1 2

1 1.1 1.2 1.3 1.4

1.1

pH

HPLC

(/)

pH

1.2 1.2.1

1.2.2 (1) 1000020000r/min

(2) -15-20401000105 Pa2000105 Pa 90

(3) pH 37pH815 SDS

1.2.3 pHpH

1) ()DNA-(KClNaClNH4Cl(NH4)2SO4)

2) pHpHpH=68pH 3) 05 4) pH 5)

1.3

1.3.1 pH

pH (Polyethyene glycol, PEG)

1.3.1.1

-COOH -NH2 -OH 1100nm

(NH4)2SO41)(04)0 (g/100ml) 0 20 80 100 (NH4)2SO4 70.6 75.4 95.3 103 Na2SO4 4.9 18.9 43.3 42.2 NaH2PO4 1.6 7.8 93.8 1012)7543)23mol/L(NH4)2SO44)

(mg)

10 20 30 40 50 60 70 80 90 100

1) 2.53.02530mg/mL2) pHpH3) 04

1.3.1.2 ()

520mg/mL

pHpH5

1.3.1.3 pH

1.3.1.4

1.3.1.5 HOCH2(CH2OCH2)nCH2OH (n4)(Polyethylene GlycolPEG)600020000 PEGPEG

1.3.2 (MwCO) 10KD 1 BaCl2 (NH4)2SO41AgNO3 NaClKCl

1.3.3

4104Pa5001411044104 7105Pa10-10035105140105Pa10

pH1050

pH1-149012 (cm3/(cm2h))()()

1.3.4

OOC-40 0.1mmHg -60 0.01mmHg100580

1.3.4.1 pHpH 15 mg/mL

1.3.4.2 2 cm -

1.3.5

1.3.5.1 (1) (2)

(3) ()

1.3.5.2 (1) HPLC(2) 1/101/6

() -20-70

1.4 1013

pHpH

-5-20-70HCl9050601530 min

(14mol/L)MgSO4NaCl(NH4)2SO4

2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9

(1) .(2) (3) :

2.1 (Enzyme Activity) mol/s2.1.1 2.1.2 /

(Molar Catalytic Activity) Vmax=K0[E] K0=Vmax/[E]K0 (Kcat)n

(1/s) (1/s) 600,000 100 25,000DNA 5 2,000Trp 2 1,000 0.5

2.1.3 () (Specific Activity) IU/mg 2.1.4 pH2.1.4.1 (Stopped Method) SDS

+ O2 + H2O2H2O2:

4- 435nm A435 4-H2O2 () 5

ATP -6-P--6- -6-P + NADP+ 6-P- + NADPH + H+ 340nm

(2) NH3CO2

3H

2.1.4.2 (Continuous Method)

+NAD+ +NADH+H++NADH +NAD+340nmNADH340nmNADH(Glycosidase)Methylumbelvifery + glucoside + Methylumbeviferone (Methylumbelviferone) + mgATP 6-P- + ADP + H+pHpH405nm

(pH) (Aldolase) 1,6-3-P- (Triose Phosphate Isomerase) (Coupling Enzyme)-1-P- (Glycerol-1-Phosphate Dehydrogenase) NADH NAD+

1-P-

2.1.4.3 515 ()pH (v) (E) v (E)

[S][E]/ 5min10min

2.1.4.4 : O2+ O2+2H+ H2O2 + O2 O2 O2 O2 SOD (ESR) (NMR)SODSOD50%SOD 1 (1) (SOD)

pH8.250mmol/L Tris-HCl4.5ml2520min45mmol/L10mol/L HCl10l1cm 30s 325nm 0.070 OD/min 10l 10l 4.5(u/ml) = {([(0.07 - )/0.07]100%)/50%} (/) = (u/ml)

(2)400nm 5mmol/L 200ulpH4.60.2mol/L 0.1mol/L 200ul3710minl100ul37 15min0.5mol/L Na2CO3 1ml () 400nm A 1mol1

