Is the KD value an absolute value?

Hi,

Thank you very much for your last help.

I have one thing curious about KD.

The KD value is usually known as a value that indicates the property of a substance.

However, there is a difference in the KD value among the articles or other experiments.
The difference of KD value between different article or experiment somtimes shows more than 10 times or over even when binding between the same substances is measured.

I wonder if I'm in the wrong.
If you have any reference to the articles about this things, it would be of great help if you let us know.

Thank you for reading.

First what is the equilibrium dissociation constant (KD)? It is the quotient of the dissociation rate and association rate constants (see formula).

From the affinity plot one can deduce that a particular KD (say 1 nM; 1.10-9 M) can be calculated by many different kd and ka pairs. Thus knowing the KD only gives you a feeling over the overall interaction strength but it is impossible to determine if this is due to a high association rate or a slow dissociation rate.

Although the rate constants ka and kd are specific for a specific ligand-analyte pair, they are independent of the concentration of both ligand and analyte. However, the rate constants are dependent on the buffer composition, pH, and temperature of the solution (1). In most cases a different interaction environment give a slightly different answer. For instance some experiments are done at 20°C, 25°C or 37°C and this can give a huge difference {Navratilova, 2007 #1652}. But when experimental conditions are closely monitored the results are comparable (2-5). Therefore, it is important to keep the experimental conditions constant and to describe these in publications.

In addition you should bear in mind that SPR is a biological measurement with its own noise and errors. Just like an ELISA where a intra assay variance of 10% is acceptable, the precision of SPR should not be overestimated.


1. Andersson, K., Choulier, L., Hamalainen, M. D., et al.; Predicting the kinetics of peptide-antibody interactions using a multivariate experimental design of sequence and chemical space. J.Mol.Recognit. (14) 1: 62-71; 2001.
2. Cannon, M. J., Papalia, G. A., Navratilova, I., et al.; Comparative analyses of a small molecule/enzyme interaction by multiple users of Biacore technology. Analytical Biochemistry (330) 1: 98-113; 2004.
3. Katsamba, P. S., Navratilova, I., Calderon-Cacia, M., et al.; Kinetic analysis of a high-affinity antibody/antigen interaction performed by multiple Biacore users. Analytical Biochemistry (352) 2: 208-221; 2006.
4. Navratilova, I., Papalia, G. A., Rich, R. L., et al.; Thermodynamic benchmark study using Biacore technology. Analytical Biochemistry (364) 1: 67-77; 2007.
5. Rich, R. L., Papalia, G. A., Flynn, P. J., et al.; A global benchmark study using affinity-based biosensors. Analytical Biochemistry (386) 194-216; 2008.

Dear Arnoud,

Thanks for your kind advices.

Your information and articles help me a lot, thanks.

but I'm still confusing about KD.

I performed experiment for measuing my Ligand-Analyte KD, and I finally gain KD values about nanomolar.

But in another article, they measured KD about 10^-11M strength.

I use Amine coupling method, and they use Protein A method which wash out analyte and ligand.

Is this difference can effect KD?

If I don't understand your adivce, I'm sorry for that.

Thanks for reading

The method used to measure the kinetics (via amine coupling or capture via ProtA) should not have influence on the kinetic constants. However, when the protein A method is used, the ligand dissociation should be taken into account when fitting the ligand analyte interaction; e.g. decaying surface model.
The main source of error in the curves is probably the concentration analyte used. This is because the analyte concentration directly influences the association rate. If the actual analyte concentration is higher than expected then the association rate constant is higher than expected and the equilibrium dissociation constant becomes lower.

But the difference between the nM-range (10^-9) and 10^-11 M seems a bit big for a concentration mismatch but stranger things happen. As stated before, all experimental conditions (buffer, temperature) can have effect s on the kinetics.

(search for equilibrium in the forum and read the post for extra background)

Kind regards
Arnoud