Biochemistry I Lecture 11 Sept 23, 20051Lecture 11: Ligand Binding MeasurementsRecommended reading: Ligand Binding Handout by Dr. RuleKey Terms:• Association constant• Dissociation constant• Equilibrium dialysis• Scatchard equation & Scatchard plotMeasurements of the binding affinity and binding stoichiometry between molecules andmacromolecules provide essential information on topics that range from mechanism to specificity.In the case of enzymes, binding of the substrate necessarily preceeds the catalytic event.Energetics: The formation of a ligand-protein complex can involve any and all of the fundamentalforces in biochemistry:Energetic Term Native Protein StabilityContribution to Ligand BindingConformational entropy -------Hydrophobic effect ++++Van der Waals interactions +++Hydrogen Bonds ++Electrostatic interactions +11.1 Fundamentals of Ligand Binding:• Ligands collide with their targets at a rate constant of kon. Usually this is diffusion limitedand occurs at about 108 sec-1M-1.• The ligand leaves its binding site with a rate constant that depends on the strength of theinteraction between the ligand and the binding site. Rate constants for dissociation (koff)can range from 106sec-1 (weak binding) to 10-2 sec-1 (strong binding).• The equilibrium constant for binding is given by:† Keq=[ML][M][L]=konkoff= KAwhere [ML] is the macromolecule-ligand complex, [M] is the concentration of free macromolecule,and [L] is the concentration of free ligand. For example, M could be an Fv fragment and L couldbe DNP.KA is the term for the equilibrium constant for association. Note that Keq is no longerdimensionless as it was for protein folding, so remember to work in units of molarity-1.It is very useful to define a dissociation constant, KD:† KD=[M][L][ML]=1KAKD has units of molarity and is the ligand concentration where [M] = [ML]. The KD serves asimilar role as the acidity constant, marking the point of half-saturation.Biochemistry I Lecture 11 Sept 23, 20052• Ligands with low KD values bind tightly.• Ligands with high KD values bind weakly.The fractional saturation, Y, is defined as:† Y =[ML][M] + [ML]=[ML][MT]Y is just the amount of macromolecule with bound ligand [ML], divided by the total concentrationof macromolecule [MT].Using the expression for Keq and KD:† Y =KA[L]1 + KA[L]=[L]KD+ [L]Y varies from 0 to 1. A plot of Y versus [L] is referred to as a saturation binding curve.When [L] = KD, Y = 0.5.Measurement of KD: The dissociation constant, KD, is obtained by measuring Y as a function offree ligand concentration [L]. Once the KD has been determined for a particular macromolecule-ligand combination (e.g. antibody and DNP) then it is possible to predict the fractional saturationat any ligand concentration.11.2 Equilibrium Dialysis: It is common to use equilibrium dialysis for this measurement. Adialysis membrane will allow small ligands to pass through, but will retain macromolecules suchas proteins (M) as well as protein-ligand complexes (ML).The following figures show how the equilibrium dialysis experiment can be used to determine theconcentrations of M, L, and ML at binding equilibrium.Model reaction: M + L ML, or schematically:At the start of the measurement, the protein (M) is present only in the left cell of the dialysischamber. The small molecule (L) is present only in the right cell. The left and right cells areseparated by a semi-permeable membrane, through which only the ligand can pass. Forillustration we will use concentration units of “balls/box” instead of mol/liter.Starting concentrations: (b/B):Left cell: [ML]=0; [L]=0; [M]=4.Right cell: [ML]=0; [L]=12; [M]=0.+Biochemistry I Lecture 11 Sept 23, 20053When equilibrium is reached, the concentration of free ligand [L] will be the same in both cells.However, because the protein can bind the ligand, the concentration of total ligand will be higherin the left cell.Equilibrium concentrations (b/B):Left cell: [ML]=2; [L]=5; [M]=2. Observable [LT]=7.Right cell: [ML]=0; [L]=5; [M]=0. Observable[LT]=5.Experimentally, the ligand concentration inside [L]in andoutside [L]out the dialysis bag is measured.The total amount of macromolecule, [MT], is also known.To calculate the fractional saturation, Y, it is necessary to determine the following:[L] =[ML] = [L]in – [L]out =[M] + [ML] =[MT]=† Y =[ML][M] + [ML]=A complete binding curve is generated by measuring Y at different ligand concentrations.11.3 Example of Data Analysis:1. Acquire binding data as a function of ligand concentration. To do so you need a method ofmeasuring [ML]. This could be by radioactivity (i.e. radioactive ligand) or by measuring changesin absorption of the protein due to binding of the ligand.The following are some example data of Y as a function of [L]:2. Estimate KD from the binding data.3. Plot the data on a Saturation Binding Curve: Equation plotted: † Y =[L]KD+ [L]KD is just the concentration of [L] that gives Y = 0.5(half fractional saturation).[L], mMY0.020.200.050.370.10.570.20.740.40.840.60.931.00.981.00.80.60.40.2000.20.40.60.81.0Binding CurveY[L], mMBiochemistry I Lecture 11 Sept 23, 200544. Plot the data on a Scatchard Plot:From the saturation equation, the following rearrangement can be made to give a usefulanalytical tool, the Scatchard equation:† Y[L]=-1KDY +1KDA plot of Y/[L] versusY will give a straightline with a slope of–1/KD. This is a usefultransformation of theoriginal hyperbolicbinding curve to asimple line, from whichthe dissociationconstant can be readilyobtained.This transformation essentiallyfacilitates:• Interpolation of the data in the region of [L]=KD• Extrapolation to infinite ligand concentrationSlope of line = -1/KD = -KAScatchard Ploty = -11.388x + 12.0220246810120 0.2 0.4 0.6 0.8 1