2.2 2.2.1

2.2.1.1(1) (2) (3) 2-15(4) N2/CO237(5) (6 )

2.2.1.2 (1) EDTATrisMgCl2(2) 25-30 35-403-52-3h(3) (4) -1,4-(5) pH

2.2.2 2.2.2.1 pH pHpHpH pH 4-6

2.2.2.2 0.020-0.050 mol/L0.15mol/L NaCl2.2.2.3 0-4372.2.2.4 1-52.2.2.5 ,LysC

- Cyt C0.145mol/LTCA TritonTweenTeepolSDS0.1 8

Al(OH)3Ca3(PO4)2

(%) 1.210.611.3 2.410.123.8 3.811.233.9 8.411.970.6 10.411.689.7 9.911.685.9 2.910.926.6

2.2.3 1-l0m1.03

200mlpH 6.4

0.26% (PAM) 200-300, 328-429l/L, 25%Al2(SO4)3, 0.65-0.84%, PAMAl2(SO4)3 (), 400-500, 0.0047% PAM, 105 l/L1.5-2.0%AlCl36H2O 1%, PAM 60l/L, Na2HPO4 1.5%, CaCl2 2.5%, pH 8.00.8ml/5min4.5ml/5min

29ml/5min

24ml/5min

5, 500L/h - 1000mlpH 6.9

- 1000ml 8471 100mlpH4.0 Al2(SO4)3 25ml, 0.47%,200 PAM (0.13%) 300ml, 294l/L, 1.7%, PAM 0.35% (350l/L)30% 4ml, 700PAM 0.2% (74l/L)20ml23min20ml35min

1, 81.5ml/min1.7,

0.1-1.5

pHpH Duolite S3011pH5.555pH5.50.5-1 (Na2SO3NaHSO3)

2.2.4 0.1-1

1/4pH2.2.4.1 pHpH 7NaH2PO4pH3.5

2.2.4.3 pH2.2.4.2

.2.4.4 Sephadex G-250G-502.2.4.5 (1) (2) (3)

2.3 MnCl20.1-0.2410,500r/min.2.3.1

(1)(2)(3)(4)(5)

(IU/ml)(mg/ml)(ml) 2.3.2 1993, 25(1):27 (Agave americana) 30Sephadex G-50SP-Sephadex C-50:

(ml)(mg)(IU)(IU/mg)(X)(%) 1. 11036564682.10177.30- 2. 10145.547179.84324.401.83 3. Sephadex G-506523.338810.341665.689.40 4. SP C-504011.436563.453207.3218.10 5. -6.2829642.344346.3424.50

2.3.3

2.3.3.1 pH

(1) 50(2) 3-50mg/ml(3) (4) 0-44 (5)pH pH0.2pHpHpI

(6) Ca2+Zn2+Co2+Ni2+Cd2+Cu2+Mg2+Mn2+(7)

(1) (2) pH 2.3.3.2

(3) 1mg/ml104(4) (5) pH(6)

(1) (2) (3) 10(4) 2.3.4

(0-4) pH pHpH 2.3.5 (1)

SH--GSHDTT Ca2+-Mn2+Cl-1 (2)

2.4 2.4.1 (Dialysis)

(Ultrafiltration)

(1) 0.2mm,4-20a b

5%a)b)

40

2.4.2 (20,000r/min) (80,000r/min) (ac)ac (W, 1/S) (r, cm) ac W2r2 W2 n/60 n ac42n2 r/3600R.C.F g(g980cm/s2) R.C.F 4 2n2 r/3600 980 R.C.F11.18 10-6n2r

()(r)(0)(g/cm.s)(t)Xt(cm), Xb(cm)

2.4.3 (Gelfitration) (Gelfitration Chromatography) (Molecular-Exclusion)(Moleculai-Sieve Chromatography) ()

