SPR singe concentration kinetics

Hi all,
I've got a question regarding kinetics and affinities.
I understood that we can calculate k_on and k_off from the curve progression of a single analyte concentration and the value for K_D can be calculated by division of these values.
But I also saw that people inject different concentrations of analyte solution and the K_D value is the concentration at R_max1/2.
Could you please tell me what is the difference between these two K_D values? I want to screen many analytes on a polymer gold surface. Do I need to analyze always different concentrations of one analyte to calculate K_D or can I prepare one concentrations for all analytes (e.g. 100 µM) and compare the calculates binding affinity K_D.
How important is the order of kinetics (first, pseudo first, second) when we calculate kinetics? I think I am dealing with a pseudo first order kinetic.

Hi Manfred,

A classical SPR experiment consists of several analyte injections at different concentrations preferably between 0.1x <K_D <10x. When the sensorgram is analysed with a global fitting the k_a, k_d and the Rmax are calculated (fitted). The K_D is calculated from the k_d/k_a. When the analyte injection is long enough then equilibrium can be reached where the total response is not changing any more. At equilibrium the number of association events is the same as the number of dissociation events. Only at equilibrium there is a relation between the response and the analyte concentration. And then you can make a plot of the concentration vs the response (steady state affinity). The K_D is the analyte concentration giving ½ Rmax ( www.sprpages.nl/data-fitting/models/equilibrium-analysis ).
So both ways you can get the K_D and there is no difference but only via the first option provides the k_a and k_d

In general when you have a compound bound to a surface and flow an analyte over it you can use the pseudo first order kinetics.

You could inject a single concentration for your analyte but be aware that the Rmax is dependent on the size of the analyte. I would advise to measure at least two analyte concentrations per analyte to have a better estimate/constrain on the kinetic parameters.
You can do some dissociation ranking, looking only at the dissociation rate, since this is analyte size independent and also independent of the initial starting response.

I know many people want to compare compounds on the K_D. However, in my opinion it is better to compare compounds by the k_a and k_d using an affinity plot.

Kind regards
Arnoud

Hi Arnoud,

thank you very much for your reply.
I am looking for a way to analyze how good different analytes bind on my polymer coated gold substrate and I think it is a good idea to do some dissociation rankings since I want to screen many compounds.

Regarding the k_a values. Do I understand you correct that I can calculate k_a by injecting a single concentration of an analyte and wait until the responsive units stagnates (equilibrium) or which concentration is used to calculate k_a? So can I compare the k_a values of different analytes at one concentration?

If not why?

I think there is a detail missing to make it better understandable for me.

Thank you for your expertise.
I appreciate every good advise.

Hi Manfred,
The equilibrium analysis is not capable of determining the k_a. To calculate the k_a and k_d you have to fit the association and dissociation curves. Comparing association rate values (k_a) is rarely done. In general, we are more interested in compounds that slowly dissociate because that determines the time that the compound is bond.

I suggest you explore the sprpages - look at kinetics and sensorgrams section - to get more insight in how kinetics work. I also can recommend the sprpages book

kind regards
Arnoud