In the lab we used iodoacetate, with the structure shown below, to irreversibly
ID: 518136 • Letter: I
Question
In the lab we used iodoacetate, with the structure shown below, to irreversibly block the thiols of ribonuclease A after reducing the disulfides of the native protein. When this reagent reacts with a thiol, it is actually the thiolate ion that is the reactive species. As a consequence, the rate of reaction depends on the fraction of the thiols that are in the ionized form. For the following, assume that the pKa of a cysteine thiol is 8.3 and that the second-order rate constant for the reaction of the ionized form of a thiol is 10s^-1 M^-1. A) Suppose that you want to carry out a cystein-modification reaction with a protein known to contain a single Cys residue. The reaction will be carried out at a pH 8. At this pH, what fraction of the Cys thiols will be in the reactive form? B) If you were to determine the second-order rate constant for this reaction at pH 8, based on the total concentration of iodoacetamide in the reaction, what value would you expect to find? C) In the reaction, the protein concentration will be 50 pM. Suggest a reasonable concentration for the iodoacetate that will ensure that the reaction kinetics will be well approximated as pseudo first-order. Briefly explain how you decided on this concentration. D) What will the value of the pseudo first-order rate constant be under these conditions? E) Using the iodoacetate concentration you chose in part c, calculate the minimum time for the reaction that will ensure that 99% of the protein molecules will have reactied with iodoacetamide. F) Suggest an experiment that you could carry out to determine the extent to which the reaction has gone to completion. Be sure to describe the possible results of the experiment and how they would be interpreted. Describe any important conditions of the experiment that must be specified and any important controls.Explanation / Answer
Answer for (A):
We can get fraction of cysteine in reactive form or ionized form by using
Henderson-Hasselbach formula:
pH = pKa + log [(base)/(acid)]
In the present case
pH = 8.0
pKa = 8.3 and substituting these values we may get fraction of ionized form of cysteine which can be shown as below:
Cys-SH <===========> H+ + Cys-S-
Accordingly
8 = 8.3 + log [Cys-S-/ Cys-SH]
log [Cys-S-/ Cys-SH] = 8 - 8.3 = -0.3
[Cys-S-/ Cys-SH] = 10-0.3 = 0.5
Fraction of Cys-S-/ Cys-SH = 0.5
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Answer for (B):
For a general reaction:
A + B -------------------> C
The rate law is written as, Rate = k[A]x[B]y,
where k is the rate constant; x and y are the rate orders with respect to individual reactants. The rate orders x and y are not related to the reaction coefficients. Their values must be experimentally determined from a set of kinetic data.
In the present case the rate constant equation will be, if reaction is considered to be second order w.r.t w.r.t. protein and iodoacetamide
Rate = k [iodoacetamide]2 [protein]2
if protein concentration is ignored in case of large excess of it then
Rate = k [iodoacetamide]2
Rate = 10 s-1 M-1
k = ka of cysteine
pKa of cysteine = 8.3
Therefore Ka = 10-8.3 = 5 x 10-9
[Iodoacetamide]2 = Rate/ka = 10/5x10-9 = 2 x 10-9
[Iodoacetamide]= 4.47 x 10-5 M
Total concentration of iodoacetamide in the reaction = 4.47 x 10-5 M
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