B2

Enzyme Analysis

1

a.

b.

(albumin)

(immunoglobulin)

c.

1.1 Biuret

carbonyl carbonyl biuret ( mg) Tris Biuret BCA (bicinchoninic acid)

1.2 Lowry

biuret Folin-Ciocalteau phosphomolybdic-phosphotungstate ( 0.1 mg)

1.3 UV

ECX 2005 139

a. 280 nm 200 nm 280 nm

B2

(molar extinction coefficient)

b. E (1%, 280 nm) 4~15 ( 10) E 10 280 nm 1 1 mg/mL E b c

(c % 100 mL b 1 cm)

c. E 10 280 nm 1 1 mg/mL

1.4 Coomassie Blue (dye binding) Bradford Method

Coomassie Brilliant Blue G-250 (CBG) 595 nm ( g)

1.5

peroxidase heme 403 nm ()

1.1 (-N-C-C-N-C-C-) (Lys, Arg, Tyr )

140 ECX 2005

NC C N

N

N

CC

C CN

CC C

CNCC N C

CNCC

N CC

O

O

O

OO

O

OO

O

R

R R

R

R

R

H

H

H

H

H

H

H

H

HH

H+

Cu2+

OH...phsophomolybdic-phosphotungstate

Lowry Method

Biuret Method

UV Absorbance

280 nm

206 nm(carbonyl)

(aromatic)

CC

CCCNH3

+

LysCC

C

NH2+

Tyr

Coomassie Blue G

HNC

Special Binding Groups (heme)

(carbonyl)

(metal)

Arg

NH2

O

ActiveSite

Fe N=NOH

SO3-

NH

SO3-

SO3-

CuCu

N=N

OH

SO3-

NH

SO3-

SO3-

B2

2

2.1

a.

2.1

(1) () --

(2)

(3) ( pH ) ()

(4) ()

(5) ()

b.

(Vmax)Km pH

2.2

(t) (P) P/t (vo)

2.2.1

(t) (P) (P/t)

a.

NAD+ NADH

2.1

NAD+

NADH

10 x Km

ECX 2005 141

B2

240 280 320 360 400

340

NA

Ethanol NAD+ Acetaldehyde NADH H+

NADH 340 nm ( 2.2)

b.

(P) (Q) 340 nm

S P Q 2.2 NADH

c.

(invertase, IT)

+ IT

d.

HPLC (PPC)

e. (manometry)

Warburg

f.

pH pH

g. HPLC

HPLC

2.2.2

a. 3~5% TCA () pH

b. (100) 10 min ( RNase)

142 ECX 2005

D

NADH

+

Wave length (nm)

B2

c. 1% SDS protease K SDS

d. EDTA

e.

f. (cold)

2.2.3 (continuous-reaction)

a.

340 nm

b.

2.2.4

a.

(Pi)

(Glc)n-Glc + Pi (Glc)n + Glc-1-P () (Glc)n + Glc-1-P (Glc)n-Glc + Pi ()

(Pi) Pi

b.

(primer) Glc-1-P

(amylose)

( 2.3 A)

ECX 2005 143

2.3

Glc-1-P

BA

SP

A

SP BAPh

Glucose+Pi

B

B2

c.

-amylase (BA) Glc-1-P phosphatase (Ph) ( 2.3 B)

()

2.3

2.3.1

a. pH 2.1

2.1

pH

Formate 3.0 ~ 4.5 Citrate 3.0 ~ 6.2 Acetate 3.7 ~ 5.5 Phosphate 5.8 ~ 8.0 HEPES 6.5 ~ 8.5 Tris 7.1 ~ 8.9 pH Borate 8.1 ~ 9.0 Carbonate 9.7 ~ 10.7 Universal 2 ~ 12 pH

b. ( 2.2)

2.2

144 ECX 2005

NaN3 (sodium azide) 0.01% EDTA, EGTA 0.1~1 mM -Mercaptoethanol 1~10 mM Dithiothreitol (DTT or DTE) 1~5 mM BSA (bovine serum albumin) 0.1~10 mg/mL Tween-20, Triton X-100 0.5~0.05%

B2

Glycerol, glucose 50% Urea 6~8 M PMSF, TPCK, TLCK, benzamidine

c.

pH pH (Tris)

d.

pH

2.4

2.4 e. Stock solution

pH

2.3.2

a.

b. (aliquot) () -

4

c. (-20) 50%

d.

2.3.3

a.

()

ECX 2005 145

1 mM 10 mM 100 mM 1 M

B2

b.

