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Short answer: The affinity constant KD (equilibrium dissociation constant) is the concentration of one binding partner at which half of the other partner is bound at equilibrium. It is expressed in molar units, and a lower KD means tighter binding. For a simple one-to-one interaction, KD = kd/ka, the ratio of the dissociation and association rate constants.
Definition and scope
For a protein (P) binding a ligand (L) to form a complex (PL), KD = [P][L]/[PL], where the terms are the equilibrium concentrations of free protein, free ligand and complex (Bio-Rad Bulletin 5538). Because it is a concentration, KD carries units of molar (M), and values are most often reported in nM or µM. The same quantity is called the dissociation constant, binding constant or simply “binding affinity KD” in methods sections; these are the same measurement, not different ones.
Reading the number: when the free ligand concentration equals KD, half of the binding sites are occupied (GraphPad Prism guide, law of mass action). If the receptor has high affinity for the ligand, KD is low, because it takes only a low ligand concentration to occupy half the sites.
Affinity and KD: which way round?
Affinity and KD run in opposite directions. Tighter binding gives a smaller KD. The association constant Ka is the reciprocal, Ka = 1/KD (FIDA Bio knowledge base), so Ka rises as binding gets tighter. If a paper quotes an “affinity constant” without saying which, check the units: M means KD, M-1 means Ka.
How KD relates to kon and koff
At equilibrium, KD is related to the association rate constant ka (also written kon) and the dissociation rate constant kd (koff) by KD = kd/ka (Bio-Rad Bulletin 5538). High affinity therefore means a low KD, which comes from a high ka (fast on), a low kd (slow off), or both.
The units differ. Per the GraphPad guide, Kd is in M, kon in M-1 min-1 and koff in min-1. Lowercase kd (a rate constant) and uppercase KD (an equilibrium constant) are easy to confuse in text where subscripts are lost.
Why the kinetics add information
Because KD = kd/ka, two rate constants can change together and leave KD unchanged. Bio-Rad notes that such changes may be overlooked if only KD is measured. Two interactions with the same KD can therefore differ in how fast they reach equilibrium and how long the complex lasts. Equilibrium methods such as equilibrium dialysis and immunoprecipitation give KD but not real-time kinetics (Bio-Rad Bulletin 5538).
Occupancy at multiples of KD
The GraphPad guide gives the fraction of sites occupied at equilibrium for ligand concentrations expressed as multiples of Kd. This is the most practical use of the number when designing a titration series.
| Free ligand concentration | Sites occupied at equilibrium |
|---|---|
| 1 × KD | 50% |
| 4 × KD | 80% |
| 9 × KD | 90% |
| 99 × KD | 99% |
Occupancy climbs slowly past KD: going from 50% to 90% takes nine times the concentration, and 99% takes about a hundredfold. A concentration series that only spans values below KD never shows the plateau, and the FIDA Bio knowledge base notes that without a plateau in the curve the KD fit is unreliable.
Related concepts at a glance
| Symbol | Name | Unit | Direction for tighter binding |
|---|---|---|---|
| KD | Equilibrium dissociation constant | M | Lower |
| Ka | Association (affinity) constant, 1/KD | M-1 | Higher |
| ka / kon | Association rate constant | M-1 time-1 | Higher (fast on) |
| kd / koff | Dissociation rate constant | time-1 | Lower (slow off) |
Where it matters in the lab
Several methods report KD, and the site covers them individually: surface plasmon resonance and bio-layer interferometry measure kinetics and derive KD from the rate constants; isothermal titration calorimetry measures binding in solution; and fluorescence polarization assays need a tracer concentration chosen relative to KD. The FIDA Bio knowledge base notes that methods differ mainly in whether one partner must be immobilised. For inhibitors, the Cheng-Prusoff equation converts IC50 to Ki, a related but distinct constant.
In cells, Bio-Rad points out that concentrations must be in the same range as KD for a significant interaction, and a lower KD needs a lower concentration. So a KD is best read next to the concentrations of the partners in the system under study.
Common misunderstandings
“KD is a fixed property of a molecule pair”
Bio-Rad states that competitors, ions, cofactors, pH and molecular crowding can promote or inhibit binding, and that interactions should be studied under controlled, relevant conditions. A KD belongs to a pair of molecules under stated conditions, so compare values only when buffer, pH, temperature and method are comparable.
“A bigger number means stronger binding”
The opposite holds for KD. A 10 nM KD is tighter than a 100 nM one. Only Ka rises with affinity.
“Same KD, same behaviour”
As shown above, different ka and kd pairs can give an identical ratio.
FAQ
What is binding affinity KD?
It is the equilibrium dissociation constant: the concentration of one partner at which half of the other is bound. Lower values indicate higher affinity.
What is the relationship between affinity and KD?
They are inversely related. Affinity is high when KD is low, and Ka = 1/KD.
Is a lower KD tighter binding?
Yes. A low KD means a low ligand concentration is enough to occupy half the binding sites.
How do I calculate KD from kinetics?
Divide the dissociation rate constant by the association rate constant: KD = kd/ka, converting units so the result is in molar.
Sources
- Bio-Rad, Bulletin 5538 (surface plasmon resonance application note): bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_5538.pdf, read 2026-11-08.
- GraphPad Prism curve fitting guide, key concepts: the law of mass action: graphpad.com, read 2026-11-08.
- FIDA Bio knowledge base, What is KD: fidabio.com, read 2026-11-08.
