BIOC 3560 Lecture Notes - Lecture 3: Binding Constant, Hyperbolic Function, Partial Pressure

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26 Oct 2018

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= [l] at which 50% of the ligand-binding sites are occupied. Occupancy = fraction of total binding sites which are occupied. Larger kd = high affinity between the ligand and protein. The fraction of ligand-binding sites occupied, y, is a hyperbolic function of ligand concentration, [l]. When the graph begins to plateau, it means that the proteins are almost fully saturated. For o2-binding proteins, we use the following (oxygen is a gas): po2 = partial pressure of o2. P50 = partial pressure of o2 in the air above the solution at which half the ligand-binding sites are occupied. Protein conformation is critical for specificity of protein-ligand interaction: Co binds free heme with 20,000x greater affinity than o2. Co binds heme in myoglobin (mb) with only 40x the affinity of o2 (a similar effect as seen in hemoglobin) This is due to steric hindrance between his e7 and co, and a favourable h-bond between o2 and his e7.

Related Questions

Answer the bold text. Briefly explain answer.

2. What is the dissociation constant or Kd? How does the Kd relate to the affinity of a protein for its ligand? Does the number of sites occupied by a ligand increase linearly with the [L]? What is fraction or percent bound? How does percent bound relate to the Kd? When I say that the Kd is 20uM what does this mean in terms of [L] and occupied binding sites? How is Kd affected by the rate constants governing the ligand binding event? What is the relationship between Kd and changes in free energy? What changes in free energy would you expect to see for a protein with a high affinity for its ligand?

Dr Vlad is testing the effects of two drugs named D1 and D2 on a protein P found in the blood. He does several tests to characterize how the drugs interact with protein P. He tests them separately and determined that one protein P molecule has only one binding site for D1. Similarly, one protein P interacts only with one molecule of D2.

a) (2pts) First, Dr Vlad, tests the interaction of D1 with P in his lab. At equilibrium: the concentration of free D1 is 10 mM, and the concentration of free P is 50 mM

Given that the affinity constant is for this interaction is Kd=40 mM under these experimental conditions (pH salt, temperature, etc), what is the concentration of complex PD1 at equilibrium?

b (2pts) Then Dr Vlad adds more D1 drug is added to the mixture. When the equilibrium is established again, the concentration of free D1 is now 40 mM. What percentage of P remains free at this new equilibrium? Explain. Note: you need to know that the experimental conditions (pH, salt, temperature, etc) in part b are the same as in part a

c (2pts) The experimental conditions used by Dr Vlad in the lab reflect very closely the conditions in the blood, he can therefore use his data to calculate what happens in the blood. Using his available data, what is the concentration of free drug D1 in the blood when 50% of the protein P in the blood is bound to the drug? Explain your reasoning.

d (2pts) Now Dr Vlad considers the same protein P binding to drug D2. The experimental conditions are the same as those used for D1 above (points a and b).

P and D2 are mixed together: at equilibrium, the concentration of free P is 100 mM and the concentration of free D2 is 100 nM, the concentration of PD2 complex is 10 mM.

Which drug D1 or D2 has the highest affinity for protein P? ________

Explain how you get to that answer

e) (2pts) Dr Vlad accumulated lots of data on how D1 and D2 bind to P. In addition to Kd values (defined or determined above), he also found that the ka value for D1 binding to P is 10^-6 M^-1s^-1 and the ka value for D2 binding to P is 10^-5 M^-1s^-1. Which drug has the fastest association rate for P? And how much faster is that rate compared to the other drug? Explain briefly without calculations.

BIOC 3560 Lecture Notes - Lecture 3: Binding Constant, Hyperbolic Function, Partial Pressure

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