Binding

1
Binding
Old based on mass action various definitions of
equilibrium constants Better based on
thermodynamics Newest based on modified GC p.f.
1
2
Binding
Need Ligand activity (M is dilute and treated
as ideal solution) saturation ML/(MML)
2
3
Binding
Need Ligand activity saturation
ML/(MML)

• Spectra of M and ML differ
• Excess L is present as ML

3
4
Binding
Single-site binding curves
4
5
Binding to macromolecules
Multiple-site binding Independent and not
equivalent Independent and equivalent DEPENDENT
Second site with lower affinity
5
6
Net charge 18
COOH ? COO H HISH ? HIS H Phe-OH
? Phe-O H NH4 ? NH3 H
High ionic strength
low
Number of H ions titrated per molecule
Net charge 11
pH
Titration curve for ribonuclease at 25C and
three ionic strengths. (from Tanford and
Hauenstein, JACS, 1956 see also Tanford,
Physical Chemistry of Macromolecules, Wiley,
1961, pp. 554-)
6
7
Binding to macromolecules
Multiple-site binding n independent and
equivalent sites
(unfortunate distinction) 1. Site binding
constant sites are distinct Stoichiometric
binding constants 2. for one site 3. for i
sites
7
8
Binding to macromolecules
Fit model to data Experiment ltiLgtexpt , ln
aL,expt Model binding constants,
dependencies Compute ltiLgtmodel for each ln
aL,expt Least squares fit minimize S(ltiLgtmodel
ltiLgtexpt)2 May weight points according to their
reliability
8