first submitted to J.Theor.Biol. in March 95; also rejected
from Biochem.Journal; no response from Trends in Pharm.Sci.; published in Internet in July 96
# Refining Cheng-Prusoff equation
**A generalisation of the Cheng-Prusoff formula relating affinity constant
and the concentration of inhibitor giving 50% inhibition has been derived
for the case when concentrations of ligands are not in a great excess.
It shows that methods ensuring precise solving binding equations (including
computer approaches and equations derived in the present work) can not
be used if these "Cheng-Prusoff conditions" of ligands' excess are not
observed. The trouble is that beyond these conditions analysis becomes
unreliable: the error contained in estimations of necessary parameters
appears abruptly multiplied in the calculated affinity constant.**
**Short demonstration: an amazingly simplified Cheng-Prusoff
eqn.**
Derivation of all equations in the article is based on a single mathematical
trick of introducing new variable **f** - the fraction of free binding
sites in the absence of inhibitor. That is, **f** equals to approx.
unity when the concn. of labeled ligand is small; and it approaches zero
when this concn. is big and labeled ligand (tracer) almost completely saturates
binding sites.
As a demonstration of the amazing effect of this substitution let's
use it to rewrite famous Cheng-Prusoff equations:
** I**_{50}=K_{d
}(1+
T_{0}/K_{d}^{*})
[1]
where **I**_{50 }is the concn. of inhibitor
giving 50% inhibition; **K**_{d }- dissociation constant of
inhibitor-binder reaction; **K**_{d}^{*}- dissociation
const. of tracer-binder reaction and **T**_{0 }is the total
added concn. of the tracer.
So, the new variable fraction **f **of free binding sites is defined
as: **f=(S**_{0}-B_{max}^{*})/S_{0}
where **S**_{0} is the total concentration
of binding sites and **B**_{max}^{* }is the bound
concn. of tracer in the absence of inhibitor (i.e. the maximal signal on
inhibition curve)
Then, in the absence of inhibitor and under the Cheng-Prusoff conditions
the mass action low is written as:
**B**_{max}^{* }=T_{0}(S_{0}-B_{max}^{*})/K_{d}^{*}
Now we replace **B**_{max}^{*} with new variable
**f**that
is **B**_{max}^{*}=(1-f) S_{0
}and
**(S**_{0}-B_{max}^{*})=f
S_{0}
**(1-f)S**_{0}=T_{0}f S_{0}/K_{d}^{*}
after obvious rearrangements:
**1-1/f=T**_{0}/K_{d}^{*}
**1/f=1+T**_{0}/K_{d}^{*}
comparing this with eqn. [1] we see that a much simpler form of Cheng-Prusoff
equation is just:
**I**_{50}=K_{d}/f
[2]
This eqution is mathematically equivalent to Cheng-Prusoff, i.e. it
was derived with the same assumptions and it is valid inder the same conditions.
But this form has substantial practical advantages:
1. Physical meaning of this equation is much more clear: **I**_{50} deviates from affinity
diss. constant if the tracer saturates considerable fraction of binding
sites.
2. Procedure of rigorous using eqn. [2] remains essentially the same
-- as a rule, we still should conduct both inhibition and saturation experiments.
But now we may just ESTIMATE one parameter instead of MEASURING two.
**S**_{0} is the total concentration of binding sites (the
same in inhibition and in saturation experiments, of course); **T**_{0}
is the concentration of tracer used in the inhibition experiment. Note,
that signal **B**_{max}^{*} on inhibition curve
should not ideally coincide (as in this picture) with signal produced by
**T**_{0} concentration of tracer on saturation curve because
of the larger inter-experimental measurement error.
Fraction **f** should be understood on this picture as measured
in units relative to **S**_{0}; that is, here it is equal
approximately to 0.5
3. Eqn. [2] also opens an opportunity to estimate fraction **f** in alternative
experiment when obtaining saturation curve is impossible. This situation
occurs in a not too important immunological problem of measuring "true"
affinity constants, for which monstrous method proposed by Friguet is now
most widely used. Read about it in the full text version.
The article itself derives a generalization of Eqn.[2] valid beyond Cheng-Prusoff conditions which is just:
**I**_{50}=S_{0} + K_{d}/f [3]
where **S**_{0} is total concn. of binding sites.
Unfortunately, this equation proves that it can not be used practicaly beyond "Cheng-Prusoff conditions":
To get K_{d} we have to subtract **S**_{0} from **I**_{50}
Under "Cheng-Prusoff conditions" S_{0} is negligibly small. If it is not small,
the measurement errors contained in values for **I**_{50} and **S**_{0}
appear abruptly muliplied in **I**_{50}-S_{0}. If **S**_{0}
and **I**_{50} values become closer measurement errors may even lead to negative estimate for K_{d}
**So far my works are more frequently plagiarized than cited. Therefore I expect that equations [2] and [3] must have been stolen as well.
I would greatly appreciate information about publications plagiarizing these equations. If you know such instance please write me to dyuryev@yandex.ru
** |