2.4.3.1 (1) 1000-500000Sephadex G ()Gg () 10Sephacyl-SN,N-Sephadex-LH

(m)(mg/g )(mg/g )(h)(Mw)(Mw)20100 G-1040-120

(2) N,N-Bio-Gel PP-1000 ()

(m)(Mw)(mg/g )(mg/g )(h)20P-250-400150-40200-20001.532-42P-450-400150-40800-40002.44.82-42P-50-400150-401000-60003.77.42-42P-1050-400150-401500-200004.59.42-42P-3050-400150-402500-400005.711.410-123P-6050-400150-403000-600007.214.410-123P-10050-400150-405000-1000007.515.4245P-15050-400150-4015000-1500009.718.4245P-20050-400150-4030000-20000014.729.4435P-30050-400150-4060000-40000018.036.0485

(3) D-3,6-Sepharose2B4B6BBDNASepharose CLDio-Gel A

(m) (%) 6B40-2101104 41066 4B40-19010421074 2B50-25010421072 A0.5m

pH4-9pH2.2.4.3.2 A280VeRs

Rs/Rs1.2Rs ( ) fpr2/L, fPr1/4-1/201/100-1/500.1%

2.4.3.3 (Vt)(VoVt31 (Ve)(Vt-Vo)80VoVt30-3555 8-109010

2.4.3.4 0.1mol/HCl0.1mol/LNaOH002NaN3

2.5.1 (Ionic Stength)(Salting Out) :2.5 .

O697g/L25767g/L).1000 4mol)100m1S1S2ml(V) VVo(S2-S1)/(1-S2) 025SlS21L:

%20253035404550556065707580859095100

(%)1L *0106134164194226258291326361398436476516559603650697579108137166197229262296331368405444484526570615662105381109139169200233266301337374412452493536581627152654821111411722042372713063433814204605035475922002755831131431752072412763123493874274695125572502756841151461792112452803173553954364785223002856861171481812142492853233624024454883502857871181511842182542913293694104534002958891201531872222582963353764184502959901231561902262633023423835003060921251591942302683083485503061931271611972352733136003162951291642012312796503163971321682052447003265991341712097503266101137174800336710313985034681059003470950351000* 0

%10202530333540455055606570758090100

(%)1L *05611414417619620924327731335139043047251656166276710578611813715018321625128832636540644949459269420295978911231551892252623003403804245206192530496193125158193230267307348390485583301930629412716219823527331435644954633124374107142177214252292333426522353163941291642002382783194115064031639713216820524528537546945326599134171210250339431503366101137176214302392553367103141179264353603469105143227314653470107190275703572153237753611519880771579079* 25

pHpHPIpH5pH601mg/ml100ug/ml(200ug-1mg)/m1 .,

2.5.2 (Dielectric Constant). 2.5.2.1 0-15-20

2.5.2.2 Sumner32.(m1) V=Vo(S2-S1)/(S-S2)VVoSS1S2

2.5.2.3 5-100.05molZn2+Ba2+pH0.05-0.02molZn2+. pHpHPI

2.5.2.4 pH(Isolectric PointpI)pHpH.pIPIpHpHPHPIpH

2.5.2.5 1964Polson(PFG)(PEI)(SDS)PEG6,0004,00020(W/V)pHPEGEcoRPEIPEI0.2mol KClDNAKCl0.6molEcoR

2.5.2.6 (PAA)PAA

PAA + PAA- PAA- + Ca2+ PAA-Ca2++ PAA-Ca2+ + SO42- CaSO4+ PAA

PAA

2.6 2.6.1

2.6.1.1 .5-10

5-1010.13-5 pH4-6)5NaCO3pH6.5-7.0NaCl1/570-85

2.6.1.2 Ca2+50g/L0.5h1mol/L EDTA0.1mol/L NaOH1mol/LHCl

2.6.1.3 (Ion-Exchange Chromatography) pH(1)

