Ki vs IC50 vs EC50: Reading Peptide Affinity and Potency Data
Open almost any pharmacology paper on a research peptide and you will meet three numbers: Ki, IC50 and EC50. The question of Ki vs IC50 vs EC50 matters because the three are often lined up in the same table, quoted in the same sentence and compared as if they measured one thing. They do not. One describes how tightly a molecule binds, one describes how much of it is needed to block something under particular conditions, and one describes how much is needed to switch a response on. This guide explains each in plain terms, how they relate, and why the lot and the concentration arithmetic behind your own in-vitro work affect the numbers you generate.
Affinity: Kd and Ki
The equilibrium dissociation constant, Kd, is the ligand concentration at which half of the available binding sites are occupied once binding has settled. It is expressed as a concentration, and smaller means tighter: a Kd of 2 nM reflects much stronger binding than one of 200 nM. Kd is usually measured directly with a labelled ligand in saturation experiments.
Most peptides are not labelled, so their affinity is usually inferred indirectly. A labelled reference ligand is allowed to bind the receptor, the unlabelled test peptide is added over a range of concentrations, and the concentration that displaces half of the reference signal is read from the curve. That value is a binding IC50.
A binding IC50 is not a constant of the molecule. Use more reference ligand, or a tighter-binding one, and the IC50 of the same test peptide rises. The Cheng-Prusoff equation corrects for this in the simple competitive case: Ki equals the IC50 divided by (1 plus the reference ligand concentration over the reference ligand’s Kd). The corrected value, Ki, is an estimate of the test compound’s own affinity and can be compared across laboratories in a way raw IC50 values cannot.
Ki vs IC50 vs EC50 at a glance
| Value | What it describes | Depends on | Comparable across labs? |
|---|---|---|---|
| Kd | Affinity, measured directly | Molecule, receptor, buffer and temperature | Broadly yes, with the same receptor and conditions |
| Ki | Affinity, inferred from competition | As above, plus validity of the correction | Broadly yes |
| IC50 (binding) | Half-maximal displacement of a reference ligand | Reference ligand identity and concentration | Only within one assay design |
| IC50 (functional) | Half-maximal inhibition of a measured response | Agonist used, its concentration, the cell system | Only within one assay design |
| EC50 | Half-maximal activation of a measured response | Cell system, receptor density, readout, timing | Only when the system is matched |
Potency: what EC50 actually reports
EC50 comes from a functional assay. Instead of asking how many receptors are occupied, it asks how strongly a cell responds: second-messenger accumulation, calcium signalling, reporter output, receptor internalisation and so on. The EC50 is the concentration that produces half of the maximal effect in that assay.
Because the readout sits downstream of binding, EC50 absorbs everything in between. Receptor density matters most. A cell line engineered to express a receptor at high levels may reach a full response while only a small share of receptors are occupied, a situation often called receptor reserve. In that system the EC50 can fall well below the Ki. In a native tissue with sparse receptors, the same peptide may show an EC50 close to, or above, its Ki.
That is why an EC50 without its cell line, receptor species, readout and incubation time is hard to interpret. It is a property of the peptide in a particular experiment, not of the peptide alone.
Efficacy is a separate question
Potency tells you where a concentration-response curve sits along the concentration axis. Efficacy, often reported as Emax relative to a reference agonist, tells you how high it climbs. A partial agonist may be very potent but never reach a full response. Two peptides with the same EC50 can therefore behave quite differently, and a table listing EC50 alone hides that difference.
Pathway preference
Many receptors signal through more than one pathway, and some ligands activate one route more than another. When that happens, EC50 values are pathway-specific. Two groups reporting different EC50 values for one peptide at one receptor may both be right if they measured different outputs.
Why your lot and your arithmetic change the number
Every one of these values is a concentration, so it can only be as accurate as the concentration you think you prepared. For labs running potency or binding work across many plates and weeks, a few practical points matter:
- Net peptide content: a lyophilised solid contains counter-ions and water as well as peptide. If you calculate molarity from gross weight and the solid is, say, 80% peptide, every concentration is overstated by a quarter and every EC50 or Ki you report is shifted by the same factor.
- Lot consistency: using a single lot for an entire potency campaign removes one source of variation. When a new lot is introduced, run a bridging curve against the old one before mixing data.
- Record keeping: log the lot number, the certificate details you relied on and the concentration basis beside every potency value in your notebook or database.
- Log-scale averaging: potency values are usually summarised as pEC50 or pKi (the negative log of the molar value), because they are roughly log-normally distributed. Averaging raw nanomolar values overweights the larger ones.
Bulk Peptides products undergo independent HPLC and purity testing. We make certificates available for some products, and each vial’s cap and crimp colour identifies its matching certificate, which is useful when a long assay series draws on one lot.
A checklist for reading published values
- Is the number affinity (Kd, Ki) or function (EC50, functional IC50)?
- Which receptor species and which cell system or tissue?
- For a binding IC50, which reference ligand and at what concentration?
- Which readout, and after how long?
- Is the maximal response reported alongside potency?
- Do any ratios or selectivity claims compare like with like, for example Ki against Ki from one assay format?
A selectivity ratio built from a Ki at one receptor and an EC50 at another is not a meaningful comparison. When all six questions have clear answers, the underlying numbers are usually more informative than the headline summary.
These notes cover in-vitro pharmacology and literature interpretation only. Bulk Peptides supplies research compounds for laboratory use, never for use in people or animals.
