Anda di halaman 1dari 155

: .

2009
:

1.

,
2.

10

,
3.

20

,
,
4.

28

,
,
5.

37

,
,
6.

49

,
7.

- ANTITPETH KAI MH ANTITPETH


ANATOH

53

,
8.

PTENE AIMATO

62

,
9.

OOTIKO POIOPIMO YKOZH TON OPO AIMATO ANIXNEYH


YKOZH KAI KETONOMATN TA OYPA

73

,
10.

81

,
11.

94

,
,
12.
i

AIMOAIPINOAEIE - ENZYMA EPYPN AIMOAIPIN

106

,
13.

DNA 117
,

14.

AMATOKOIKE IIOTHTE KAI HEKTPOOPHH DNA

127

,
,
15.

ii

137

iii

iv

1
YATIKA IAYMATA
O , ,
()
. K '
, pH .
,

()

. ,

pH
. E
H+, pH ()
.
:
, .
pH
pH (pHmeter ). T
H2O. E pH
.
P
T
. E
pH
.
, ..
NaHCO3-H2CO3.
H pH
pK
pK, HendersonHasselbach :
pH = pK + log

[MA]
[]
= pK + log
[HA]
[]
1

pH 5 (pK=4,76)
.
E Henderson-Hasselbach:
pH = pK + log

[]
[]
[]
5 = 4.76 + log
log
[]
[]
[]

[]
[]

= 0.3

=2

pH = 5 CH3COOH
CH3COONa
.. 0.2 M : 0.1 M 1 M : 0.5 M.
T
pH . - 0.1 M, pH
4.5 0.1 M .

,
pH .
A .. 0.1 M
0.1 M.
E' :
C1 Co1 = 0.1 M

(0.1 M). (C2 Co2)
C2/Co2 = 2. E Vo1 Co1 = Vo2 Co2 V1
C1 = V2 C2 :

C1 V1
2=

C2
C2

V2
C1 V1
Vo2

V1

Vo1

V1/Vo1 = 2. A pK

,

, pH
.

T
,
:
1.

M (Mlarity). E
1000 ml ( ) M. 1 M
BaCl2 1 (mole) BaCl2, . 208 g BaCl2 1000 ml
.
. M
( ) .

2.

K (Nrmality). E
1000 ml N. 1 N
BaCl2 104 g BaCl2 (
, 2
) 1 .

3.

% (% w/v). E
100 ml .

-3

-6

, mM (10 mle/l), M (10 mole/l)


nM (10-9 mole/l).
A
T
. ,
( )
,
,
.
:
V1 C1 = V2 C2
V1 C1 ( ml)
V2 C2
. H moles/l /l % W/v
.
3

: 5% NCl 2 ml
0.25% NaCl. X V1 C1 = V2 C2 V1
C1 5%
V1 (ml) 5 = 2.0 (ml) 0,25 V1 = 0.1 ml
0.1
ml 5%.
(A)
- 0.1 M (pH 5) 0.2
M .
M pH
H pH :
.

M ( pH-meters). (T A,
K.3, ).

M ( ). O
.
- H. T
H
X A

H+ +
X B


H . M Henderson-Hasselbach (pH = pK +
log [ -]/[H]) []/[H]
pH, .
A .. pK = 5 pH 4, pH 5 pH 6 []/[H]
1/10, 1 10 .
pH pK
(H), pH > pK ()
().
pH 4-6 [-]/[H]
. E H pK 5
pH pH 4-6. pH
pH = pK 1.
4

O
pH pH 1-10.5.
, pH ,

pH.
INAKA 1
EIKTH

pK

pH

1.5

1.2 - 2.8

2. K 4.0

3.0 - 4.6

3. E

5.6

4.8 - 5.6

Ko

4. K

6.7

6.0 - 7.6

5. E

7.9

6.8 - 8.4

6.

9.4

8.3 - 10.1

1. K


EIPAMA 1. pH A
pH
A. pK
pH ( 1)
pH .



pH

1 ml A ,
.
. A ,
pH A.
A .. No 4 , 1 :
[H] > [-] []/[H] < 1 log []/[H] < 0
K pH = pK' + log []/[] pH < pK
A , , pH
pK . ,
1
pH .
B. 5 ml A pH
pH.
EIPAMA 2. - (0.1 M,
pH 5)

, 0.2
, 0.2
pH
6

10 ml ,
0.2 M .
pH pH.
3. P -

pH .
NaOH 10 ml
A pH
5 ( pH) 10 ( ).
E NaOH
pH, 10 ml H2O. T
.

NaOH, 0.1
( )

1.

2
. 0 NaOH 0.1
N. M NaOH
. ml 0.1 N NaOH
( ) ;

2.

10 ml H20 , pH pH
H20
A. ml 0.1 N NaOH
;

AOTEEMATA
1
A
(H ) . (pH > pK pH
< pK).
EIKTH

XPMA

1. K

pH

pK

2. K

pH

pK

3. E

pH

pK

4. K

pH

pK

5. E

pH

pK

6.

pH

pK

pH A
.

pH;

; ;
2
Y ml CH3COOH (0.2 N) CH3COONa (0.2 N)
CH3COOH/CH3COONa (0.1 M, pH 5), (pK
CH3COOH = 4.76).
3
ml 0.1 N NaOH
;
ml 0.1 N NaOH
H2O; E .

E 1
8


A ( 2) ;
E 2
K pH. pH
in vitro
. H pH :
o

B pH

7.6

6.0

P A

5.0

4.5

5.6


A;
E 3
- Helix pomatia
. H pH 4.5
pH 6.2.
,
4.3-6.5
; .

2
AMATOKOIA-AMATOTOMETPIA
A ( 1)
.
E ,
,


.
A
.

.

.

1
O

. A
(UV),
(Vis) (IR) . A
10

, . T

, max
( 2).

,
(OD)

NAD+ (max 260 nm)

NADH (max 340 nm)

250

300

350

,
(nm)

. T max. O

. M
.. pH .
H
.
NOMO LAMBERT - BEER
T
,
. O
Lambert-Beer :
log Io/ = k C l
:
I


11

H log Io/I (absorbance, )


(ptical density, OD) A OD, . A
C l = 1 cm, k
( AM, EM)
(extinction). O -1cm-1
, 1 M, l = 1
cm max. , NADH mM = 6.2 -1cm-1
NADH, 1 mM, 6.2 l = 1 cm
= 340 nm. A 100 ml
, k E1% A1% (
1% l = 1 cm). O EM E1%
:
EM = E1%/10 M
, ,
pH. To

( ).
H I/I (transmittance) T. H
log T = A. E
Lambert-Beer, A/C
. O
max
,
.
TO AMATOTOMETPO
(. 3) :
-

,

,

(),

(detector) ..

12

, ,
.

T ,
.

3
O
1.

3 ,
.

2.

K
. (300 400 nm) ,
(400 - 700 nm) . H

.

3.

E .

4.

P
, T = 0 % OD =

5.

. A
. A ,
.

6.

: T (Blank)

,
. E A+B+
B , ""
A .

13

T = 100 % OD = 0.
7.

K
H

( 2). T
OD. H :
.

,
.
,

.


. B
OD. OD
.

. T
max.

H

max. M :
.

Lambert-Beer.


, . B

. T
. H
14

(molarity, moles/l) (w/v %).


K B ,
moles/l ().


.
E . E
Lambert-Beer, .
H . A'
.
X
. A :
.
B OD
. H
.
E
,
. E
C, C/OD .
CM/ODM = C/OD = K
:
CM

(. )

ODM
C

OD
O

, .

