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

The amount of total ligand present is calculated as follows:

c=A/El

= 0.310/5640 M-1 cm-1

= 5.49 x10-5 M

= 0.0549 mM

The total unbound ligand is

=0.118/5640 x 1

= 2.09 x 10-5

= 0.0209 mM

The amount of bound ligand is calculated as,

= Total ligand - unbound ligand

= 0.0549 - 0.0209

= 0.034 mM

Ratio of bound ligand and bound receptor is 1:1. Therefore, there are 0.034 unbound receptors.

Kd = [R unbound] X [L unbound] / [RL bound complex]

= (0.034 x 0.034) / 0.0549

= 0.021

Kd = 0.021 mM

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