Dowex#704#724#732101101AmberliteCC-50

: pH: . (2.5 - 4.5mmol/gpH

pHpHpH

pK(mmol/g)DEAE-9.1-9.50.1-1.1TEAE-100.5-1.0GE-0.2-0.5pHECTEOLA+7.4-7.60.1-0.5DEAE-Sephacel1.40.1DEAE-Sephadex A-253.50.5 A-50QAE-Sephadex A-25-2-3.00.4 A-50DEAE-Sepharose0.130.02CM-3.6P-1-2pHSE-6.0-6.5pHCM-Sephadex G-252.24.50.5 G-50SP-Sephadex G-252.30.3 G-50CM-Sepharose CL-6B0.120.02

(2) : pIpHpIpHpIpH pI69pH6pH9pH

(3) pHpHpIpHpI1pH

(4)

0.5mol/L0.5h0.2mol/LpH

pH1/41/315pH/pH/pH

pHpHTrispI1 pHGlypI1 pH

.

pH pIpI

2.6.22.6.2.1 (electrophoresisEP)

2.6.2.2 18081937Tiselius A121948

2050601967 Shapiro A LSDS-7090

2.6.2.3(1)

NaCl Na+ + Cl

H+

(2)(mobility)QE F = QE (1)FStock F = 6 r v (2)vrStocksF

F=F(1)(2)

3 mE (V/cm) t(s)d(cm) (1 V/cm) 4

(3)(4) Q m = (5) 6r (4)cm2s-1V-1

(3)vEJK

v = mE= mJ/K (6)vEmJmK

2.6.2.4 (1) (zone electrophoresis, ZEP) (3-2a)(2) (moving boundary electrophoresis, MBEP)(3-2b)(3) (steady state electrophoresis) (isotachophoresis, ITP) (isoelectric focusing, IEF) (2c2d)

a b c d a b c d

2.6.3 (Chromatofocusing) (pH7-9)pHPHpHpH6-9pH9 (pH) (pH6-9)pH7 (pH)pH7-97-9pH7-9pHpH7-9pH

(1)(2)pH(3)pHPH5-10ml(4)pH(5)

pH10.5-9PBE118pH 1110.5-8PBE1180.025mol/L -HClpH8.0 Pharmalytic pH8.0-10.5 HCl1:451.511.513.010.5-7PBE118-HClpH7.0 Pharmalytic pH8.0-10.5 HCl1:452.011.513.59-8PBE 94pH9.40.025mol/L -HClpH8.0 Pharmalytic pH8.0-10.5 HCl1:451.510.512.09-7PBE 94-HClpH7.0Polybuffer 96-HCl1:102.012.014.09-6PBE 94pH9.40.025mol/L -CH3COOHpH6.0Polybuffer 96- CH3COOH1:101.510.512.08-7PBE 94pH8.30.025mol/L Tris-HClpH7.0Polybuffer 96-HCl1:131.59.010.58-6PBE 94pH8.3 0.025mol/L Tris- CH3COOHpH6.0Polybuffer 96- CH3COOH1:133.09,012.08-5PBE 94Tris- CH3COOHpH5.0Polybuffer 96(30%)+Polybuffer 74(70%) - CH3COOH1:102.08.510.57-6PBE 94pH7.4 0.025mol/L CH3COOHpH5.0Polybuffer 96- CH3COOH1:3.07.010.07-5PBE 94pH7.4 0.025mol/L HClpH5.0Polybuffer 74- HCl1:82.511.514.07-4PBE 94pH7.4 0.025mol/L HClpH4.0Polybuffer 74- HCl1:82.511.514.06-5PBE 94pH6.2 0.025mol/L HClpH5.0Polybuffer 74- HCl1:102.08.010.06-4PBE 94pH6.2 0.025mol/L HClpH4.0Polybuffer 74- HCl1:82.07.09.05-4PBE 94pH6.2 0.025mol/L HClpH4.0Polybuffer 74- HCl1:103.09.012.0