15

M
H :
1.

T KMnO4
max.

2.

T (CmM-OD)
KMnO4 .

3.

T KMnO4 mM
,
.

4.

T mM max KMnO4

1 cm.

1. x mM
KMnO4

Y x mM KMnO4
H2O

I.
I
A
H 2O
x mM KMnO4

5 ml

5 ml

. P
440 nm. T
0. T
. E
20 nm
600 nm.
. K
max.
16

2. K KMnO4

H2O
Y 1 mM KMnO4

6
II.
INAKA II
A
A
H2 (ml)
1 mM KMnO4 (ml)

6.0

5.4

5.1

4.8

4.5

4.2

0.6

0.9

1.2

1.5

1.8

M .

1 . Y KMnO4
.
M KMnO4.

17

AOTEEMATA
1.

N A OD x mM
KMnO4.
INAKA A.

KMnO4

2.

(nm)

OD

(nm)

OD

(nm)

OD

400

.......

480

.......

560

.......

420

.......

500

.......

580

.......

440

.......

520

.......

600

.......

460

.......

540

.......

N
KMnO4.

3.

N max KMnO4.

4.

N B OD KMnO4
.
INAKA B. A KMnO4 max

5.

A/A

KMnO4 (mM)

OD

A/A

OD

KMnO4 (mM)

..........

........

..........

........

..........

........

..........

........

..........

........

..........

........

N KMnO4
.

6.

N x mM KMnO4
.

7.

N mM KMnO4 max.

18

1.

KMnO4, x/2 mM,


KMnO4, x mM,
.
.

2.

. 1
320 nm, 365 nm, 382 nm 500 nm. 1
242 nm, 355 nm 382 nm.
1 1
; .

3.

. 1
320 nm, 365 nm, 382 nm 500 nm. 1
242 nm, 355 nm 382 nm.

; .

4.

5.

, pH;
.

19

3
AMINOEA
:
.


Rf.


O

. A .

A . -
CO2,
. .

OH

OH

OH

O
NH2

R C COOH + 2
H

C
C

+ CO2 + R C O

C N C

N
H -

.
E
A (R )

. :
. A Sakaguchi. H
.
.
20

. A Millon. E
.
.
. A . H
NOH .
M NOH N2S
PbS.
M HCl PbS
.
N2S + Pb(CH3COO)2
PbS + 2HCl

PbS + 2CH3COONa

PbCl2 + H2S

280 nm
. ,
,
280 nm.
,
. 280 nm
.

(Thin Layer Chromatography, TLC),
.
50 G. E. Stahl.
.
.
TLC :
.

21

TLC
.
,
() () .

,
. ,
.
.


. ,
, . ,
, , . ,
,
.
. ,
, (
) ()
.
().
.
. ,
,
, , , ,
, , .
5 .
, ,
.
Rf . H
, .

Rf =

22

Rf
:
.


.
.
:
:
.

, .
Rf
.

. ,
.

.
.
,
.

23


1.


:
(pro),
(arg),
(tyr) (trp),
(cys),
(gly).
6 ml 2 mg/ml.

(
),

pro

Sakaguchi

arg

cys

280 nm
OD > 0:
tyr trp
24

OD = 0:
gly

.
. 1 ml
.
Sakaguchi. 1 ml 1-2
NOH 10%, 4-5 - 1% 2-3
(NOCl).
.
Millon. 1 ml 2-3
Millon (20 g Hg, 10 ml HNO3 HNO2). T
.
A . 1 ml 5 ml
10% NaOH.

15

min.

10

(Pb(CH3COO)2). . ,
1 ml HCl H2S.
280 nm.
. 280 nm
.
2-3 ml .
2.

, ,
0.25 mg/ml

- 7:3
2%
Silica Gel 60

().

25

. ,
,
.
1.5 cm.
. ,
. ,
( 5mm).
, ,
.
2 cm
.
.
, ,
110C
. ,

. , Rf
.

26

. Rf
.

27

4
PTENE
E pH
M
. O
( ). T
pH .
. pH
(
=0). To pH , pI. pH
pI . E pH
pI .
pI
.
pH pI .
. H
pH (6.3-6.6).
, pH. E pH
4.7, pI , ,
, .
E
O 35C .
, 35C
60-70C. M
.
E
O ,
Hg, Pb, Cu Zn.
HgCl2.
E

28


. M . O
, , .

.

1.

. T
Sephadex () Biogel () . H
.
E
. (Vo)
(Vi). O

:
V. = Vo + Vi
H .

. T .

T ,

29

.
Vo . T ,

, .
Vo + Vi . A
,
.
O Ve : Ve = Vo + Kd.Vi

Ve:
Vo:
Vi:
Kd:

O Kd 0 ()
1 ()
. O Kd .
H
,
.

) . M
( ),
.
H
0.5 10 cm. T
10 cm 1 m.
) T .
.

.
'
. H
, .
H .
30


.
) E . H
.

. O
.
M .
) T . T
,
.
. H
.
) . A

. H . K

, . ,
,
.
2.

.


.
,

SDS-

N, N--bis-

(SDS-PAGE). ,
bis-
31

. SDS ( )

. ,


.



, .


(Coomassie brilliant blue,
..),
.

SDS-


. -
( ) ,
SDS, .
.
.
, pH

.
( pH +)
( pH- +)
.
pH
( )
. pH
.
.
32


.
(
) . ,
SDS. ,
.


(.. ).



.

(Western blotting).

33


1. K

, 0.5
, 0.5
, 0.5%

5
. :
log 0.6 = -0.2218

log 2 = 0.3010

log 1.2 = 0.0792

log 2.8 = 0.4472

log 3.4 = 0.5315

pK CH3COOH: 4.7
A

A
1

0.5M CH3COOH, (ml)

3.4

2.8

2.0

1.2

0.6

0.5M CH3COONa, (ml)

0.6

1.2

2.0

2.8

3.4

0.5%, (ml)
pH (
Henderson-Haselbalch)


pI .
2. K .

' 2 ml
. T
.
3. K HgCl2


HgCl2
34

2 ml 3-4 HgCl2.
.
. ;
4. K Esbach.


Esbach ( , 2% , 1%)

ml

ml

Esbach.

A . O
. H

.
5.


NaCl
, 1
Esbach

Sephadex G-15, 1.6 40 cm

NaCl 1N CH3COOH.
H CH3COOH 1 N. H
. 3 ml
.
H Esbach NaCl
.

35

1.

2.

3.


HgCl2 .
;

4.

Esbach .
5.


.
NaCl;
NaCl ( x
y,
).

36

5
YATANPAKE
[C(H2O)]x.
() () :
i.

: 3 9 .
, , ,
.

ii.

: .
A , .

iii.

: .
, .

,
.
(
) D L . n
2n . ..
4 (22), D L D
L .

D L
.
D-, D-,
L- L-. D L

. D-
D(+), D-
D(-).
. D- D C-2.

..
,
37

,
.

, ,
.
A
1.

A Benedict ( )
M


. H Fehling
Benedict. T Benedict Cu++
. T CuSO4.
Cu++ Cu(OH)2,
. H Cu++
Cu2O
. K , ,
.
,
.
H Benedict ,
50-80 mg/100 ml.
( ).

.
2.

E
H , HCl,

5-

5-

,
. H 5-
. ,
. (POOXH!
T , )

38

E
2.

5-

A Molisch.
, ,

. -

.
.
2.

A Seliwanoff
To Seliwanoff HCl. H

HCl

5- + 32

5- +
2.

A Bial.
T Bial HCl, FeCl3 . H

+ 32

H
.
3.

