Determination of Ka and Kd

Hi Arnoud,

I am new to SPR, My question is that how to calculate kinetic parameters Ka and Kd (from sensorgram) and affinity parameters KA and KD for Straptavidin-Biotin interaction? What are the steps I have to go through?

Hi John,

First of all some definitions about how to call and write the kinetic constants.



Note that the k is in italic and the subscripts with the rate constants are small letters (not italic) and with the equilibrium dissociation constant it is a capital D. The relation between these three is:



We are all aware that there are many different ways to describe these constants but this is the only correct notation. In addition, do not use the KA since it wil confuse your results.

When you have a curve and you fit this curve with a 1:1 langmuir model, you will get the k_a and k_d as fitted parameters and you can calculate the K_D as described above.

I am not sure what you mean with a streptavidin-biotin interaction, but since this interaction is highly stable I would not try to measure it because the dissociation rate is outside the instrument specification.

If you want more information, please start with reading the kinetics section on the SPR-pages (or buy the book of which a new version is available at www.sprpages.nl/contact-spr-pages/sprpages-book ).

Kind regards

Arnoud

Thank you, this book has all the information about curve fitting ?? for determining Kd and Bmax, should i consider whole sensorgram or just baseline to association phase, thats what i am confused about.

Hi John,

The book contains all the information to fit sensorgrams. But if you want to learn some curve fitting concepts you can also read the curve fitting guide at ( www.graphpad.com/data-analysis-resource-center/ ). For a better understanding of the sensorgrams, follow the Sensorgrams tutorial at the SPRpages.

To answer your question. To calculate the K_D or k_d (you confused the symbols) and the Rmax (the maximal response) the complete sensorgram should be used, preferable a set of curves with different analyte concentration in order to get a robust result.

Kind regards

Arnoud

Is the curve fitting possible in origin pro8 software for determining these association and dissociation constant??

Thank you

I am not familiar with this software. Probably you have to define the fitting model yourself.

Arnoud