()

John Newport Langley (1852-1925)

(receptive)

Paul Ehrlich (1854-1915)

(lateral chains, receptors)

 -
(receptor-ligand binding)
3

(~ 12 )

125

(specific radioactivity)

(~ 60 )
(.. )

 -
(specific binding)


( )

(c)

1. -


.. 15
,
,

15

2.


-



Ci/mmole
Ci / (dpm: desintegration per
min) / (cpm: counts per min)

minimum 1.000 cpm 10
Ci/mmole 100 fmoles/mg

M (non specific binding)

,


(100-1.000 fmoles/mg .)


(rapid
filtration under vacuum)


(1.000-10.000
)

+ 1000X

R+L* RL*

= +
RL*

L*

B (Bound)

(cpm)

[RL]

[C]
(nM)
F (free), L (ligand)

(cpm)

(min)

[ ] mg/ml

22 C ( ) :
37 C ( ) :
4 C : ,

pH

7 - 8

Scatchard

[F]

max [F ]
B=
K d +[ F ]

max (maximal Binding):

Kd (dissociation constant)


[3] 5- 16,2 Ci/mmole,
2 mg
10-5 5-
22o C 30
F
[3]5(F)
(nM)

(T)
(cpm)

(NSB)
(cpm)

TB

NSB

SB = TB-NSB

B/F

1717

432

1285

1285

1.5

2241

648

1593

1062

2655

865

1790

895

2.5

3163

1081

2082

833

3781

1279

2502

834

4451

1729

2722

681

5023

2161

2862

572

5571

2594

2977

496

1717

432

1.5

2241

648

2655

865

2.5

3163

1081

3781

1279

4451

1729

5023

2161

5571

2594

6502

3026

6502

3026

3476

497

7018

3458

7018

3458

3560

445

7491

3891

10

8023

4323

7491

3891

3600

400

10

8023

4323

3700

370

10000

NSB

max
5000

B/F (x 103)

(cpm)

-1/KD

max/2

Bmax
KD
0

10

F (nM)

B (cpm x 103)

 ( Hill, 1910)

max
d

- in vitro
- in vivo

max
C50


d = C50

C50<<<d

max

C50 (effective concentration)




(
)

,

( )

log []

R- Receptor ( )
R- Receptor ( )
L (Ligand)

 ( )
( )

 /
(competition experiments)

(, inhibitor)

..
(competitive inhibition)
(non competitive inhibition)
(uncompetitive inhibition)

IC50 (Inhibitory Concentration)

50%

inhibition constant
Ki = IC50
2

IC50
? ?

2 ( )