. , ,
.
.
, ,
39

.

.

E .
O ,

,
.
, ,
l
c
.

. A
1 dm 1 g/ml 25C
, D N (589.3 nm).
[] 25
= /l c
D
.

X .
K

.
E
. T
- -
. K
(..
-D- ). ,
,
. O

X
,
, ,
. O

.
40

D-

-23.1

-23.7

L-

-54.0

+104.5

+175.0

D-

+92.0

+19.0

-20.0

D-

+113.4

+52.2

+19.0

D-

+144.0

+80.5

+52.0

D-

-21.0

-92.0

-133.5

D-

+34.0

+14.6

-17.0

D-

-7.7

+8.9

+54.0

D-

-43.4

41

-
-


1.


Benedict

Seliwanoff
Bial

6 ml :
, , , .
,
.
1.

A Benedict ( )

2.5 ml Benedict 0.5 ml


. 5-50 min.
,
.
2.

2.

A Molisch

2.5 ml , 4-5
Molisch . ,
, 1.5 ml
,
. ,
, /. E
,
. (T
).
2.

A Seliwanoff
42

2.5 ml Seliwanoff
2-3 .
15 min. H
.

Molisch


Benedict

Bial

Seliwanof

2.

A Bial

2.5 ml Bial 0.5 ml


. 30
min. O .
2. A

43

H ,
,
. ,
( A. 8). T
10 sec
.
3.

1 ml
.
. K
.
4.

30 ml , 10 ml , 10 ml




: 4 KnO4-Na2CO3 4:1
Whatman 1, 13 22 cm

A
44

. H
:
50 ml "
". T .
Whatman N 1 ( ) 2
cm 22 cm.
. M ,
5 mm, "
" A
(). E ,
, ,
.

,
. T

. H 80 .
B ,
. P
. O
Rf Rf . T Rf

.

45

E .
Rf ratio () front ().
A
.
,
Rf . M
.
5.


, 10%
HCl

Y ,
. H

,
.
P : . M
.
. M
(1) . T
(2). H
21= []T.
M :
.
A . M
HCl
(). A
5 min
().
46

Y (
) ().
(. ).

[]DT:

(g/100 ml)
.

(w/v)
() . K
.
[]D. A
[]T
( ).
A []20 52 ()
.
) X .
) X .

47

AOTEEMATA
A :
.


.
;


, .
.

X. . Rf
; ;

48

6
IIIA
M
. A .
E
.
H ( )
. ,
. T
(, ,
) , .
OMH KAI TAINOMHH TN IIIN
T: E .
. X
, . H
, ,
-.
,
.
(): ,
. H
. K
(LDL
HDL).
(): E ,
. H

.
: . '
.

49

KO MEPO
, ()
.
.
T
'
.
T .
EH IIIN-POIOPIMO ENEPOTHTA IAH
T .
A ,
.
, .

.
T


. T
(.. ),
- .
O
,
.
1. X

B , 37C, pH 7.2
, 10%
, 22%


X
NaOH, 0.1 N
50

: X
. 22% 200 ml/100 g
30 min. A . T
.
T :

50 ml

50 ml

50 ml

2 ml

2 ml

1 ml

A (.)

2 ml

1 ml

1 ml

A 37C. 10 ml
0, 10, 20 40 min 10 ml .
T NOH 0.1 N . K
NOH .
2.


. E .

M
X
NaOH, 0.05 N

, 10%
T 4 .

H
X
N

2 ml

2 ml

2 ml

2 ml

1 ml

1 ml

6 ml

6 ml

5 ml

7 ml

51

2 ml

2 ml

2 ml

A (.)

A 37C 3 min 10 ml
.
T .
NaOH .
ANIXNEYH KETONOMATN
T (, , - )
.
, .
,
.
(, , )
.

. T
H2O2.




ml .
0.5 ml . T 2 ml
. H
.

52

7

ANTITPETH KAI MH ANTITPETH ANATOH
ENIKA
O
"" ,
. T -OH ,
-SH .


.
,
. pH
.
.
,
.

.
,
.
,
.

katal. katal mol
(mole s-1) .
katal.
U (enzyme unit)
mol 25C
.

( )
.
53

(vo) ,
.
.

.
,


( 30 s 1 min) ( 5
10 min). ,
A y
x.
t = 0. H
dA/dt .


. A
.
:
1.

M 30 s , 5 10 min.

2.

x
y A.

3.

X .

4.

E dA/min.

5.

A dA/min

min.

7.
.

A katals.

M .

(A1) .
M t
(A2). H A = A1 - A2
t.
A/t
. Y

.

54



(.. , , EDTA, )
.
, ,

,
.

H , ,
,
.
.
.
.
,
. ,
. :
1.

.
.

2.

.

.

3.

.
- (ES).

E .. -SH
,
. H
- .
-SH

.
A,

.
.
55

56


1. A -
H :
H2N-CO-NH2 + H2O

(NH4)2CO3

A
. T
pH
.
T (SH)
Salyrgan :
Enz-SH + HO-Hg-R

Enz-S-Hg-R + H2O

H
-SH .

O ( )

R:

, 1M
Salyrgan, 0.5 mM

COO -

H
H H

, 2 mM

H
C

C N C C CH 3
H H O

CH 3

:
A

1 ml

1 ml

1 ml

Salyrgan

1 ml

1 ml

1 ml

H 2O

2 ml

1 ml

4 ml

4 ml

4 ml

!
57

15 min. .
2. M -M .
T
.
RCOOH + HOCH2CH2N+(CH3)3

RCOO-CH2CH2N+(CH3)3 + H2O

,
(E.C.3.1.1.7)

(E.C.31.1.8).

. T
pH 7.2
. H
.

,
.
H
. E
,
. E pH
.
H .

o N+

.



-OH

( 1).

1
58

H
. O
. H ()
( -OH
). I
-OH
. O
( 2).

2
O
. H
, , . A,
. A,
,

.
H 2.5 0.6 U/ml.

H
.
CH3COSCH2CH2N+(CH3)3 + H2O

CH3COOH + HSCH2CH2N+(CH3)3

H
(-SH) Ellmann
(DNTB: Dithio-BisNitroBenzoic acid = - ).

O
59

I , 0.156 M
: 0.0055% --
DNTB, 0.25 mM.

1.

:
A

DNTB (ml)

2.9

2.9

2.9

H2 (ml)

0.2

0.05

(ml)

0.05

I (ml)

0.1

0.1

O (ml)

0.05

0.05

2.

412 nm 1 .

3.

2
30 sec 3 min.

4.

3
30 sec 3 min.

5.

Y katal/ml o

- 1.36 104 M-1cm-1.

6.

Y % 3.


:
katal /ml = OD/sec 1/A B
:
A

[1.36104]

[0.0031] moles
3.1 ml

ml [1/0.05]
katal/ml

!
60

AOTEEMATA
A :

.
1.

1 ( ):

2.

2 ( Hg):

3.

3 ( Hg
):

M : M .
1.

Y Units/ml

2.

Y %.

61

8
PTENE AIMATO
T :
.

A. A
O2 CO2. H
: 15.4 1.5 g/100 ml , 14.2 1.5 g/100 ml
.

- . O

(.. ).
O

:
1.

H , pH, ,

2.

H , , , , .,

3.

H ,

4.

H .
, , ,

. 6-8 g/100 ml
. E , '
. O

(-) .

.
:
1.

K (M ).

2.

K (M Cohn).

3.

Y.

4.

H.
, (

), :
62

, 1-, 2-, - - (. 1).



.

