Hi, Can someone break this question down into steps for me please? I have tried

Question

Hi,

Can someone break this question down into steps for me please? I have tried to do it myself and would like to see if my working out and answer is the same. Thanks.

Question:

A candidate drug molecule has an absorption peak at 470nm with an extinction coefficient of 5640 M-1 cm-1. Preliminary experiments with its protein targets demonstrate that these parameters are not changed substantially upon binding. 1ml of a solution containing 50uM protein and 100uM drug candidate is dialysed to equilibrium against a physiological buffer. At equilibrium, the dialysis sac contains 1.1ml of solution with an A470 310 in a spectrophotometer with a path length of 1cm, while the A470 of the external buffer is 0.118. Assuming that the protein has only 1 drug binding site, and that binding the ligand doesn't change its absorbance, calculate the Kd for this interaction.

Notes: For every bound ligand, there is a bound receptor, 1:1 ratio                     Formula for Kd =        [Ligandfree] x [Receptorfree] / [Ligandbound Receptorbound]

Explanation / Answer

Yes, It is framed correctly.

The extinction coefficient is Molar absorptivity or Molar Extinction Coefficient is the path length determined per CM at a particular wave length.

The above given case is a 1 to 1 stoichiometry.

In this 1 :1 interaction, where receptor is the protein and ligand is the drug.

It is an equilibrium dialysis, where receptor (protein) and ligand (drug) are separated by a semipermeable membrane (the dialysis sac) and system attains equilibrium.

Under the standard experimental conditions, where the steady state is achieved . The binding equilibrium between receptor and ligand obeys the law of mass action, the the formula given for dissociation constant Kd is correct.

Usually under defined conditions of solvent, temperature and pH, the molar absorption coefficient of a compound at a particular wave length is constant.

The equilibrium constant can be writtenand calculated in terms of association constant or dissociation constant.

Ka = [RL] / [R] [L]

Kd = [R] [L] / [RL]

Association constant (Ka) and dissociation constant (Kd) are inversly proportional.

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