Law of Mass Action and Binding Assays

Question 1

What is the equation for law of mass action?

Answer 1

[L] + [R] <———> [LR]

Question 2

What conditions are required for law of mass action?

Answer 2

1. Reversible binding
2. No coopertivity (+ve or -ve) : The ability of 1 mol. to bind affecting how the rest will bind.
3. No change in L or R

Question 3

What is Kon and its unit?

Answer 3

Association rate constant
unit: M^-1Sec^-1

Question 4

What is the rate of association?

Answer 4

[L][R]Kon
(reagents * constant )

Question 5

What is Koff and its unit?

Answer 5

Dissociation constant
Unit: Sec^-1

Question 6

What is the rate of dissociation?

Answer 6

[LR]Koff
(reagents * constant)

Question 7

What is equilibrium binding?

Answer 7

rate of association = rate of dissociation

Question 8

What is Kd?

Answer 8

Measure of affinity
Equilibrium dissociation constant

Question 9

What is the formula of Kd?

Answer 9

Koff/Kon

Question 10

Why do we use equilibrium dissociation and not equilibrium association?

Answer 10

Units

Question 11

What is Rtotal?

Answer 11

Rfree + Rbound

Question 12

How can we explain Kd in terms of receptors?

Answer 12

[L] binding 1/2 the receptors

Question 13

Relationship between Kd and Affinity

Answer 13

Lower Kd means better affinity

Question 14

What are the components of ligand binding studies

Answer 14

1. Ligand
2. Receptors
3. Buffers
4. Variants

Question 15

Explain the component: ligand

Answer 15

Must be labeled either with radioisotope or fluorescence.
Isotopes used: 3H 14C 125I
Problems: Half-life and specific activity.