, ,
,
.. ' ,


1: .
.
. -
.


.
, , .
() :
50% (3.5-4.5
g/dL).
, ,
, , Ca++ Cu++
.

2:

.
()
:

( ), , (),
( , ), ( )
.
1-: 90% 1- 1-,

(serpins: serine proteases inhibitors)
() . , 1-
, ,

63

1-

.
H 1- (1- ) ,

.
().
32 .
(
).
Down 18.
2-:
( 1:1) .
. .


(
..
). Wilson,


-: (
Fe3+/ ) , ,
.
, ,
. -
.

.
H C- (C-reactive protein-CRP)
.
6 (L-6)
. .
- ,

64

(V, VII, IX, XI, XII XIII), , ,


..
-: ()
CIq. ,

).
-
, , ,
.. -

().

65


T :
1.

2.

3.

4.

5.

6.

1. (
)
O
. ()

5 : , 1, 2, .
(
) .

,
. M
,
Ponceau S., Coomassie, Amido Black .. O
.

, Tris- pH 8.6, 0.1% NaN3


1% Tris- (
)
1:4
Amido Black 4% 10%
10%

.
.

66

.
.

5 l
.
.

80 olts 30.
.

, ( 60C)
.

. ,
-.

520nm.
2.
( )
M
N2SO4 24%. N2SO4
, .

O
N2SO4, 24%

:
24% N2SO4

5 ml
67

0.25 ml

A :

2 ml

. A
. 10 min 2.500
rpm. M ,
, . H
.

.
3.
O
(Biuret). H
Cu2+ . E ,
-CO-NH2
,
. O

.
.

NaCl, 0.9%
O
A (8 g NaOH, 15 g , 5 g CuSO4, 5 g KJ, H2O
1000 ml)

A . 5 ml 0.9% NaCl 0.25 ml

:
A

0.9% NaCl (ml)

2.0

2.0

2.0

2.0

A (ml)
A (ml)

A . A 30 min.
68

(540 nm) .
Y g/100ml
.
4.
.

N2SO4, 24%
Y
N2SO4
A

15,
. B

(2 ml).
.
:
A

2.0

2.0

2.0

2.0

24% N2SO4 (ml)


Y (ml)
A (ml)

A . A 30 min.
(540 nm) .
Y g%
.
.

.
T

. O (g%) (g%) = (g%).


5.
T C2+.
,
. O
,
69

0.5 M, pH 6.4
NaCl, 0.85%
(Simplastin) H2O
CaCl2, 1%
NaOH, 3%
A

( EDTA).
1.0 ml 30 ml 0.85% NaCl, 1 ml 0.5 M
0.5 ml 1% CaCl2. 0.2 ml
15 min. H
. B
. B ,

.
,
.
2.0 ml 3% NaOH 2 ml
' . B
37C . M 30 min
2 ml NaOH 3% 2 ml
g% .
: 0.2-0.4 g/100 ml .

70

AOTEEMATA
.

A :
1.

.
.

O :

2.

3.

4.

A :

71


0.6

0.5

OD

0.4

0.3

0.2

0.1

0
0

g/100 ml

72

9
OOTIKO POIOPIMO YKOZH TON OPO AIMATO
ANIXNEYH YKOZH KAI KETONOMATN TA OYPA
YKOZH AIMATO
H 8-12
70 - 110 mg%. O

.
, -
.
. ( 126mg%
200mg%) ,
(, , , )
.
.

126 mg%
,
.
M
Y . O
.

,
.
,
, ,

(.. , , ,
..) .

73



. ,
, ,
,
,
(, , , ..)
. 75 g 250-300
ml 5 min.
0
, 1, 2, 3 4 .
6 . ...
, ,
, .
6 .
: H
.
1.

( 0 ).

2.

X 75 g 250-300 ml
.

3.

A 5 30, 60, 120, 180, 240 min.

4.

Y 0, 30, 60, 120, 180


240 min
. A
.

140 mg/100 ml.


A
. K
(.. 75 mg/100 ml),
.
, .

(.. 220 mg/100 ml).
74

mg%

126

2 h 75 g

200

<126

2 h 75 g

140 <200

>110 <126


.
2.

.
YKOZH OYPN
T
.

, 160-180 mg% (.. ),
,
.
M
O

. A ,
Glukotest, .
M
.
KETONOMATA OYPN
A
, - .
E
75

().
M
O
,
, -. E
.

76


1. (
GOD-POD)
A : T
H2O2. T H2O2

. M
.

-D- + O2 + 2

(GOD)

2H2O2 + + 4-

D- + H2O2

(POD)

+ 4H2O

, 100 mg/dl 5.55 mmol/l


:

200 mmol/l, pH 7.5

O (GOD),

>15 mmol/l

Y (POD),

>3 mmol/l

4-,

0.3 mmol/l

5 mmol/

EDTA
M
, ,
() :
T

20 l

()

20 l

2.0 ml

2.0 ml

2.0 ml

10 min .
OD 510 nm.
.O
:
77

OD/OD 100 = mg /100 ml (mg %)


2.

6 : 0, 30, 60,
120, 180, 240 min. 8 , ,
.
(: ).
.
3. A Glukotest

Glukotest

1 min
.
. E

.
4. A
M Rothera ( ketostix)
A
T ,
, .

ketostix ,

78

.
.
M Gerhardt

H . T
.

10%

2 ml . T
.
. K
, .
E
, .
H Rothera.

79

AOTEEMATA
1.

2.



(mg%)

3.

(mg%)

N ( )

..........

..........

30 min ( )

..........

..........

60 min

..........

..........

120 min

..........

..........

180 min

..........

..........

240 min

..........

..........

X
N ( )

80

Glukotest

Ketostix

Gerhardt

..........

..........

..........

10
IOPTENE AIMATO

T (, , )
, ,
(200.000 - 10.000.000)
- ,
, A, B, C, D . O
. H
,
"", .

.
E
,
,

, , .
O

.

.
Y


-, -, -IV, B-100, B-48, C-I, C-II, C-III, D, E Lp().

-
. Apo A-I Apo B

. Apo B/ Apo A-I
,
.

81

:

.
O (risk factors)
: 1) , 2) , 3) ,
4) , , stress,
. M ,
. H
,
.

. ,
, .
T
O
.

.
O :
1.

2.

B ,
,
,
. A
,
. M 4
:
1) T ( ), 2) --, 3) 4) -.
O
, (scanning) ,
,
82

. K
.

(density g/ml) .
,
.
M 4 :
1.

X d 0.94 g/ml

2.

VLDL (Very low density lipoproteins: )


d 1.006 g/ml

3.

LDL (Low density lipoproteins: ) d


1.063 g/ml

4.

HDL (High density lipoproteins: ) d


1.21 g/ml.

H
I.
INAKA I: T
M

M
K

B-48, C, E



(VLDL)

B-100, E


(LDL)

B-100


(HDL)

T :
1.

X: A (90%) ().
,
.

2.

VLDL (--):
(60%). H
83

, LDL.
3.

LDL (-): VLDL ( IDL). To


(47%) ( ),
. O LDL 3/4
.
.
. O LDL Friedewald, :
LDL = O /5 - HDL (mg %)

4.

HDL (-): . T
(50%) (28%).

( ). H
HDL x
HDL .
HDL
. H HDL (HDL1, HDL2, HDL3)

. T,
HDL
. /HDL
5 .
M

,
,
( ), D.S. Fredrickson
() ,
.O.Y. ( O Y) .
, () -,
5 ( )
I, II, III, IV V. H
II.