2.7 2.7.1

2.7.1.1 (Sepharose CL-4B6B)(1) (2) .(3) .(4) pH,

2.7.1.2 (1) -10-8 mol10-4 mol

1-20umol/ml(2) --DL-DL-

2.7.1.3 pH1-520-30mg/m110m1/cm2h

(1)Amp-Sepharose(2)pHpH

Con A--D- -D-(--D-)(0-0.5mol/L); pH3; (0.1mol/L, pH6.5)Con A, (--D-)(0-0.1mol/L)N---D- (T-)(N---D-)N---D- (T-)Poly (u)-Poly A, A-mRNA(90%)Poly (A)-Poly u, u-hnRNAmRNARNA(90%)RNALysinerRNA(0.2mol/L) (0.05-0.3mol/L NaCl)Cibacron (Blue Dextran)(>50)2-NAD+(0-0.2mol/L); (0-1mol/L NaCl); pH5-AMP 2,5-ADPNAD+ NADP+NAD(P)+ATPNAD+, NAD+(0-0.2mol/L); (1mol/L); pH

pH-0.5mol/L NaClpH8.50.1mol/L Tris-HClpH8.50.5mol/L0.1mol/L pH4.5pH4.52.7.1.4 1

0.1mol/L NaOH1100-150mg250-300mg)NaOHpHpHpHll3010-20minBrCNpH5-10

R-NH2 (2-3min412h41mol/L 4h412h

2mol/LNa2CO352g BrCN2min

1.33-6mol/L45-508h0.1mol/LNaOH50m1100ml0.3mol/L HCl10m1 1mol/LNaNO22-5min0.3mol/L HCl0.1mol/L41-2h2mol/L NH4Cl lmol/L NH4OH(pH8.8)4h

X

A. NH2(CH2)nNH2--N-BrCN-NH-(CH2)n-NH2100ml100m1(100mol/L)5mol/LNaOH pH630pH45h2ml0.1mol/L NaOH2430-40min

2L1L300ml300-400ml300mlN-

-NH2410min6hpH5-8.51mmolGlypH92h

B.

(1) (Micrococcus lysodeikticus)Separose 4BDBrCNpH1068mg100-20080(2) 218090

(3) NAD+NADP+ATPAMPBrCN

NAD+40502.7.2 (1) 5mm(2) 5mm(3) 5-6cm

2.7.3 2.7.3.1BrCN

2.7.3.2 2.7.3.3 2.7.3.4 2.8 2.8.1 -7010-15min- -70-pH

2.8.2 pH-pH3--

2.8.3 -pH

2.9

190519521956(Golav)-5060,

,15pH

2.9.1.1 HPLC (High Performance Liquid Chromatagraphy, HPLC) 2.9.1

(1) A, B)150-450kg/cm2 HPLC

10-30cm2.1-4.6mm5-10um60HPLC

2.9.1.2 HPLC HPLC2.3m0.1m)100ml/minHPLC

2.9.2 (Size Exclu Chromatogyaphy, SKEC(GFC),8010-15um5-400nm500025-30nm5,000-5,000,000

pHpH6.5-8.01ml/min100 1. 2. 3.4. 5.C

2.9.3 (Ion Exchange Chromatogmphy, IEC)IEC IECIECIEC-

.pHpHpH 5-8pH 5.51mol/L KH2PO4pH 8, 0.5mol/L NaAcpHpIpHpIpIpHpIpH

.pH80

2.9.4 (Reversed Phase Chromatography, PRC)PRC-pH 3-7HPLC

2.9.4.1 C18C8CNC18C18C2C18C18CN10,0005-10nm20,00020-50nn

2.9.4.2

(TFA)(HFBA)2.9.5 (Hydropholic Interraction Chromatography, HIC)

HICHICHIC RPCHICHICPRCHICHICpH6-7pH