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

Dialysis sac: c=A/El 0.310/5640 M^1 cm^-1 * 1cm = 5.5*10^-5 (total ligand)
External equilibrium: 0.118/5640 * 1 = 2.0922*10^-5 (Unbound ligand)
Convert to mM
Total ligand - unbound ligand = bound ligand
55-20.9= 34.1
Ratio of bound ligand and bound receptor is 1:1
so that means there's 20.9 unbound receptors.
Kd = [R]unbound * [L]unbound/ [RL] bound complex

(20.9*20.9) / 55 =7.942
Kd= 7.942 mM

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