84

INAKA II: T Fredrickson


X

12h

<220 mg%

<150 mg%

T I

<260 mg%

>1000 mg%

,

,


, -

,
LDL, VLDL,
HDL

T II

>300 mg%

<150 mg%

LDL

T II

>300 mg%

150-300 mg%

LDL VLDL

T III

350-500 mg%

350-500 mg%

,
,

(VLDL), HDL

T IV

<260 mg%

200-1000 mg%

-,

VLDL,
HDL

T V

>300 mg%

>1000 mg%

,

,


-, VLDL, HDL

:
O T I
.
O T II (II II) --
. X LDL (-)
. T

. (O II
).
O T III (--) .
O T IV ( )
. X
,
. K
.
O T V ( ) .
H
,
,
. E

85


, ,
.
,
, (), (RIA),
(), (FIA), (ELISA) ..
H
,
.

86

. O
( ) .
1.
O .
()
pH 8.6, (
).
5 : (-) , (LDL), -
(VLDL), (Lp) (HDL) (+). M ,

, Fat red.
( )
12 , 5 mg
Na2EDTA/ml ( ).
. ' ,
2 .
' , - -
28 4. --
5 .
4 .

, Tris- pH 8.6, 0.1% NaN3


1% Tris- (
)
4:5
Fat red 7B (5 ml stock 0.25 ml
0.2M NaOH, )
70%

.
.
87

.
.

5 l
.
.

3-5 min
.

100 volts 25.


.

, ( 60C)
.

. ,
-.
(dennsitometry)
520nm.

- (DL)
-- (VLDL)
- (LDL)

2.

. (%)
22 - 46
0 - 27
47 - 71
0

)
A
E + H2O
X + O2

X +

4- + H2O2
88

2H2O2 + 4- +

4(-) + 4H2O

A:
O


, 30 mmol/l, pH 6.5
4--, 0.25 mmol/l
, 25 mmol/l
Y (POD), 2.5 kU/l
X, 0.07 kU/l
X, 0.05 kU/l
O , 0.05%

:
T

O ()

0.02 ml

0.02 ml

2.0 ml

2.0 ml

2.0 ml

20 min 20-25C ( 10 min 37C).


546 nm
.
Y: O :
T mg/100 ml = 200 OD /OD
O 200 mg/100 ml.
200 mg/100 ml .
E 260 mg/100 ml .
3. HDL - M Burstein
A
M ,
89

, VLDL LDL. M
HDL .


A :

0.55

mmol/l,

, 25 mmol/l)
A


K: :

0.5 ml

1.0 ml

A, 10 min
10 min 4.000 .
T HDL.
.

: :

0.2 ml

0.2 ml

( )

0.2 ml

2.0 ml

2.0 ml

2.0 ml

A 20 min 20-25C.
546 nm, .
Y: O HDL :
T HDL mg/100ml = 150 OD/OD
E :
A

90

<35

35-45

>45

<45

45-55

>55

4. ( )
A
O
.
T + H2O
+ ATP

3- + O2

+
3- + ADP

H2O2 +

2H2O2 + 4- + 4-

K + 4H2O
(E )

H 505 nm
.


: , 200 mg/dl 2.8 mmol/l
A:

P Tris, 40 mmol/l, pH 7.5, 4-, 5 mmol/l,


, 5 mmol/l, ATP, 1 mmol/l, , 600 U/l,
, 200 U/l, , 200 U/l, ,
0.05%, 4-, 0.4 mmol/l.

0.02 ml

2.0 ml

0.02 ml
2.0 ml

2.0 ml

A, 10 min 20-25C ( 5 min 37C). M



510 nm,

91

Y: H
:
T mg/100 ml = 200 OD /OD
:

<150 mg %

150-200 mg%

()

>200 mg%

A ().

92

AOTEEMATA
AKHH 1
1) T ..............................................................................
( < 200 mg%)
2) T ..........................................................................
( < 150 mg%)
3) ...........................................................................................
4) T ................................................................................
5) H :
:

X................................................

(.T. 10%)

-- (VLDL)..................

(.T. 20%)

- (LDL).............................

(.T. 40%)

- (HDL)............................

(.T. 30%)

Z: N ( Fredrickson)
( , )
.
A:
AKHH 2
M ( HDL)
.
A:

93

11

. -
T ,
.
B
. H
, (GPT), ,
- .
T
NH3+

H C COO- + C COO-

C COO- +

R2

R1

NH3+

O
R1

H C COOR2

-
- .

. ,
- - (GOT)

(GPT),

. T ,
. A
(6 40 .)
,
.
- (SGPT)
K

L-

94

L-

.
H GPT
.

E
. (, , )
pH 9, (, , ) pH 4.9 - 5
pH 5.5 - 6.
A
B , , , .
. ,
.
X
120
. M
.E.. , .
H
.
. E ,
,
(). H
(
Van de Berg).

.
-UDP--


( Van de
Berg). To
,
.
H
, .
H
95

,
.
H ,
,
. O


.
-
H -
, (14) .
.
(1 6)
.
H ,
. E
.
T
. T
, .
A :
1.

K
.



.

2.

3.

( ).

.

.
.
. ,
96

.
, .

.
.
, .
,
.
.
(LDH)
,
.
(ALP).
-.
(LDH)
LDH :
+ ADH + H+

+ NAD+


, , .
140.000 4 35.000 .
( Heart) ( Muscle).

.
5 LDH,
:
LD1 (H4), LD2 (H3M), LD3 (H2M2), LD4 (HM3), LD5 (M4)
LD1 LD2 .
LD1
LD1/LD2. LD1 LD2
, ,
.
LD2 LD3
,
.
LD4 LD3,
97

.
, LD5 LD4
.
.
120-140
U/ml 5 :
LD1: 17-27%
LD2: 28-38%
LD3: 19-27%
LD4: 5-13%
LD5: 5-16%
,
. , LD1/LD2
<0.76,
>1.
LD5 Vmax
NAD+,

(, ). LD1, ,
, Vmax
.
LD1
LD5. LD1 LD5.
LD1

LD5

69-75

31-37

0.1510-3

1.210-3


pH 8.6 (cm)
(%)
50C (min)
(mol/l)
V0 10C

98

1. X SGPT (M Reitman-Frankel)
A
H
NADH,
- (GPT) (LDH)
- + L-

GPT

LDH

+ NADH + H+

L- +

L- + D+

O
:

(pH 7.4)

80 mmol/l

L-

0.8 mmol/l

LDH

1.2 U/ml

NADH

0.18 U/ml

18 mmol/l

' :
O

0.4 ml

2 ml

A 340 nm
0, 1, 2 3 . .
GPT U/l /min 952.
( ):

22 U/l
17 U/l

2. (M
BESSEY)
A

SGPT: serum glutamate-pyruvate transaminase,



99

H -
- .

O
Y ( - MgCl2).

:
Y

2.00 ml

0.05 ml

A (25C) 1 min. A
405 nm. E 1,
2 3 min.
(A/min) .
Y: H :
U/l (25C) = 2751 A/min
2751
,
.
( ):
E

50 - 190 U/l (25C).

400 U/l (25C).

3.
A
H ..
. H
H2O .
"" Van den Berg. H

.
"" Van de Berg.

