond

C-o

Acetylated

cysteine

扯破Cleaving多肽链

TABLE3-7RieSpecificityofSomeCommon

MethodsforFragrncntingPolypeptideChains

Reagentfdio<a0cdisourcerOeavo^powits1

Trypsinlys.Arg(C)

(bovinepancreas)

SubmaxiHantsprotease〃(C)

(mousesuomanllary0and)

Clvnotr>psinPhe.gM(C)

(bovinepancreas)

StapftyJooocct/saureusV8proteaseAsp.Glu(C)

(bacte«iumS.aureus>

AspWproteaseAsp.Glu(N)

(bactenumPseudomonas^agi)

RopjmPh«,Trp.Tyr(N)

dxxeinestomach)

LndopcoteioaseLysClys(Cl

(bacteriumLysobacter

enzymogeres)

Cy<aix)eentxonudeM«(C)

•AJm即THflaacxcym9tobmrrw^am⑷AmMMiUbM

cawnercMsources.

咯5加埠gprtflMiy但W6OOHlortfwBON统or

0m：pwodetmSoccirsonrthetthecabonyl(C)orttie

aniro«N)MedZgc“edsmnoaodmMLn.

某些proteases蛋白酶只把破临近特殊氨基酸残基的肽键

Orderingthepeptidefragments

ProcedureResultConclusion

h心。lyt•:ASH2I*11Polypeptidehas38

IVHWIQMH9C2J_1$2dminoaddresidues.Tryp・

D4IK~T1JrwillckavethreUes

t2J4V1(atoo*R(ArgIandtwo

FiIMdv2

PolypeptideK(Ly$Htogivefour

G3PI

trnnts.Cy»no9«nbromide

willclMveattwo

M(M#t)togiveth，”

reectwHtiFDNfe;hydrolyM；fragments,

2(4-0inHroptwnylglut«matvE(Glu>杯imino-

rXuc«detectedtecminalresidue.

<Skuffid«

tendsIlfp/eseotl

©

deeve.0；®CASMALIK，力p&cdataminoterminus

5•URItb«9lnswithE(Glu).

©EGAAYHOFEPIDPR

()pU<Xatcarboxylterminus

DCVMSO

o-becauseitdoesnotendwith

YLIACGPM1KR(Aro)orK(Lys).

(Move“MM9M

bwnitff；tepervftfr”nwntxki)EGAAYHDFEPIOPRGASM4i)ov»rljipswith

¥6―0•g6dm.c

,C2\TKEXVHSD(；And^)Mawing

Mel|C)

XJ)ALNCYLIACGPMthemtob«ordertd.

@®0©

匕JsAYHXM”%同鹏UypMvHSD:由蹙

Aminoacidsequencescanalsobededucedbyothermethods

Aminoacid

sequence(protein)Gin-Tyr-Pro-Thr-lle-Trp

I11111111Ii]

DNAsequence(gene)CAGTATCCTACGATTTGG

■・w.

8d。"

Investigatingproteinswithmassspectrometry

光谱测定法

uilMS3Xecw

-V-*"jL^J

100

I

E75

U5O

W2CS

A

A

e