mesters wrote:
Yes!, there is:
the fraction of occupied protein with substance can be calculated: S /
(S + Km) with S being the concentration of the compound.
So, if S = Km, half of the sites are occupied (it follows from
Michaelis-Menten theory).
But- one warning (perhaps obvious but I think worth pointing out):
S here is the _free_ concentration. The total concentration you have
to add must include the bound ligand also. To achieve 50% saturation
you need to add 50% of the protein concentration plus 1 x Km.
In enzyme assays, where the enzyme may be picomolar, the amount
bound is often insignificant. But in crystallization experiments
the protein is often hundreds of micromolar, and Ki's < 1 uM,
the Ki becomes irrelevant- just add stoichiometric ligand and
a little extra.
Ed
In order to saturate the enzyme for 90,90909 % with the compound:
1) S = 10 x Kd (concentration of S at least 10 times the Kd)
and
2) S > P (total concentration of S must be larger than total
concentration of protein or "binding sites")
Depending on the solubility of the compound, this is not always
possible. In such a case, you need to use DMSO and/or add solid compound
to the protein solution and leave it for quite some time for the
compound to finally bind to the protein.
- J. -
yangliuqing wrote:
Hello,everyone,
I have a question for cocrystallization, is there some relationship
between Km value and substrate concentration when making
cocrystallization? How can I know the substrate is enough for binding?
Thank you very much!
liuqing
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