O
-:

-A ( + HCl)
100

-B ( N)
-: 1.5 ml HCl/100 ml H2O
M

(
ml):
A

T O

H 2O

1.8

4.3

1.8

0.2

0.2

0.2

0.5

0.5

0.5

2.5

2.5

-A
M

30 min .
540 nm.
OD 0.26. A
mgr % .
T 0.26
. ,
.
O : OD 0.26 = mg %
: OD 0.26 = mg %
: mg % o mg % .
( )
O

0.3 - 1.4 mg/100ml

0.2 - 0.8 mg/100ml


0.1 - 0.4 mg/100ml

4. - (M Wohlgemuth)
A
H - ,
37C, 1 mg
. (
101


NaCl, 0.9%
, 0.1% ( )
, N/10

X "" 10 .
1 ml NaCl. 1 ml .
1 ml
. A 1 ml
. 9. A 9 1 ml
. O , . .
M 2 ml (. 2 mg
) "" 37C 30 min.
23 . T
.
Wohlgemuth.
O :
M Wohlgemuth = 2 (
).
H 1/2, 1/4, 1/8, 1/16,
1/32 ...
3o 4 .
( )
4 - 32 /ml Wohlgemuth.
5. LDH

, LDH
.
, .
,
102

.
LDH
, (4)
R ,
(4) R .
LD1 (H4)
(+), LD5 (M4) (-). (LD2, LD3
LD4)
.


8

LDH

() NAD NADH,

2
1

,
:
+ NAD+

LDH

+ NADH + H+

NADH + PMSox

NAD+ + PMSred

PMSred + NBTox

NBTred

PMS: phenazine methosulfate


NBT: nitro-blue tetrazolium
red: (reduced)
ox: (oxidized)
PMS NBT .

1% Tris-, pH 8.6,
0.1% NaN3

!
103

: Tris- pH 8.6, 0.1% NaN3


:

Tris-, pH 9.0, NAD+ 10 mg/ml, NBT 4


mg/ml, PMS 2 mg/10 ml

: 5%

1.

2.

3.

,
.

4.

5.

,
.

6.

5 l .
( ),
.

7.

8.


(-).

9.


20 100 V.

10.

,
.

11.

15 .

12.

13.

, .

, .
(
, densitometer).

104

1.

SGPT .

2.

3.

, .

4.

( Wohlgemuth) -/ml ;

5.


. . ,
,

.

105

12
AIMOAIPINOAEIE - ENZYMA EPYPN AIMOAIPIN
A.

A
E (Hb) ,

.
.
T Hb .
K ,
.
Hb : , , A, G, , . E
,
mRNA .
HbA (22) 97%,
HbA2 (22) 2.5% HbF (22) 0.5%.
A: M
.
M -
E - .
-: ' -. T
- -. O -
-.
-: - -
,
- -, .
.
- . E -
4,
.
106

HbF HbA2.
.

HbA2
O HbA2 -

( ,
, , Ht, Hb, ). H
,
.
H ,
,

. O - , ..
. O
.
H
.

H
. O ,
Hb,

.
pH ,

Hb
. O

. A
pH

.

HbA2
Hb . T HbA2

HbA2.
.

6-
H 6- (G6PD)

107

, ,
, , . E
( ).
K 6- 6- ,
NADP+ NADPH + H+.
. H
NADPH
,

.
G6PD
. T

. A NADPH,
(GSH).
.
,

(. B J.M. Berg, J.L. Tymoczko, L. Stryer, 5 ,
. 571).

108


:
1.

2.

HbA2

3.

G6PD

1.
O
.
.
,
, pH,
.

: 1% Tris
0,01% merthiolate
: Tris Titriplex Boric acid + merthiolate
: Amido Black 0,4% 10%
: 10%
: 0.2% + Titriplex + KCN


(, EDTA ).

100 l 5 ml ,
.

( ) 300 l . (
,
).

1.

109

2.

,
.

3.

4.

T .
,
.

5.

T 5 l
20.

6.

7.


,
24 min 100 Volts.

8.

,
( 60C) 5 min
.

9.

.
10.



()
580 nm.

2. HbA2

300 mg DEAE 0.2 M


0.01% 0.1%
110

A HbA2 0.2 M

A : 0.1% Triton X-100
: (EDTA )

A ( ).

:
50 l 250 l
. A ,
,
. M 5 min.

E :
1.

T , . A
.
A , .

2.

A
.

3.

T , .

4.

T 100 l (
),

5.

A 100 l

( : 15 ml).

6.

A .

7.

M
2.5 ml .

8.

A
HbA2 ( 30 min). T HbA2.

9.


( : 3 ml).

10.

A ,
.

11.

HbA2 ,
415 nm
( HbA2 ). O
111

( ). Y HbA2
:
ODHbA2 100

= HbA2 %

5 OD.
:
ODHbA2:
HbA2
OD.

.
5:
, 15
ml 3 ml (15/3=5).
HbA2 1.5-3.5%.
3. G6PD

A
H G6PD 6- (G6P) 6-P-,
NADP+ NADPH:
G6P + NADP+

G6PD

6-P- + NADPH + H+

P , 50 mmol/l pH 7.6, EDTA 5 mmol/l.


NADP+, 10 mmol/l.
G6P-Na, 31 mmol/l.
, 0.02%.
NaCl, 0.9%

M /ml. 0.2 ml 2 ml
NaCl 0.9% 3 . 10 min 3000
rpm. A 0.5 ml
, 15 min 4C .
X 0.05 ml .

112

3.00 ml

NADP+

0.10 ml

0.05 ml

A, 5 min 25C

A

0.05 ml

365 nm ( ).
,
1, 2 3 min.
(A/min) G6PD, o
:
E G6PD = A/min 54857 mU/ml
O
ml . 4.900.000/l 4.9 109
/ml 4.9.
G6PD: 131 13 mU/109 .
4. G6PD

T NADPH G6PD
, -. T
. .


Y G6PD :
6- , 4.6 mmol/l
NADP+, 0.13 mmol/l
-, 0.55 mmol/l
P Tris-HCl, 0.3 mol/l, pH 8.5 (37C)

113

X ,
EDTA.
1.

A . , 50 l
2.5 ml . A
5 min .

2.

K 0.5 ml .

3.

1 ml .

4.

E 2 ml .

5.

T 37C,
. M , (G6PD)
. A
.

5.

K Fe2+ Fe3+
. H
,
:
Hb (Fe2+) + K3 (Fe(CN)6)
M + KCN

M (Fe3+)
K

A Drabkin ( K3[Fe(CN)6] KCN)


O

2 :
A

Drabkin (ml)

A (ml)

0.02 ( )

, ,
Drabkin. A 15 min.
(540 nm) . Y
Hb g/100 ml .
7. A ()
114

H ,
, . H
. E
, .
H
-,
,
,
. O2
H2O2 '
().

, 33%
A , 5%
Y , 3%
O


1 ml
2 ml
4 ml H2O2
5 ml .
A . H o
. H
.

115

AOTEEMATA
1.

2.

HbA2 .

3.

N G6PD/109 .

4.

G6PD
.


0.6

0.5

OD

0.4

0.3

0.2

0.1

0
0

6
1

12

15

g/100 ml

116

18

21

24

DNA

13
DNA

.

DNA
: DNA ( ).

,
DNA DNA.
DNA ( )
. DNA
.
, (D-2) .
DNA
.
DNA .
DNA ,

. DNA
,
.
DNA, ,

,
(>80C)
pH.

DNA .
:
.

DNA

DN

DNA
117

DNA

DNA.

, SDS ( )
, EDTA ( ).
, DNA
, NaCl (, ).
DNA,
( ).
DNA .
DNA, DN,
SDS ,
EDTA, ,
Mg2+, .
B.

DNA
DNA

.
.
.
DNA .

.
,
().
. DNA
, DNA
(blund ends), DNA
,
DNA (sticky ends).

-20C 50% .
.