(1) pH

( SDS )

(2) cofactor

cysteine -SH EDTA -SH

(3)

PMSF (phenylmethylsulfonyl fluoride) Ser PMSF

(4)

c.

(1) 4

(2)

(3) BSA

(4) ()

(5)

(6) ()

2.3.4

146 ECX 2005

(activity unit) pH 1 mole

B2

3

3.1

3.1.1

a.

(mobility)

( mV) ( )

b.

pH pH pI pI () pH ( 3.1)

c.

pH 8.3 pH pI 8.3

d. ()

pH

3.1.2

( 3.2)

a. (moving-boundary electrophoresis)

ECX 2005 147

++ANODE CATHODE

Friction

Voltage

Charge

- -

76

89

10

543

11

+ -0Net Charge of Protein

Environmental pH

Isoelectric point, pI

3.1 pH

B2

- + b. (zone electrophoresis) Cellulose

Protein Denatured

(band)

(1)

() ()

(2) () cellulose acetate

(thin-layer electrophoresis, TLE)

(3)

(starch gel electrophoresis) (polyacrylamide gel electrophoresis, PAGE) (agarose gel electrophoresis)

c. (1) (isoelectric focusing)

(2) (peptide mapping)

(3) (Western blotting) (immunostainning)

(4) (preparative electrophoresis)

(5) (immunoelectrophoresis)

(6) (capilliary electrophoresis) HPLC

(7) Pulse field gel electrophoresis DNA

3.1.3

a. 100~500V

b. (1620) (810)

c.

3.1

()

(TLE) Cellulose acetate

Partial Denatured

Starch Gel

Serious tailing

148 ECX 2005

3.2

B2

3.2

PAGE PAGE

3.2.1 PAGE

a. (disc-PAGE)

Disc-PAGE PAGE

SDS-PAGE

b. SDS (SDS-PAGE)

SDS SDS-PAGE SDS-PAGE (denatured) (native)

c.

pI 8.3 pH SDS -SDS-PAGE

3.2.2 PAGE

3.2.2.1

a. (monomer) (acrylamide)H2C=CH-CO-NH2

Acrylamide Bis

b. (bridge) Bis [N,N'-methylene-bis(acrylamide)]

c. (free radical) (ammonium persulfate, APS) riboflavin ( BB2)

d. TEMED (tetramethylethylenediamine)

3.2.2.2

a.

ECX 2005 149

b.

B2

c.

3.2.3 PAGE

3.2.3.1

3.2

pH

1 () Tris-glycine 8.3 -

2 Tris-glycine 8.3 -

3 Tris-HCl 6.9 5%

4

Tris-HCl 8.3 5~20%

5 () Tris-glycine 8.3 -

a. 3, 4 pH (3) pH (pH 6.9, glycine pI) (disc)

b. 3.3 (4) (3) (2) (1) (5) ()

c.

3.2.3.2

( 3.4)

Glycine ( pH > 6.9) ( pH = 6.9 zwitterion)

a. 3.5A

Gly

b. -- pH pH 8.3-6.9-8.9 Gly pI 6.9

150 ECX 2005

Sample

Running gel

+

-

Stacking gel

(1)(2)

(3)

(4)

(5)

3.3

3.4

Glycine: Negative charged No net charge

Chloride ion:

Proteins:

B2

c. 3.5B Gly () Gly

d.

e. 3.5CGly

3.2.3.3

3.6 disc-PAGE SDS-PAGE

a. X, Y, Z X > Y > Z pH (8.3) X Y (pI < 8.3) Z (pI > 8.3)

3.3 Mobility

Protein QuaternaryStructure

MolecularMass (D)

pI Native PAGE SDS-PAGE

X Tetramer (40,000)4 5.8 Y Monomer 88,000 5.2 Z Monomer 60,000 9.3

b. native-PAGE X, Y Z Z X (160 kD) Y

c. SDS-PAGE X, Y, Z SDS SDS ()

ECX 2005 151

A CB

+ +

8.3

6.9

8.9

3.5

B2

d. SDS X, Y, Z

Z Y

e. X Y Z SDS-PAGE SDS (40 kD) Y Z

f. SDS-PAGE SDS X (160 kD)

3.2.4

a.

ammonium persulfate, TEMED, acrylamide APS APS

b.

Bis acrylamide

c.

() -mercaptoethanol acrylamide

d.

