Ligand Binding and Dissociation Constant Quiz

Ligand Binding

• Ligands can bind strongly or weakly to receptors (proteins).
• Strong binding involves numerous interactions (e.g., H-bonds) between the ligand and receptor.
• Weak binding involves fewer interactions, often weaker forces.
• The dissociation constant (K_D) represents the ligand concentration at which half the protein's binding sites are occupied.

Calculating K_D

• K_D = [P][L]/[PL] (where P=protein, L= ligand, and PL=protein-ligand complex)
• K_D can be calculated from a binding isotherm.
• Saturation (fractional saturation equal to 0.5) is a sign of specific binding.
• K_D = [L] when fractional saturation = 0.5
• A low K_D value indicates high affinity between the ligand and its receptor
• A high K_D value indicates low affinity between the ligand and its receptor.

Importance of K_D

• Low K_D values (pM, nM) indicate high ligand affinity for its receptor, meaning the ligand will stay bound longer.
• High K_D values (mM) indicate low ligand affinity.
• Drugs typically have high affinity for targeted protein receptors to achieve desired effects, Drugs may also bind to off-target proteins.
• Off-target effects usually have lower affinities, requiring higher drug concentrations for impact.

Calculating K_D (Graphical Methods)

• Measuring binding over a wide concentration range (nM, μM, mM) can compress data.
• Plotting [PL]/[P] against [L] yields a hyperbolic curve.
• A semi-log plot (log [L] vs. [PL]/[L]) creates a straight line in Scatchard plots

Calculating K_D (Scatchard Plot)

• Scatchard analysis rearranges the basic binding equation to [PL]/[L] = -1/K_D[PL] + [P]_total/K_D.
• Plotting [PL]/[L] against [PL] yields a straight line with a slope of -1/K_D, and the y-intercept giving [P]_total/K_D, enabling calculation of the protein concentration [P]_total.
• The Scatchard plot is used when the concentrations of the protein and ligand are not known.

Calculating K_D (Retinoic Acid)

• Retinoic acid binds to charcoal to separate bound and free retinoic acid and determine K_D
• Scatchard analysis can be used in retinoic analysis for [PL]/[L] vs [PL].
• This method uses charcoal to separate the bound from unbound form of the ligand from the receptor.

Myoglobin

• A monomeric protein in muscle tissue.
• Main oxygen-carrying protein in muscle.
• Gives meat its red color due to bound iron (Fe²⁺) in a heme group.
• Solved by John Kendrew in 1957.
• Combines eight helices with a heme group (containing Fe²⁺) chelated by porphyrin.
• Exhibits hyperbolic oxygen binding curve with a high affinity for oxygen.

Haemoglobin

• A tetrameric protein in red blood cells.
• Transported oxygen from lungs to tissues and some CO₂ to the lungs
• Shows sigmoidal oxygen-binding curve due to positive cooperativity.
• Two conformational states (R and T states)
• Cooperativity enhances oxygen unloading in tissues. Oxygen binding to one subunit facilitates binding to the subsequent subunits.
• Several factors affect hemoglobin's oxygen binding, including pH (Bohr effect), carbon dioxide, and 2,3-BPG (2,3 bisphosphoglycerate).

The Hill Plot

• Used to quantify the cooperativity of binding to hemoglobin and myoglobin.
• Hill Plot involves plotting log [f/(1-f)] against log pO₂ and calculates the Hill coefficient (n).
• n=1 indicates no cooperativity in ligand binding, while n>1 indicates positive cooperativity (e.g., oxygen binding to hemoglobin).
• A lower n value indicates negative cooperativity (e.g. the binding of one ligand hinders the binding of other ligand).
• Allows to determine the degree of cooperativity.

Other factors influencing haemoglobin Binding

• Protons, carbon dioxide, and 2,3-BPG are allosteric regulators of haemoglobin.
• Lower pH (higher H⁺ concentration) reduces haemoglobin's oxygen affinity, which is crucial for oxygen release in tissues and is called the Bohr effect.

Summary of Key Takeaways

• Different methods to calculate K_D for ligand-receptor interactions.
• Understanding myoglobin's and hemoglobin's oxygen binding characteristics.
• Role of cooperative binding in haemoglobin oxygen transport.
• Influence of pH and other factors on hemoglobin's oxygen affinity.
• Application of Hill plots in understanding cooperative ligand binding.