(PCR)
PCR (Polymerase Chain Reaction)

DNA (, cDNA).
118

DNA

PCR (the cycling reactions):


PCR,
30 40 . PCR
,
. :
1.

(Denaturation)

2.

(Annealing)

3.

(xtension)

PCR (Principle of the PCR)


1.

(denaturation) 94C.
, DNA

. .
2.

(annealing) 42-62C.

( rown).
( )
DNA, DNA
. ,
.
3.

(extension) 72C.

119

DNA

Taq DNA .
,
( )
. ( ) o 3
( dNTP's 5 3,
- 3' 5' ).

94C x 5 min ( )
94C x 45 sec ()
54C x 45 sec ()

35-40

72C x 2 min ()
72C x 10 min ( )
4C
DNA (target DNA)
PCR (
DNA, , , , ,
(Primers )
PCR (PCR primers) DNA (15-30
) DNA-
(DNA target region) . PCR 3-OH
DNA dNTPs.
DNA
DNA.
Taq DNA (Thermus Aquaticus, Taq), -
- .
, DNA
, ( ),
(dNTPs) , MgCl2, Taq DNA
pH .

DNA
10 PCR

200 mM Tris-HCl pH 8.4, 500 mM KCl

dNTPs

25 mM

MgCl2

50 mM

(25 )

(25 )
120

DNA

Taq DNA

5 U/l

dH2O
PCR
1.

2.

PCR (500l) :

1 (25 M)

1 l

0.5

2 (25 M)

1 l

0.5

dH2O*

38.8 l

10 PCR

5 l

gCl2 (50mM)

1.5 l

1.5 mM

dNTPs (25mM )

0.4 l

0.2 mM

DNA (1g/ml)

2 l

Taq DNA (5U/l)

0.3 l

50 l

1.25 U

3.

PCR. :
95C 5 min (, denaturation)
95C 1 min (, denaturation)
57C 1.5 min ( , annealing)

35

72C 2 min (, extension)


72C 10 min (, extension)
4C min
4. , 4C,
PCR .
5. (2%) 0.5 g/ml EtBr.
PCR
H PCR

DNA
:
.
121

DNA

.
. (DNA
sequencing analysis)
(Agarose gel electrophoresis)

. DNA
,
, DNA
(UV)

122

DNA


1. DNA


: 10 mM Tris HCl pH 8.5, 5 mM EDTA, 0.2 % SDS, 100 g/ml
100%
NaCl (~ 6M)
TE (10 mM Tris HCl pH 8,0, 1 mM EDTA)
Eppendorf

1.

40-50 mg Eppendorf.
0.5 ml .
37C 14-18 (overnight [o/n]) 55C 2-3 .

2.

0.125 ml NaCl

(~6M).

15 sec. 15 min
.
3.

.
.

4.


DNA.

.

5.

DNA
Eppendorf. DNA
50 l .
DNA
(UV) DNA ( ).


DNA , .
123

DNA

DNA
.
DNA ,
, ,
. H
, . 50-150 g
DNA, .
2.

DNA

DNA

DNA ( DNA ), 1g/ml


10 :

50mM NaCl, 10mM Tris-HCl, 10mM MgCl2, 1mM


(DTT), pH 7.9

EcoR, 10U /l, HindIII, 10U/l


(ddH2)
, 1%
, 10mg/ml
: 0.9M Tris-, 0.002M EDTA, pH 8.3
:

0.25% xylene cyanol FF, 0.25% , 40%


(w/v)

Eppendorf


UV

0.2-1 g DNA.
DNA .
1.

Eppendorf :
DNA

2 l

10X

2 l

1U (unit) 1 g DNA
, .
124

DNA

ddH2O

15 l

EcoRI HindIII

1 l

20 l

2.

37C 30 min.

3.


.
5 l
.

4.

, , (HindIII).

5.

TBE 80 Volts (5
volts/cm). UV

EcoRI

coRI

EcoRI

EcoRI

- 4.0 kb
- 3.0 kb
- 2.0 kb
- 1.5 kb

EcoRI

- 1.0 kb
- 0.5 kb

EcoRI

125

DNA

1.

DNA.
( );

2.


DNA ;

3.

DNA
HindIII, ;

4.


, ;

5.

() ()
HindIII; A

4.

126

/ DNA

14
AMATOKOIKE IIOTHTE KAI HEKTPOOPHH DNA

T ,
. T DNA RNA
(. 1) 260 nm. H

.

220
260
(nm)

300

1: DNA .
1,
.
DNA , A=T G=C
, . M
DNA.
E1M (1cm, 260 nm)

1
A
G
C
T
U
DNA
DNA

,
DNA.

127

15.4 103
11.7 103
7.3 103
8.8 103
9.7 103
7.2 103
9.0 103

/ DNA

DNA
O
. , pH
, , .
.
H DNA
80C 35% (. 2). T
. H

(E

+ E

)/2

(Tm). H Tm

%
GC .
(
pH),
, OD . O
,
Tm . A , ,
OD . A

(OD, . 2) % .
OD

, OD

1.4

:
:

1.2

OD

1.0
30

40

50

60

70

80

, C

90
Tm

2: M OD () DNA
OD 260 nm.

128

/ DNA

% =

100 OD
OD - OD

DNA ( )
. E, DNA
,
.
DNA
H
260 nm OD260/OD280
. OD260/OD280
. K DNA RNA
OD260/OD280 1.8 2.0 . A
DNA (
)
.
H DNA
H DNA ,
DNA. H ,
DNA,
. H 0.560 (kb) , 10-500
(bp) . H DNA
,
DNA
. M DNA
1 ng .
K , DNA, ,
. H
:
1.

T DNA. M DNA
.
.

2.

. DNA
129

/ DNA

.
DNA .

DNA ' :
(%)

3.

M DNA (kb)

0.3

60-5

0.6

20-1

0.7

10-0.8

0.9

7-0.5

1.2

6-0.4

1.5

4-0.2

2.0

3-0.1

DNA.
DNA .
K , DNA

,
,
DNA. Y
.

4.

. voltage
DNA voltage,

. '
5 V/cm 10 V/cm
.

130

/ DNA

EIPAMATIKO MEPO
1. DNA

DNA
: Tris-Hcl 0.05M, pH 7.0

A 40 l DNA 2.5 ml A
2.5 ml . M
260 nm A.
E OD 280 nm
.
.

1
DNA mol / :
OD = E c l

c DNA l = 1 cm

Y DNA g/ml
OD260 = 1, 50 g/ml DNA.

2. DNA

DNA 1


, 5 min .
.
% .

131

/ DNA

: OD OD
, OD OD
( ), OD
OD .
3. DNA pH

DNA 1
: 0.05 , pH 8.5
: 0.05 , pH 12.5

A 0.25 ml DNA 2.25 ml B


0.25 ml DNA 2.25 ml . T

260 nm
.
4. E DNA .

P Tris- pH 7.7
0.04M Tris- (4.84 g Tris, 2.29 ml /L)
0.001M EDTA (0.372 g/L)
1%
X

20 l ( 10 mg/ml) 100 ml

DNA
(0.25% , 20% Ficoll 100 mM
EDTA)
(ddH2O)

POOXH!

132

/ DNA


(302 nm)


X . A

(. 3).
1.

8 12 cm , d.d H2O

.
2.

H 4 ,
0.6-1 cm

3.

A 0.7 g 70 ml. To
133

/ DNA

. M
.
4.


1 mm , (
plexiglas) . O 1 5 5
mm.

5.

T 60C
.

6,7.

H 1
4C, 2
. B .