() APS

152 ECX 2005

3.6 Disc-PAGE SDS-PAGE

X Y Z x Y Z+ SDS

+ +

Native-PAGE SDS-PAGE

+-

-

B2

e.

pH ()

f.

g.

pH 6.9

h.

()

3.3

3.3.1

3.7 ()

a.

(1) (ammoniacal silver)

CBR

(2) Coomassie Brilliant Blue R-250 (CBR) CBR

Coomassie Blue R-250 G-250

b.

(glycoprotein) (3) PAS (periodic acid-Schiff's) CBR Schiff

c.

ECX 2005 153

UV 300 nm (4)

B2

d. KCl

SDS-PAGE 0.3 M KCl 4 15 min (5) SDS KDS

e.

(6)PAGE (disk)

f. (autoradiography)

154 ECX 2005

NC C N

N

N

CC

C CN

CC C

CNCC N C

CNCC

N CC

O

O

O

OO

O

OO

O

C

R R

R

R

H

H

H

H

H

H

H

H

HH

H+ -

CC

CCCNH3

+

Lys CCC

NH2+

Tyr

HNC

Arg

NH2

O

ActiveSite

N=N

OH

SO3-

NH

SO3-

SO3-

Coomassie Blue R

CCC

NH2+

Ammoniacal

silver

Ag

Glutaraldehyde

(Cys)

Ag+H N...2NHGlu

3H N...Ag...NH3+

Lys

OHUV

Absorbance(300nm)

CO H

OH OH

CO H

Periodate

Schiff's reagent

Ammoniacal silver

CN

H

CarbohydrateStaining

Ag

C

N

H

Dye3H N...Ag...NH3

+

Ser O C

Specific Binding Groups (metal,SDS-KCl)

N=N

OH

SO3-

NH

SO3-

SO3-

OO

3.7

B2

g.

p.72~74 (gel dryer) (scanner)

3.3.2 (IEF)

Ampholyte pI ampholyte ampholyte pH pI pH (pH = pI) IEF pI

3.3.3

IEFSDS-PAGE

disc-PAGE

3.3.4

a. (nitrocellulose) ( 3.8A ) (nylon)

b. ponceau amido black (immunostaining) ( 3.8B)

c.

ECX 2005 155

B

CoomassieBlue

Staining PonceauStaining

A

+

Gel

-

HRP

Filter Paper

Nitrocellulose

3.8

B2

4

4.1

a.

b. (molecular weight marker)

(kD)

Thyroglobulin (669, 330); ferritin (440); catalase (232); immunoglobulin G (160); lactate dehydrogenase (140); serum albumin (67); ovalbumin (43); lactalbumin (14.4)

c.

(peroxidase 405 nm heme )

d. Blue Dextran 2000 ( 2,000 kD)void volume (Vo) Blue Dextran 0.1~0.2 M (NaCl)

e.

(1) Vo

(2) HPLC FPLC

4.2

a.

156 ECX 2005

disc-PAGE SDS-PAGE SDS-PAGE

B2

b.

pI disc-PAGE SDS-PAGE

c. (marker)

d.

disc-PAGE pI (4~6 )

4.3

4.3.1

a. (S)

b.

4.3.2

a. (cDNA)

b. (post-translational modification) ()

4.3.3

ECX 2005 157

B2

5

( Tris )

5.1 N- C-

N-

a. N- C-N- dansylation dansyl HCl dansylation N- ( 5.1)

b. polyamide (TLC plate) dansyl UV 20 dansyl HPLC

c. N- (blocked)

d. ( chymotrypsin)N-

e. C- carboxylpeptidase C-

5.2

a. 6 N HCl 4 N methanesulfonic acid 110 24 hHPLC

b. metallothionein Cys Cys

c. HCl tryptophan glutamine asparagine glutamic acid ( Glx) aspartic acid ( Asx) ( Tris) HPLC

158 ECX 2005

5.1 dansylation N-

Dansylation

HCl hydrolysis 2-DChromatography

N-terminal

TLC plate

UV

N-

B2

5.3

5.3.1 cDNA

(reverse biochemistry) cDNA

GCG ()

a.

(SWISSPRO)

b.

c.

signature signature PEST (KDEL)

d.

5.3.2 Edman

a. Edman

dansylation N- PITC (phenylisothiocyanate) N- PTH Edman N- HPLC Edman ( 5.2)

ECX 2005 159

5.2 Edman degradation

N-1 2

1

1

2

2

PITC

PTH-

Second cycle

Amino acidanalysis

PTH-

PTH-

N-

Peptide

B2

b.

c. (1)

(2)

(3) prosthetic group

(4)

d.

