I am working with this protein which gives fluorescence after bonding with its chromophore. I have measured the absorbance change after addition of protein to the chromophore to follow the reaction. The reaction occurs in 2 steps - Binding step and the Bonding step. Is it possible to find the Kd value of the binding step from the absorbance curves ? How am I suppose to find the value of Kd?
Do you know whether there is a fluorescence signal for the binding step? If so, and of the bonding step is not too fast, you may be able to use rapid kinetics (stopped flow) to measure the Kd of the binding step.
There so fluorescence for the binding stage but the bonding reaction is slow and has fluorescence. So I have to calculate the concentration change of the chromophore throughout the reaction for example? and am possible to calculate kD? Adam B Shapiro - Thank you very much for the help !
It's a bit complicated because there are two sequential steps:
1. A + B A.B (Kd)
2. A.B => A-B (kreact)
If you measure the rate of absorbance change of the chromophore as a function of protein concentration, the rate is not just related to the Kd, but also kreact. You can measure the combined 2nd-order rate constant kreact/Kd. Measuring Kd, however, requires having an absorbance signal for the binding step. If there is no signal for that step, it may not be possible to measure Kd.
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