8,9,10. A
, .
( 1 L) 2-3 mm.

30 l 5 l
. H
.
H
:
.

DNA ( 48.6 kb)

DNA , Hind III,


II 23, 9.4, 6.6, 4.3, 2.3 2 kb.

DNA .

DNA DN I.

DNA PCR.
T
K

: H
, (15 l
70 ml ).
134

/ DNA

50 100 l. K .
,
. T DNA
. E
80 Volts 45 min. M
10-15 min

(302 nm), DNA


.
T () DNA ,
. T (), DNA Hind III
. T () DNA
.
E . T
(), DN I
. T ()
DNA PCR
.

POOXH! M ' .
135

/ DNA

AOTEEMATA
1.

A DNA 1.

2.

OD260/OD280 . T
DNA ;

3.

Y % OD ( 2).
% DNA ;

4.

Y DNA
3. DNA ;

5.

DNA
(DNA DN I) (DNA
Hind III).

1.

H RNA 25C 0.8. M


, OD = 1.1. T
; M
;

2.

K DNA A B
OD260/OD280 1.8 1.4, .
; A
;

136

15


, ,
:
1.

(RADIOIMMUNOASSAY-RA)

2.

(IMMUNORADIOMETRIC ASSAY-RMA)

3.

(ENZYME IMMUNOASSAY-EIA)

4.

(LUMINESCENE IMMUNOASSAY-LIA)

5.

(ELCTROCHEMILUMINESCENCE-ECL)

6.

7.

(FLUORESCENCE POLARIZATION IMMUNOASSAY-FPIA)



S R :
k1
S+R

S-R

(1)

k2
k1 S - R k2
.
S R ,
:

a (mol/)-1 1/.
1.

(RIA)


(S)
(R), :

137

S+R

SR

(1)

S R
(S*), :
S

S-R
+R

S*

(2)
S*-R

S:

S*:

S 125 3.

R:


S S* :
.

S S*,

()

S S*.

S S*
'
R.

S R.
R S S*.

(2) S
S*
R.
S ( ),
S* R
S*-R
S*-R. S
S*-R.
S*-R ( S*
)
(cpm).

138

S (standard)
S*-R ,
(x)
(y) (cpm).

(. 1).

1.6104
1.4104

(U/ml)

cpm

15300

cpm/min

1.2104
1104

10

11095

8103

20

9150

610

40

6050

4103

80

4050

2103

160

1950

7800

40

80

120

160

200

(U/ml)

1. . A
..
7800/min, 24 U/ml. T
.
2.

(IRMA)
IRMA RIA -

(S + R S-R), RIA R
. ,
(

125

3) R* ( ),

S ,
R-S-R*
S (
RIA).
IRMA :

139

S+R
SR + R*

SR
R*-SR

(3)

(R)
(coated tubes) (beads).
S-R *R'-S-R
R R* .
,

RIA.
S* S*-R R IRMA
S* S*-R ,
:
.

S S*
(charcoal) ,
S*-R, ( S-R),
.

() (- )
.

( ).
(Fe)
S*-R-Fe
.

,

R
( IRMA).
S,
, ,
S-R .
R* ,
.
R*
140

R-S-R*,
S.
( ) (coated
tubes) ().
O-R-S-R*
.
3.

()

(IRMA) ()
. ,
-.
(beads)
R , S
R + S

R-S.

R'
(R' - ).
:
R+S

R-S

RS + R-

RSR-E

RS-R-E +

(4)

(R'-E)
R-S-R'-E.
,
, (
) (S).
IRMA
(x)
S (y) (OD)
(standards)
.

141


RIA ( ),
.
4.

(LIA)
(hn)

. ,
, .

(H22) ,
, , , , ..
,
, , , .. ,

22/

5- + 2 + 2 + hn

(5)

To
, (in
vitro) ,
(.. )
(hn),
.
R-S-R'-E
(3) .. ,
.. --, (22),
( 5).
,
IRMA , ,
(y) (mv)
cpm
.

RIA
(Luminescence immunoassay) LIA.

142

:
R+S

R-S

R-S + R-E

R-S-R-E

R-S-R-E +

5.

22/

(6)

+ (hn)

(CL)
ECL
. LIA ( <1 pmol/l)

(t) (Au) (Ru)
. ECL
IRMA 2
(-)
(620 nm)
.
,
R+S

RS

RS + R-Ru++

RSRRu++

:
, (),

+.
Ru++ Ru+++. :
+

e- + TPA+

Ru++

e- + Ru+++

IRMA (Immunochemiluminometric
Assay) ICMA.

143

+ (+)
+

+ +

,
*Ru++
+ Ru+++

+ *Ru++

*Ru++ Ru++
*Ru++

Ru++ +

To Ru++
.
(620 nm)
S.
ECL
( LIA )

.
6.


.
(
) ,
-
.

-. (
0).
,

. (
0
).

144

,

.
.

(..)

(/) ()
- .
.., ..
.. 380-800 nm,
.. <380 nm >800.

.. 400-620 nm.

.
/
, ,
2-, - ,
(HbA1C)
.
7.

(FPIA)

(Fluorescence
polarization immunoassay - FPIA). H FPIA

.
. . RIA

(,
). FPIA
.

145


.
8.

, (microdialysis
tehnique)
, ,
(<20.000 Da) , ,
( ..) ( ,
, , ..).

( 3 cm 0.6 mm)
(Ringer acetate)
(0.3-5 /),
.


.


.

146

EIPAMATIKO MEPO
EIPAMA 1. P (IRMA)
(CEA)

(-CEA)
, 125I
(standards) CEA (0, 5, 10, 35 70 ng/ml)

2104

cpm

1.5104

1104

5103

10

20

30

40

50

60

70

80

CEA (ng/ml)

2: CEA
IRMA. A o CEA
5.1 ng/ml.

1.

5 ml (50 l)
, .

2.

3.

(50 l)
.

4.

E 37C.

5.

K .

147

6.

3 ml
4-5 .

7.

M -.

8.

K (y)
(cpm) -
(x) standards (. 2).

EIPAMA 2. E (EIA) CEA

1 (-CEA).
,
.
P Tris (Tris buffer).
(standards) CEA (0, 4, 10, 30 60
ng/ml).
Y , -2HCl (OPD) .
P - OPD.
1 N .

N OPD . E
. K
OPD 5 ml - 15 min
OPD .

1.

T 100 l ,
.

2.

100 l Tris .

3.

4.

E , 45C .

5.

A 3 ml
. K
.

6.

200 l .

7.

E , 45C .
148

8.

E 5, 3-4 .

9.

300 l OPD.

10.

E 30 min (15-30C).

11.

1 ml 1 N .

12.

M 492
nm, .

13.

K (. 3)
CEA .

14.

E , .
1.6
1.4

(OD)

1.2
1.0
0.8
0.6
0.4
0.2
0

12

16

20

CEA (ng/ml)

3: CEA EIA. A
..
0.31, 3 ng/ml.
EIPAMA 3. A

CEA

(ICMA)


1
CEA (standards) 0.3, 6.25, 150 400 ng/ml
,

, --, H2O2 0.1 M Tris-HCl, pH 8.0

149

1.

(50 l)
, .

2.

(150 l) 2
, .

3.

E 1 37C.

4.

A 0.5 ml
( )
.

5.

250 l , --, H2O2.

6.

M
2-20 min.

100

80

60

40

20

100

20
0

300

400

CEA (ng/ml)

4: CEA
ICMA
7.

K ,
(y) (mv)
standard
standard ( ) (x)
standards. ( 4).

8.

A
. (H

).

9.

K
.
150