BIOC2000 Lecture 24: Lecture 24
Lecture 24: Quantifying binding
interactions
Quantifying analysis of ES complex formation
Rates of association and dissociation are determined by rate constants, kon
(or k1) and koff (or k-1)
capital K – equilibrium
constants; lower case k – rate
constants
The equilibrium between bound and free is determined by an equilibrium
constant:****
Ka and Kd are inverse of eachother
Will an enzyme, which binds a
substrate very tightly, have a large or
small Kd? Small – because they are
very well bound
Which will be larger, koff or kon? kon
In biochemistry, Kd is more relevant
Enzyme-substrate complex can be generalized to any protein-ligand
complex:
1. enzyme-drug complex; lots of drugs target enzymes for effect
2. Hormone-receptor complex
3. Antigen-antigen complex e.g. peptide binding to an antibody
4. DNA-transcription factor complex where DNA is ligand and TF factor
is the protein
find more resources at oneclass.com
find more resources at oneclass.com
Quantifying binding requires some way to detect bound vs
free ligand or protein
- Radiolabelled ligand and a way to separate bound from free
Radio-labelled centre on a molecule has very little effect on its binding to a
protein
There’ll be an equilibrium established between bound and free-forms
(unbound) form of the ligands to the proteins – how to separate?
Proteins are typically much bigger than ligands so filtration will separate
these 2 groups
We can then get (a) bound ligands and (b) non-bound ligands and quantify
amount of ligand in each group; working out how much radiolabel is there
If the experiment was done at different concentrations of ligand:
y: amount of
bound ligand
x: different
concentrations
of total ligand
Total binding
increased as
[ligand]
increased.
non specific target – e.g. binding to walls of the container or other proteins
nonspecifically, much looser interaction with a higher KD
Total combinding curve can be broken into 2 components:
(1) specific binding / saturated component of binding – binding that
reaches a maximum and plateaus at a specific point
(2) non-specific binding – increases with concentration of ligand
Specific binding interactions are saturable – there are only a certain # of
binding sites, if you keep increasing amount of ligand then you reach a
point where no more ligand can bind because all the binding sites are
saturated/bound
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Rates of association and dissociation are determined by rate constants, kon (or k1) and koff (or k-1) capital k equilibrium constants; lower case k rate constants. The equilibrium between bound and free is determined by an equilibrium constant:**** Small because they are very well bound. Which will be larger, koff or kon? kon. Quantifying binding requires some way to detect bound vs free ligand or protein. Radiolabelled ligand and a way to separate bound from free. Radio-labelled centre on a molecule has very little effect on its binding to a protein. Proteins are typically much bigger than ligands so filtration will separate these 2 groups. We can then get (a) bound ligands and (b) non-bound ligands and quantify amount of ligand in each group; working out how much radiolabel is there. If the experiment was done at different concentrations of ligand: y: amount of bound ligand x: different concentrations of total ligand.