(1)

(2)

()

5.4

a.

( 5.3)

b.

160 ECX 2005

B2

5.4.1

a. Trypsin (Lys, Arg); Chymotrypsin (Phe, Tyr, Trp); Sa protease (Asp, Glu)

b. CNBr (Met)

5.4.2

a. /

90

b. HPLC

HPLC

c. SDS-PAGE

SDS-PAGE

5.5

5.5.1

(280 nm) 1.3

5.5.2

a. X

b. NMR ()

ECX 2005 161

5.3

Proteases Cutting sites

B2

6

ELISA

( Harlow E, Lane D (1988) Antibodies,

A laboratory manual. Cold Spring Harbor Laboratory)

6.1

a.

b.

() ( carrier BSA KHL hemocyanin)

c.

() hapten () carrier

d.

() carrier (PC/GENE)

e.

f.

162 ECX 2005

B2

6.2

a.

6.1A

(TiterMax)

b.

6.3

a.

ELISA ELISA 5,000 ( 5,000 ELISA 50% )

b.

6.1A pristane NS-1

c.

ECX 2005 163

6.1

BALB/c

2

(Trial Bleeding)

0

4

6

8

NS-1 Cell10

Ascites Fluids12

14wk

spin down cells ()+ 2X mL PBSammonium sulfate (AS) fractionation 0-40% sat.spin down pellet

Pelletresuspended in 40% AS

spin down pellet

Pelletdissolved in X mL PBSdialysis in PBS,three changes

spin down precipitate

IgG (stored in freezer)

+ glycerol (equal volume)Supernatant

X mL (X = 1 - 10)

BALB/c

A

B

(10 cells)6Pristane (0.5 mL)

TiterMax

Freund'sIncomplete Adjuvant

Antigen (50 mg/mouse) 0.5 mL

Freund's Complete Adjuvant

B2

d.

(Ig) ( 6.1B)

6.4

a.

b.

(CNBr-Sepharose) ( 6.2)

c.

d.

e. (ELISA)

164 ECX 2005

CNBr-Sepharose

SDS-PAGE

Ab

H

L

Coupling

6.2

B2

7

7.1

Central Dogma Central Dogma

(1) 7.1

(2) 7.1 2000

ECX 2005 165

Protein Technology

327

323

Basic Protein Techniques

373

363

Protein Purification

M1220

380

M1230

Protein Analysis

368

375

365

364Proteome

374

Protein Structure & Function

Protein Engineering

Protein Function

Protein Structure

Protein Engineering

Biochemical Engineering

356

7.1

B2

(3)

7.2

7.2.1

(what you see, what you get)HPLC

7.2

(1) SDS-PAGE

(2)

166 ECX 2005

7.2

MALDI-TOF

(1) (2)

(3)

(4)

B2

(3)

N-

HPLC

(4)

HPLC FPLC LC

7.2.2

(1)

Edman degradation 10~100 pmole

PC/GENE

(2)

LC LC/Mass

7.3

Genomic Project

proteomeProteome

7.3.1 proteome

(1) proteome proteome hexokinase glycolysis TCA cycle

ECX 2005 167

(2)

B2

(3)

7.3.2 proteome

(1) (marker protein)

(2)

168 ECX 2005

Blackstock WP, Weir MP (1999) Proteomics: quantitative and physical mapping of cellular proteins. Trends Biotech 17: 121~127

1 1.1 Biuret 1.2 Lowry 1.3 UV 1.4 Coomassie Blue (dye binding) Bradford Method 1.5

2 2.1 2.2 2.2.1 2.2.2 2.2.3 (continuous-reaction) 2.2.4

2.3 2.3.1 2.3.2 2.3.3 2.3.4

3 3.1 3.1.1 3.1.2 3.1.3

3.2 3.2.1 PAGE 3.2.2 PAGE 3.2.2.1 3.2.2.2

3.2.3 PAGE 3.2.3.1 3.2.3.2 3.2.3.3

3.2.4

3.3 3.3.1 3.3.2 (IEF) 3.3.3 3.3.4

4 4.1 4.2 4.3 4.3.1 4.3.2 4.3.3

5 5.1 N-C- 5.2 5.3 5.3.1 cDNA 5.3.2 Edman

5.4 5.4.1 5.4.2

5.5 5.5.1 5.5.2

6 6.1 6.2 6.3 6.4

7 7.1 7.2 7.2.1 7.2.2

7.3 7.3.1 proteome 7.3.2 proteome